Sample records for oligonucleotide array cgh

  1. Design Considerations for Array CGH to OligonucleotideArrays

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    Baldocchi, R.A.; Glynne, R.J.; Chin, K.; Kowbel, D.; Collins, C.; Mack, D.H.; Gray, J.W.


    Background: Representational oligonucleotide microarray analysis has been developed for detection of single nucleotide polymorphisms and/or for genome copy number changes. In this process, the intensity of hybridization to oligonucleotides arrays is increased by hybridizing a polymerase chain reaction (PCR)-amplified representation of reduced genomic complexity. However, hybridization to some oligonucleotides is not sufficiently high to allow precise analysis of that portion of the genome. Methods: In an effort to identify aspects of oligonucleotide hybridization affecting signal intensity, we explored the importance of the PCR product strand to which each oligonucleotide is homologous and the sequence of the array oligonucleotides. We accomplished this by hybridizing multiple PCR-amplified products to oligonucleotide arrays carrying two sense and two antisense 50-mer oligonucleotides for each PCR amplicon. Results: In some cases, hybridization intensity depended more strongly on the PCR amplicon strand (i.e., sense vs. antisense) than on the detection oligonucleotide sequence. In other cases, the oligonucleotide sequence seemed to dominate. Conclusion: Oligonucleotide arrays for analysis of DNA copy number or for single nucleotide polymorphism content should be designed to carry probes to sense and antisense strands of each PCR amplicon to ensure sufficient hybridization and signal intensity.

  2. Comparative analysis of copy number detection by whole-genome BAC and oligonucleotide array CGH

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    Bejjani Bassem A


    Full Text Available Abstract Background Microarray-based comparative genomic hybridization (aCGH is a powerful diagnostic tool for the detection of DNA copy number gains and losses associated with chromosome abnormalities, many of which are below the resolution of conventional chromosome analysis. It has been presumed that whole-genome oligonucleotide (oligo arrays identify more clinically significant copy-number abnormalities than whole-genome bacterial artificial chromosome (BAC arrays, yet this has not been systematically studied in a clinical diagnostic setting. Results To determine the difference in detection rate between similarly designed BAC and oligo arrays, we developed whole-genome BAC and oligonucleotide microarrays and validated them in a side-by-side comparison of 466 consecutive clinical specimens submitted to our laboratory for aCGH. Of the 466 cases studied, 67 (14.3% had a copy-number imbalance of potential clinical significance detectable by the whole-genome BAC array, and 73 (15.6% had a copy-number imbalance of potential clinical significance detectable by the whole-genome oligo array. However, because both platforms identified copy number variants of unclear clinical significance, we designed a systematic method for the interpretation of copy number alterations and tested an additional 3,443 cases by BAC array and 3,096 cases by oligo array. Of those cases tested on the BAC array, 17.6% were found to have a copy-number abnormality of potential clinical significance, whereas the detection rate increased to 22.5% for the cases tested by oligo array. In addition, we validated the oligo array for detection of mosaicism and found that it could routinely detect mosaicism at levels of 30% and greater. Conclusions Although BAC arrays have faster turnaround times, the increased detection rate of oligo arrays makes them attractive for clinical cytogenetic testing.

  3. Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples. (United States)

    Hostetter, Galen; Kim, Su Young; Savage, Stephanie; Gooden, Gerald C; Barrett, Michael; Zhang, Jian; Alla, Lalitamba; Watanabe, April; Einspahr, Janine; Prasad, Anil; Nickoloff, Brian J; Carpten, John; Trent, Jeffrey; Alberts, David; Bittner, Michael


    Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.

  4. Spatial normalization of array-CGH data

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    Brennetot Caroline


    Full Text Available Abstract Background Array-based comparative genomic hybridization (array-CGH is a recently developed technique for analyzing changes in DNA copy number. As in all microarray analyses, normalization is required to correct for experimental artifacts while preserving the true biological signal. We investigated various sources of systematic variation in array-CGH data and identified two distinct types of spatial effect of no biological relevance as the predominant experimental artifacts: continuous spatial gradients and local spatial bias. Local spatial bias affects a large proportion of arrays, and has not previously been considered in array-CGH experiments. Results We show that existing normalization techniques do not correct these spatial effects properly. We therefore developed an automatic method for the spatial normalization of array-CGH data. This method makes it possible to delineate and to eliminate and/or correct areas affected by spatial bias. It is based on the combination of a spatial segmentation algorithm called NEM (Neighborhood Expectation Maximization and spatial trend estimation. We defined quality criteria for array-CGH data, demonstrating significant improvements in data quality with our method for three data sets coming from two different platforms (198, 175 and 26 BAC-arrays. Conclusion We have designed an automatic algorithm for the spatial normalization of BAC CGH-array data, preventing the misinterpretation of experimental artifacts as biologically relevant outliers in the genomic profile. This algorithm is implemented in the R package MANOR (Micro-Array NORmalization, which is described at and available from the Bioconductor site It can also be tested on the CAPweb bioinformatics platform at

  5. CGHPRO – A comprehensive data analysis tool for array CGH

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    Lenzner Steffen


    Full Text Available Abstract Background Array CGH (Comparative Genomic Hybridisation is a molecular cytogenetic technique for the genome wide detection of chromosomal imbalances. It is based on the co-hybridisation of differentially labelled test and reference DNA onto arrays of genomic BAC clones, cDNAs or oligonucleotides, and after correction for various intervening variables, loss or gain in the test DNA can be indicated from spots showing aberrant signal intensity ratios. Now that this technique is no longer confined to highly specialized laboratories and is entering the realm of clinical application, there is a need for a user-friendly software package that facilitates estimates of DNA dosage from raw signal intensities obtained by array CGH experiments, and which does not depend on a sophisticated computational environment. Results We have developed a user-friendly and versatile tool for the normalization, visualization, breakpoint detection and comparative analysis of array-CGH data. CGHPRO is a stand-alone JAVA application that guides the user through the whole process of data analysis. The import option for image analysis data covers several data formats, but users can also customize their own data formats. Several graphical representation tools assist in the selection of the appropriate normalization method. Intensity ratios of each clone can be plotted in a size-dependent manner along the chromosome ideograms. The interactive graphical interface offers the chance to explore the characteristics of each clone, such as the involvement of the clones sequence in segmental duplications. Circular Binary Segmentation and unsupervised Hidden Markov Model algorithms facilitate objective detection of chromosomal breakpoints. The storage of all essential data in a back-end database allows the simultaneously comparative analysis of different cases. The various display options facilitate also the definition of shortest regions of overlap and simplify the

  6. Molecular karyotyping: array CGH quality criteria for constitutional genetic diagnosis. (United States)

    Vermeesch, Joris R; Melotte, Cindy; Froyen, Guy; Van Vooren, Steven; Dutta, Binita; Maas, Nicole; Vermeulen, Stefan; Menten, Björn; Speleman, Frank; De Moor, Bart; Van Hummelen, Paul; Marynen, Peter; Fryns, Jean-Pierre; Devriendt, Koen


    Array CGH (comparative genomic hybridization) enables the identification of chromosomal copy number changes. The availability of clone sets covering the human genome opens the possibility for the widespread use of array CGH for both research and diagnostic purposes. In this manuscript we report on the parameters that were critical for successful implementation of the technology, assess quality criteria, and discuss the potential benefits and pitfalls of the technology for improved pre- and postnatal constitutional genetic diagnosis. We propose to name the genome-wide array CGH "molecular karyotyping," in analogy with conventional karyotyping that uses staining methods to visualize chromosomes.

  7. Model-based clustering of array CGH data

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    Shah, Sohrab P; Cheung, K-John; Johnson, Nathalie A; Alain, Guillaume; Gascoyne, Randy D; Horsman, Douglas E; Ng, Raymond T; Murphy, Kevin P


    Motivation: Analysis of array comparative genomic hybridization (aCGH) data for recurrent DNA copy number alterations from a cohort of patients can yield distinct sets of molecular signatures or profiles...

  8. Array-CGH testing in spontaneous abortions with normal karyotypes

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    Cleide L. Borovik


    Full Text Available In about 50% of first trimester spontaneous abortion the cause remains undetermined after standard cytogenetic investigation. We evaluated the usefulness of array-CGH in diagnosing chromosome abnormalities in products of conception from first trimester spontaneous abortions. Cell culture was carried out in short- and long-term cultures of 54 specimens and cytogenetic analysis was successful in 49 of them. Cytogenetic abnormalities (numerical and structural were detected in 22 (44.89% specimens. Subsequent, array-CGH based on large insert clones spaced at ~1 Mb intervals over the whole genome was used in 17 cases with normal G-banding karyotype. This revealed chromosome aneuplodies in three additional cases, giving a final total of 51% cases in which an abnormal karyotype was detected. In keeping with other recently published works, this study shows that array-CGH detects abnormalities in a further ~10% of spontaneous abortion specimens considered to be normal using standard cytogenetic methods. As such, array-CGH technique may present a suitable complementary test to cytogenetic analysis in cases with a normal karyotype.

  9. Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing. (United States)

    Wiszniewska, Joanna; Bi, Weimin; Shaw, Chad; Stankiewicz, Pawel; Kang, Sung-Hae L; Pursley, Amber N; Lalani, Seema; Hixson, Patricia; Gambin, Tomasz; Tsai, Chun-hui; Bock, Hans-Georg; Descartes, Maria; Probst, Frank J; Scaglia, Fernando; Beaudet, Arthur L; Lupski, James R; Eng, Christine; Cheung, Sau Wai; Bacino, Carlos; Patel, Ankita


    In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60,000 SNP probes, referred to as Chromosomal Microarray Analysis - Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner.

  10. A New Normalizing Algorithm for BAC CGH Arrays with Quality Control Metrics

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    Jeffrey C. Miecznikowski


    Full Text Available The main focus in pin-tip (or print-tip microarray analysis is determining which probes, genes, or oligonucleotides are differentially expressed. Specifically in array comparative genomic hybridization (aCGH experiments, researchers search for chromosomal imbalances in the genome. To model this data, scientists apply statistical methods to the structure of the experiment and assume that the data consist of the signal plus random noise. In this paper we propose “SmoothArray”, a new method to preprocess comparative genomic hybridization (CGH bacterial artificial chromosome (BAC arrays and we show the effects on a cancer dataset. As part of our R software package “aCGHplus,” this freely available algorithm removes the variation due to the intensity effects, pin/print-tip, the spatial location on the microarray chip, and the relative location from the well plate. removal of this variation improves the downstream analysis and subsequent inferences made on the data. Further, we present measures to evaluate the quality of the dataset according to the arrayer pins, 384-well plates, plate rows, and plate columns. We compare our method against competing methods using several metrics to measure the biological signal. With this novel normalization algorithm and quality control measures, the user can improve their inferences on datasets and pinpoint problems that may arise in their BAC aCGH technology.

  11. Whole genome amplification and its impact on CGH array profiles

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    Meldrum Cliff


    Full Text Available Abstract Background Some array comparative genomic hybridisation (array CGH platforms require a minimum of micrograms of DNA for the generation of reliable and reproducible data. For studies where there are limited amounts of genetic material, whole genome amplification (WGA is an attractive method for generating sufficient quantities of genomic material from miniscule amounts of starting material. A range of WGA methods are available and the multiple displacement amplification (MDA approach has been shown to be highly accurate, although amplification bias has been reported. In the current study, WGA was used to amplify DNA extracted from whole blood. In total, six array CGH experiments were performed to investigate whether the use of whole genome amplified DNA (wgaDNA produces reliable and reproducible results. Four experiments were conducted on amplified DNA compared to unamplified DNA and two experiments on unamplified DNA compared to unamplified DNA. Findings All the experiments involving wgaDNA resulted in a high proportion of losses and gains of genomic material. Previously, amplification bias has been overcome by using amplified DNA in both the test and reference DNA. Our data suggests that this approach may not be effective, as the gains and losses introduced by WGA appears to be random and are not reproducible between different experiments using the same DNA. Conclusion In light of these findings, the use of both amplified test and reference DNA on CGH arrays may not provide an accurate representation of copy number variation in the DNA.

  12. Identification of genome-wide copy number variations among diverse pig breeds by array CGH

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    Li Yan


    Full Text Available Abstract Background Recent studies have shown that copy number variation (CNV in mammalian genomes contributes to phenotypic diversity, including health and disease status. In domestic pigs, CNV has been catalogued by several reports, but the extent of CNV and the phenotypic effects are far from clear. The goal of this study was to identify CNV regions (CNVRs in pigs based on array comparative genome hybridization (aCGH. Results Here a custom-made tiling oligo-nucleotide array was used with a median probe spacing of 2506 bp for screening 12 pigs including 3 Chinese native pigs (one Chinese Erhualian, one Tongcheng and one Yangxin pig, 5 European pigs (one Large White, one Pietrain, one White Duroc and two Landrace pigs, 2 synthetic pigs (Chinese new line DIV pigs and 2 crossbred pigs (Landrace × DIV pigs with a Duroc pig as the reference. Two hundred and fifty-nine CNVRs across chromosomes 1–18 and X were identified, with an average size of 65.07 kb and a median size of 98.74 kb, covering 16.85 Mb or 0.74% of the whole genome. Concerning copy number status, 93 (35.91% CNVRs were called as gains, 140 (54.05% were called as losses and the remaining 26 (10.04% were called as both gains and losses. Of all detected CNVRs, 171 (66.02% and 34 (13.13% CNVRs directly overlapped with Sus scrofa duplicated sequences and pig QTLs, respectively. The CNVRs encompassed 372 full length Ensembl transcripts. Two CNVRs identified by aCGH were validated using real-time quantitative PCR (qPCR. Conclusions Using 720 K array CGH (aCGH we described a map of porcine CNVs which facilitated the identification of structural variations for important phenotypes and the assessment of the genetic diversity of pigs.

  13. Detection of clinically relevant exonic copy-number changes by array CGH. (United States)

    Boone, Philip M; Bacino, Carlos A; Shaw, Chad A; Eng, Patricia A; Hixson, Patricia M; Pursley, Amber N; Kang, Sung-Hae L; Yang, Yaping; Wiszniewska, Joanna; Nowakowska, Beata A; del Gaudio, Daniela; Xia, Zhilian; Simpson-Patel, Gayle; Immken, LaDonna L; Gibson, James B; Tsai, Anne C-H; Bowers, Jennifer A; Reimschisel, Tyler E; Schaaf, Christian P; Potocki, Lorraine; Scaglia, Fernando; Gambin, Tomasz; Sykulski, Maciej; Bartnik, Magdalena; Derwinska, Katarzyna; Wisniowiecka-Kowalnik, Barbara; Lalani, Seema R; Probst, Frank J; Bi, Weimin; Beaudet, Arthur L; Patel, Ankita; Lupski, James R; Cheung, Sau Wai; Stankiewicz, Pawel


    Array comparative genomic hybridization (aCGH) is a powerful tool for the molecular elucidation and diagnosis of disorders resulting from genomic copy-number variation (CNV). However, intragenic deletions or duplications--those including genomic intervals of a size smaller than a gene--have remained beyond the detection limit of most clinical aCGH analyses. Increasing array probe number improves genomic resolution, although higher cost may limit implementation, and enhanced detection of benign CNV can confound clinical interpretation. We designed an array with exonic coverage of selected disease and candidate genes and used it clinically to identify losses or gains throughout the genome involving at least one exon and as small as several hundred base pairs in size. In some patients, the detected copy-number change occurs within a gene known to be causative of the observed clinical phenotype, demonstrating the ability of this array to detect clinically relevant CNVs with subkilobase resolution. In summary, we demonstrate the utility of a custom-designed, exon-targeted oligonucleotide array to detect intragenic copy-number changes in patients with various clinical phenotypes.

  14. Estimation of tumor heterogeneity using CGH array data

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    Li Shengting


    Full Text Available Abstract Background Array-based comparative genomic hybridization (CGH is a commonly-used approach to detect DNA copy number variation in whole genome-wide screens. Several statistical methods have been proposed to define genomic segments with different copy numbers in cancer tumors. However, most tumors are heterogeneous and show variation in DNA copy numbers across tumor cells. The challenge is to reveal the copy number profiles of the subpopulations in a tumor and to estimate the percentage of each subpopulation. Results We describe a relation between experimental data and exact DNA copy number and develop a statistical method to reveal the heterogeneity of tumors containing a mixture of different-stage cells. Furthermore, we validate the method on simulated data and apply the method to 29 pairs of breast primary tumors and their matched lymph node metastases. Conclusion We demonstrate a new method for CGH array analysis that allows a tumor sample to be classified according to its heterogeneity. The method gives an interpretable series of copy number profiles, one for each major subpopulation in a tumor. The profiles facilitate identification of copy number alterations in cancer development.

  15. Implementation of High Resolution Whole Genome Array CGH in the Prenatal Clinical Setting: Advantages, Challenges, and Review of the Literature

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    Paola Evangelidou


    Full Text Available Array Comparative Genomic Hybridization analysis is replacing postnatal chromosomal analysis in cases of intellectual disabilities, and it has been postulated that it might also become the first-tier test in prenatal diagnosis. In this study, array CGH was applied in 64 prenatal samples with whole genome oligonucleotide arrays (BlueGnome, Ltd. on DNA extracted from chorionic villi, amniotic fluid, foetal blood, and skin samples. Results were confirmed with Fluorescence In Situ Hybridization or Real-Time PCR. Fifty-three cases had normal karyotype and abnormal ultrasound findings, and seven samples had balanced rearrangements, five of which also had ultrasound findings. The value of array CGH in the characterization of previously known aberrations in five samples is also presented. Seventeen out of 64 samples carried copy number alterations giving a detection rate of 26.5%. Ten of these represent benign or variables of unknown significance, giving a diagnostic capacity of the method to be 10.9%. If karyotype is performed the additional diagnostic capacity of the method is 5.1% (3/59. This study indicates the ability of array CGH to identify chromosomal abnormalities which cannot be detected during routine prenatal cytogenetic analysis, therefore increasing the overall detection rate. In addition a thorough review of the literature is presented.

  16. Reliable single cell array CGH for clinical samples.

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    Zbigniew T Czyż

    Full Text Available BACKGROUND: Disseminated cancer cells (DCCs and circulating tumor cells (CTCs are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape. METHODOLOGY/PRINCIPAL FINDINGS: Using the Ampli1™ whole genome amplification (WGA technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points. CONCLUSIONS/SIGNIFICANCE: The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.

  17. Medical applications of array CGH and the transformation of clinical cytogenetics. (United States)

    Shaffer, L G; Bejjani, B A


    Microarray-based comparative genomic hybridization (array CGH) merges molecular diagnostics with traditional chromosome analysis and is transforming the field of cytogenetics. Prospective studies of individuals with developmental delay and dysmorphic features have demonstrated that array CGH has the ability to detect any genomic imbalance including deletions, duplications, aneuploidies and amplifications. Detection rates for chromosome abnormalities with array CGH range from 5-17% in individuals with normal results from prior routine cytogenetic testing. In addition, copy number variants (CNVs) were identified in all studies. These CNVs may include large-scale variation and can confound the diagnostic interpretations. Although cytogeneticists will require additional training and laboratories must become appropriately equipped, array CGH holds the promise of being the initial diagnostic tool in the identification of visible and submicroscopic chromosome abnormalities in mental retardation and other developmental disabilities.

  18. A hidden Markov model-based algorithm for identifying tumour subtype using array CGH data

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    Zhang Ke


    Full Text Available Abstract Background The recent advancement in array CGH (aCGH research has significantly improved tumor identification using DNA copy number data. A number of unsupervised learning methods have been proposed for clustering aCGH samples. Two of the major challenges for developing aCGH sample clustering are the high spatial correlation between aCGH markers and the low computing efficiency. A mixture hidden Markov model based algorithm was developed to address these two challenges. Results The hidden Markov model (HMM was used to model the spatial correlation between aCGH markers. A fast clustering algorithm was implemented and real data analysis on glioma aCGH data has shown that it converges to the optimal cluster rapidly and the computation time is proportional to the sample size. Simulation results showed that this HMM based clustering (HMMC method has a substantially lower error rate than NMF clustering. The HMMC results for glioma data were significantly associated with clinical outcomes. Conclusions We have developed a fast clustering algorithm to identify tumor subtypes based on DNA copy number aberrations. The performance of the proposed HMMC method has been evaluated using both simulated and real aCGH data. The software for HMMC in both R and C++ is available in ND INBRE website

  19. Genome-Wide Screening of Cytogenetic Abnormalities in Multiple Myeloma Patients Using Array-CGH Technique: A Czech Multicenter Experience

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    Jan Smetana


    Full Text Available Characteristic recurrent copy number aberrations (CNAs play a key role in multiple myeloma (MM pathogenesis and have important prognostic significance for MM patients. Array-based comparative genomic hybridization (aCGH provides a powerful tool for genome-wide classification of CNAs and thus should be implemented into MM routine diagnostics. We demonstrate the possibility of effective utilization of oligonucleotide-based aCGH in 91 MM patients. Chromosomal aberrations associated with effect on the prognosis of MM were initially evaluated by I-FISH and were found in 93.4% (85/91. Incidence of hyperdiploidy was 49.5% (45/91; del(13(q14 was detected in 57.1% (52/91; gain(1(q21 occurred in 58.2% (53/91; del(17(p13 was observed in 15.4% (14/91; and t(4;14(p16;q32 was found in 18.6% (16/86. Genome-wide screening using Agilent 44K aCGH microarrays revealed copy number alterations in 100% (91/91. Most common deletions were found at 13q (58.9%, 1p (39.6%, and 8p (31.1%, whereas gain of whole 1q was the most often duplicated region (50.6%. Furthermore, frequent homozygous deletions of genes playing important role in myeloma biology such as TRAF3, BIRC1/BIRC2, RB1, or CDKN2C were observed. Taken together, we demonstrated the utilization of aCGH technique in clinical diagnostics as powerful tool for identification of unbalanced genomic abnormalities with prognostic significance for MM patients.

  20. High-resolution SNP array analysis of patients with developmental disorder and normal array CGH results

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    Siggberg Linda


    Full Text Available Abstract Background Diagnostic analysis of patients with developmental disorders has improved over recent years largely due to the use of microarray technology. Array methods that facilitate copy number analysis have enabled the diagnosis of up to 20% more patients with previously normal karyotyping results. A substantial number of patients remain undiagnosed, however. Methods and Results Using the Genome-Wide Human SNP array 6.0, we analyzed 35 patients with a developmental disorder of unknown cause and normal array comparative genomic hybridization (array CGH results, in order to characterize previously undefined genomic aberrations. We detected no seemingly pathogenic copy number aberrations. Most of the vast amount of data produced by the array was polymorphic and non-informative. Filtering of this data, based on copy number variant (CNV population frequencies as well as phenotypically relevant genes, enabled pinpointing regions of allelic homozygosity that included candidate genes correlating to the phenotypic features in four patients, but results could not be confirmed. Conclusions In this study, the use of an ultra high-resolution SNP array did not contribute to further diagnose patients with developmental disorders of unknown cause. The statistical power of these results is limited by the small size of the patient cohort, and interpretation of these negative results can only be applied to the patients studied here. We present the results of our study and the recurrence of clustered allelic homozygosity present in this material, as detected by the SNP 6.0 array.

  1. Phenotype in patients with intellectual disability and pathological results in array CGH. (United States)

    Caballero Pérez, V; López Pisón, F J; Miramar Gallart, M D; González Álvarez, A; García Jiménez, M C; García Iñiguez, J P; Orden Rueda, C; Gil Hernández, I; Fuertes Rodrigo, C; Fernando Martínez, R; Rodríguez Valle, A; Alcaine Villarroya, M J


    Global developmental delay (GDD) and intellectual disability (ID) are frequent reasons for consultation in paediatric neurology departments. Nowadays, array comparative genomic hybridisation (array-CGH) is one of the most widely used techniques for diagnosing these disorders. Our purpose was to determine the phenotypic features associated with pathological results in this genetic test. We conducted a blind study of the epidemiological, clinical, anthropometric, and morphological features of 80 patients with unexplained ID to determine which features were associated with pathological results in array-CGH. Pathological results were found in 27.5% of the patients. Factors associated with pathological results in array-CGH were a family history of GDD/ID (OR = 12.1), congenital malformations (OR = 5.33), having more than 3 facial dysmorphic features (OR = 20.9), and hypotonia (OR = 3.25). Our findings are consistent with those reported by other published series. We therefore conclude that the probability of having pathological results in array-CGH increases with the presence of any of the features mentioned above in patients with ID/GDD. Copyright © 2016 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

  2. Detection and precise mapping of germline rearrangements in BRCA1, BRCA2, MSH2, and MLH1 using zoom-in array comparative genomic hybridization (aCGH)

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    Staaf, Johan; Törngren, Therese; Rambech, Eva


    Disease-predisposing germline mutations in cancer susceptibility genes may consist of large genomic rearrangements that are challenging to detect and characterize using standard PCR-based mutation screening methods. Here, we describe a custom-made zoom-in microarray comparative genomic...... hybridization (CGH) platform of 60mer oligonucleotides. The 4 x 44 K array format provides high-resolution coverage (200-300 bp) of 400-700 kb genomic regions surrounding six cancer susceptibility genes. We evaluate its performance to accurately detect and precisely map earlier described or novel large germline...

  3. Analysis of Array-CGH Data Using the R and Bioconductor Software Suite

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    Winfried A. Hofmann


    Full Text Available Background. Array-based comparative genomic hybridization (array-CGH is an emerging high-resolution and high-throughput molecular genetic technique that allows genome-wide screening for chromosome alterations. DNA copy number alterations (CNAs are a hallmark of somatic mutations in tumor genomes and congenital abnormalities that lead to diseases such as mental retardation. However, accurate identification of amplified or deleted regions requires a sequence of different computational analysis steps of the microarray data. Results. We have developed a user-friendly and versatile tool for the normalization, visualization, breakpoint detection, and comparative analysis of array-CGH data which allows the accurate and sensitive detection of CNAs. Conclusion. The implemented option for the determination of minimal altered regions (MARs from a series of tumor samples is a step forward in the identification of new tumor suppressor genes or oncogenes.

  4. X chromosome array-CGH for the identification of novel X-linked mental retardation genes. (United States)

    Bauters, Marijke; Van Esch, Hilde; Marynen, Peter; Froyen, Guy


    Array-CGH technology for the detection of submicroscopic copy number changes in the genome has recently been developed for the identification of novel disease-associated genes. It has been estimated that submicroscopic genomic deletions or duplications will be present in 5-7% of patients with idiopathic mental retardation (MR). Since 30% more males than females are diagnosed with MR, we have developed a full coverage X chromosome array-CGH with a theoretical resolution of 82 kb, for the detection of copy number alterations in patients with suspected X-linked mental retardation (XLMR). First, we have validated the genomic location of X-derived clones through male versus female hybridisations. Next, we validated our array for efficient and reproducible detection of known alterations in XLMR patients. In all cases, we were able to detect the deletions and duplications in males as well as females. Due to the high resolution of our X-array, the boundaries of the genomic aberrations could clearly be identified making genotype-phenotype studies more reliable. Here, we describe the production and validation of a full coverage X-array-CGH, which will allow for fast and easy screening of submicroscopic copy number alterations in XLMR patients with the aim to identify novel MR genes or mechanisms involved in a deranged cognitive development.

  5. Insertional translocation detected using FISH confirmation of array-comparative genomic hybridization (aCGH) results. (United States)

    Kang, Sung-Hae L; Shaw, Chad; Ou, Zhishuo; Eng, Patricia A; Cooper, M Lance; Pursley, Amber N; Sahoo, Trilochan; Bacino, Carlos A; Chinault, A Craig; Stankiewicz, Pawel; Patel, Ankita; Lupski, James R; Cheung, Sau Wai


    Insertional translocations (ITs) are rare events that require at least three breaks in the chromosomes involved and thus qualify as complex chromosomal rearrangements (CCR). In the current study, we identified 40 ITs from approximately 18,000 clinical cases (1:500) using array-comparative genomic hybridization (aCGH) in conjunction with fluorescence in situ hybridization (FISH) confirmation of the aCGH findings, and parental follow-up studies. Both submicroscopic and microscopically visible IT events were detected. They were divided into three major categories: (1) simple intrachromosomal and interchromosomal IT resulting in pure segmental trisomy, (2) complex IT involving more than one abnormality, (3) deletion inherited from a parent with a balanced IT resulting in pure segmental monosomy. Of the cases in which follow-up parental studies were available, over half showed inheritance from an apparently unaffected parent carrying the same unbalanced rearrangement detected in the propositi, thus decreasing the likelihood that these IT events are clinically relevant. Nevertheless, we identified six cases in which small submicroscopic events were detected involving known disease-associated genes/genomic segments and are likely to be pathogenic. We recommend that copy number gains detected by clinical aCGH analysis should be confirmed using FISH analysis whenever possible in order to determine the physical location of the duplicated segment. We hypothesize that the increased use of aCGH in the clinic will demonstrate that IT occurs more frequently than previously considered but can identify genomic rearrangements with unclear clinical significance.

  6. Array CGH improves detection of mutations in the GALC gene associated with Krabbe disease

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    Tanner Alice K


    Full Text Available Abstract Background Krabbe disease is an autosomal recessive lysosomal storage disorder caused by mutations in the GALC gene. The most common mutation in the Caucasian population is a 30-kb deletion of exons 11 through 17. There are few other reports of intragenic GALC deletions or duplications, due in part to difficulties detecting them. Methods and results We used gene-targeted array comparative genomic hybridization (CGH to analyze the GALC gene in individuals with Krabbe disease in whom sequence analysis with 30-kb deletion analysis identified only one mutation. In our sample of 33 cases, traditional approaches failed to identify two pathogenic mutations in five (15.2% individuals with confirmed Krabbe disease. The addition of array CGH deletion/duplication analysis to the genetic testing strategy led to the identification of a second pathogenic mutation in three (9.1% of these five individuals. In all three cases, the deletion or duplication identified through array CGH was a novel GALC mutation, including the only reported duplication in the GALC gene, which would have been missed by traditional testing methodologies. We report these three cases in detail. The second mutation remains unknown in the remaining two individuals (6.1%, despite our full battery of testing. Conclusions Analysis of the GALC gene using array CGH deletion/duplication testing increased the two-mutation detection rate from 84.8% to 93.9% in affected individuals. Better mutation detection rates are important for improving molecular diagnosis of Krabbe disease, as well as for providing prenatal and carrier testing in family members.

  7. Mulibrey nanism: Two novel mutations in a child identified by Array CGH and DNA sequencing. (United States)

    Mozzillo, Enza; Cozzolino, Carla; Genesio, Rita; Melis, Daniela; Frisso, Giulia; Orrico, Ada; Lombardo, Barbara; Fattorusso, Valentina; Discepolo, Valentina; Della Casa, Roberto; Simonelli, Francesca; Nitsch, Lucio; Salvatore, Francesco; Franzese, Adriana


    In childhood, several rare genetic diseases have overlapping symptoms and signs, including those regarding growth alterations, thus the differential diagnosis is sometimes difficult. The proband, aged 3 years, was suspected to have Silver-Russel syndrome because of intrauterine growth retardation, postnatal growth retardation, typical facial dysmorphic features, macrocephaly, body asymmetry, and bilateral fifth finger clinodactyly. Other features were left atrial and ventricular enlargement and patent foramen ovale. Total X-ray skeleton showed hypoplasia of the twelfth rib bilaterally and of the coccyx, slender long bones with thick cortex, and narrow medullary channels. The genetic investigation did not confirm Silver-Russel syndrome. At the age of 5 the patient developed an additional sign: hepatomegaly. Array CGH revealed a 147 kb deletion (involving TRIM 37 and SKA2 genes) on one allele of chromosome 17, inherited from his mother. These results suggested Mulibrey nanism. The clinical features were found to fit this hypothesis. Sequencing of the TRIM 37 gene showed a single base change at a splicing locus, inherited from his father that provoked a truncated protein. The combined use of Array CGH and DNA sequencing confirmed diagnosis of Mulibrey nanism. The large deletion involving the SKA2 gene, along with the increased frequency of malignant tumours in mulibrey patients, suggests closed monitoring for cancer of our patient and his mother. Array CGH should be performed as first tier test in all infants with multiple anomalies. The clinician should reconsider the clinical features when the genetics suggests this. © 2016 Wiley Periodicals, Inc.

  8. Integrated high-resolution array CGH and SKY analysis of homozygous deletions and other genomic alterations present in malignant mesothelioma cell lines. (United States)

    Klorin, Geula; Rozenblum, Ester; Glebov, Oleg; Walker, Robert L; Park, Yoonsoo; Meltzer, Paul S; Kirsch, Ilan R; Kaye, Frederic J; Roschke, Anna V


    High-resolution oligonucleotide array comparative genomic hybridization (aCGH) and spectral karyotyping (SKY) were applied to a panel of malignant mesothelioma (MMt) cell lines. SKY has not been applied to MMt before, and complete karyotypes are reported based on the integration of SKY and aCGH results. A whole genome search for homozygous deletions (HDs) produced the largest set of recurrent and non-recurrent HDs for MMt (52 recurrent HDs in 10 genomic regions; 36 non-recurrent HDs). For the first time, LINGO2, RBFOX1/A2BP1, RPL29, DUSP7, and CCSER1/FAM190A were found to be homozygously deleted in MMt, and some of these genes could be new tumor suppressor genes for MMt. Integration of SKY and aCGH data allowed reconstruction of chromosomal rearrangements that led to the formation of HDs. Our data imply that only with acquisition of structural and/or numerical karyotypic instability can MMt cells attain a complete loss of tumor suppressor genes located in 9p21.3, which is the most frequently homozygously deleted region. Tetraploidization is a late event in the karyotypic progression of MMt cells, after HDs in the 9p21.3 region have already been acquired.

  9. Molecular karyotyping by array CGH in a Russian cohort of children with intellectual disability, autism, epilepsy and congenital anomalies

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    Iourov Ivan Y


    Full Text Available Abstract Background Array comparative genomic hybridization (CGH has been repeatedly shown to be a successful tool for the identification of genomic variations in a clinical population. During the last decade, the implementation of array CGH has resulted in the identification of new causative submicroscopic chromosome imbalances and copy number variations (CNVs in neuropsychiatric (neurobehavioral diseases. Currently, array-CGH-based technologies have become an integral part of molecular diagnosis and research in individuals with neuropsychiatric disorders and children with intellectual disability (mental retardation and congenital anomalies. Here, we introduce the Russian cohort of children with intellectual disability, autism, epilepsy and congenital anomalies analyzed by BAC array CGH and a novel bioinformatic strategy. Results Among 54 individuals highly selected according to clinical criteria and molecular and cytogenetic data (from 2426 patients evaluated cytogenetically and molecularly between November 2007 and May 2012, chromosomal imbalances were detected in 26 individuals (48%. In two patients (4%, a previously undescribed condition was observed. The latter has been designated as meiotic (constitutional genomic instability resulted in multiple submicroscopic rearrangements (including CNVs. Using bioinformatic strategy, we were able to identify clinically relevant CNVs in 15 individuals (28%. Selected cases were confirmed by molecular cytogenetic and molecular genetic methods. Eight out of 26 chromosomal imbalances (31% have not been previously reported. Among them, three cases were co-occurrence of subtle chromosome 9 and 21 deletions. Conclusions We conducted an array CGH study of Russian patients suffering from intellectual disability, autism, epilepsy and congenital anomalies. In total, phenotypic manifestations of clinically relevant genomic variations were found to result from genomic rearrangements affecting 1247 disease

  10. The communication of secondary variants: interviews with parents whose children have undergone array-CGH testing. (United States)

    Christenhusz, G M; Devriendt, K; Peeters, H; Van Esch, H; Dierickx, K


    Children with unexplained developmental disabilities or congenital anomalies are increasingly being referred for genetic diagnostic testing using array-comparative genomic hybridisation (array-CGH) and next-generation sequencing (NGS) technologies. Their parents will have to deal with the secondary variants that will inevitably arise. We conducted 16 prospective semi-structured interviews with native Dutch-speaking parents whose children had undergone clinical array-CGH testing. The interviews explored the parents' experiences, expectations and opinions, specifically regarding the communication of results. Concrete examples of 'unexpected results' were provided to help guide the discussion, differing in severity, treatability, time of onset, level of risk, and carrier status. Data was analysed using content and narrative analysis methodologies. Parental motivations for and against the disclosure of unexpected results cluster around four main themes: actionability; knowledge; context; and characteristics of the result. Most parents wished to know all types of results. Disclosure was framed within a holistic, contextual, family-wide view. Genetic counselling should aim to integrate explorations of the motivations of parents surrounding the disclosure of results with good clinical care. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Array CGH demonstrates characteristic aberration signatures in human papillary thyroid carcinomas governed by RET/PTC. (United States)

    Unger, K; Malisch, E; Thomas, G; Braselmann, H; Walch, A; Jackl, G; Lewis, P; Lengfelder, E; Bogdanova, T; Wienberg, J; Zitzelsberger, H


    The aim of this study is to investigate additional genetic alterations in papillary thyroid carcinomas (PTCs) with known RET/PTC rearrangements. We applied array-based comparative genomic hybridization (array CGH) to 33 PTC (20 PTC from adults, 13 post-Chernobyl PTC from children) with known RET/PTC status. Principal component analysis and hierarchical cluster analysis identified cases with similar aberration patterns. Significant deviations between tumour-groups were obtained by statistical testing (Fisher's exact test in combination with Benjamini-Hochberg FDR-controlling procedure). FISH analysis on FFPE sections was applied to validate the array CGH data. Deletions were found more frequently in RET/PTC-positive and RET/PTC-negative tumours than amplifications. Specific aberration signatures were identified that discriminated between RET/PTC-positive and RET/PTC-negative cases (aberrations on chromosomes 1p, 3q, 4p, 7p, 9p/q, 10q, 12q, 13q and 21q). In addition, childhood and adult RET/PTC-positive cases differ significantly for a deletion on the distal part of chromosome 1p. There are additional alterations in RET/PTC-positive tumours, which may act as modifiers of RET activation. In contrast, alterations in RET/PTC-negative tumours indicate alternative routes of tumour development. The data presented serve as a starting point for further studies on gene expression and function of genes identified in this study.

  12. Supernumerary marker chromosomes derived from chromosome 6: cytogenetic, molecular cytogenetic, and array CGH characterization. (United States)

    Huang, Bing; Pearle, Phyllis; Rauen, Katherine A; Cotter, Philip D


    Supernumerary marker chromosomes (SMC) are relatively common in prenatal diagnosis. As the clinical outcomes vary greatly, a better understanding of the karyotype-phenotype correlation for different SMCs will be important for genetic counseling. We present two cases of prenatally detected de novo, small SMCs. The markers were present in 80% of amniocyte colonies in Case 1 and 38% of the colonies in Case 2. The SMCs were determined to be derived from chromosome 6 during postnatal confirmation studies. Although the sizes and the chromosomal origin of the SMCs in these two cases appeared to be similar, the clinical outcomes varied. The clinical manifestations observed in Case 1 included small for gestational age, feeding difficulty at birth, hydronephrosis, deviated septum and dysmorphic features, while the phenotype is apparently normal in Case 2. Array comparative genomic hybridization (CGH) was performed and showed increase in dosage for approximately 26 Mb of genetic material from the proximal short and long arms of chromosome 6 in Case 1. Results of array CGH were uninformative in Case 2, either due to mosaicism or lack of detectable euchromatin. The difference in the clinical presentation in these two patients may have resulted from the difference in the actual gene contents of the marker chromosomes and/or the differential distribution of the mosaicism.

  13. Genomic analysis by oligonucleotide array Comparative Genomic Hybridization utilizing formalin-fixed, paraffin-embedded tissues. (United States)

    Savage, Stephanie J; Hostetter, Galen


    Formalin fixation has been used to preserve tissues for more than a hundred years, and there are currently more than 300 million archival samples in the United States alone. The application of genomic protocols such as high-density oligonucleotide array Comparative Genomic Hybridization (aCGH) to formalin-fixed, paraffin-embedded (FFPE) tissues, therefore, opens an untapped resource of available tissues for research and facilitates utilization of existing clinical data in a research sample set. However, formalin fixation results in cross-linking of proteins and DNA, typically leading to such a significant degradation of DNA template that little is available for use in molecular applications. Here, we describe a protocol to circumvent formalin fixation artifact by utilizing enzymatic reactions to obtain quality DNA from a wide range of FFPE tissues for successful genome-wide discovery of gene dosage alterations in archival clinical samples.

  14. [Subchromosomal microdeletion identified by molecular karyotyping using DNA microarrays (array CGH) in Rett syndrome girls negative for MECP2 gene mutations]. (United States)

    Vorsanova, S G; Iurov, I Iu; Voinova, V Iu; Kurinnaia, O S; Zelenova, M A; Demidova, I A; Ulas, E V; Iurov, Iu B


    Molecular karyotyping using DNA microarrays (array CGH) was applied for identification of subchromosomal microdeletions in a cohort of 12 girls with clinical features of RETT syndrome, but negative for MECP2 gene mutations. Recurrent microdeletions of MECP2 gene in chromosome X (locus Xq28) were identified in 5 girls of 12 studied. Probably RTT girls with subchromosomic microdeletions in Xq28 could represent a special subtype of the disease, which appears as clinically milder than the classic form of disease. In one case, an atypical form of RTT was associated with genomic abnormalities affecting CDKL5 gene and region critical for microdeletion Prader-Willi and Angelman syndromes (15q11.2). In addition, data are presented for the first time that genetic variation in regions 3p13, 3q27.1, and 1q21.1-1q21.2 could associate with RTT-like clinical manifestations. Without application of molecular karyotyping technology and bioinformatic method of assessing the pathogenic significance of genomic rearrangements these RTT-like girls negative for MECP2 gene mutations were considered as cases of idiopathic mental retardation associated with autism. It should be noted that absence of intragenic mutations in MECP2 gene is not sufficient criteria to reject the clinical diagnosis of RTT. To avoid errors in the genetic diagnosis of this genetically heterogeneous brain disease molecular cytogenetic studies using high resolution oligonucleotide array CGH (molecular karyotyping) are needed.

  15. A model-based circular binary segmentation algorithm for the analysis of array CGH data

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    Tu Shih-Hsin


    Full Text Available Abstract Background Circular Binary Segmentation (CBS is a permutation-based algorithm for array Comparative Genomic Hybridization (aCGH data analysis. CBS accurately segments data by detecting change-points using a maximal-t test; but extensive computational burden is involved for evaluating the significance of change-points using permutations. A recent implementation utilizing a hybrid method and early stopping rules (hybrid CBS to improve the performance in speed was subsequently proposed. However, a time analysis revealed that a major portion of computation time of the hybrid CBS was still spent on permutation. In addition, what the hybrid method provides is an approximation of the significance upper bound or lower bound, not an approximation of the significance of change-points itself. Results We developed a novel model-based algorithm, extreme-value based CBS (eCBS, which limits permutations and provides robust results without loss of accuracy. Thousands of aCGH data under null hypothesis were simulated in advance based on a variety of non-normal assumptions, and the corresponding maximal-t distribution was modeled by the Generalized Extreme Value (GEV distribution. The modeling results, which associate characteristics of aCGH data to the GEV parameters, constitute lookup tables (eXtreme model. Using the eXtreme model, the significance of change-points could be evaluated in a constant time complexity through a table lookup process. Conclusions A novel algorithm, eCBS, was developed in this study. The current implementation of eCBS consistently outperforms the hybrid CBS 4× to 20× in computation time without loss of accuracy. Source codes, supplementary materials, supplementary figures, and supplementary tables can be found at

  16. De novo complex intra chromosomal rearrangement after ICSI: characterisation by BACs micro array-CGH (United States)

    Kasakyan, Serdar; Lohmann, Laurence; Aboura, Azeddine; Quimsiyeh, Mazin; Menezo, Yves; Tachdjian, Gerard; Benkhalifa, Moncef


    Background In routine Assisted Reproductive Technology (ART) men with severe oligozoospermia or azoospermia should be informed about the risk of de novo congenital or chromosomal abnormalities in ICSI program. Also the benefits of preimplantation or prenatal genetic diagnosis practice need to be explained to the couple. Methods From a routine ICSI attempt, using ejaculated sperm from male with severe oligozoospermia and having normal karyotype, a 30 years old pregnant woman was referred to prenatal diagnosis in the 17th week for bichorionic biamniotic twin gestation. Amniocentesis was performed because of the detection of an increased foetal nuchal translucency for one of the fetus by the sonographic examination during the 12th week of gestation (WG). Chromosome and DNA studies of the fetus were realized on cultured amniocytes Results Conventional, molecular cytogenetic and microarray CGH experiments allowed us to conclude that the fetus had a de novo pericentromeric inversion associated with a duplication of the 9p22.1-p24 chromosomal region, 46,XY,invdup(9)(p22.1p24) [arrCGH 9p22.1p24 (RP11-130C19 → RP11-87O1)x3]. As containing the critical 9p22 region, our case is in coincidence with the general phenotype features of the partial trisomy 9p syndrome with major growth retardation, microcephaly and microretrognathia. Conclusion This de novo complex chromosome rearrangement illustrates the possible risk of chromosome or gene defects in ICSI program and the contribution of array-CGH for mapping rapidly de novo chromosomal imbalance. PMID:19105807

  17. De novo complex intra chromosomal rearrangement after ICSI: characterisation by BACs micro array-CGH. (United States)

    Kasakyan, Serdar; Lohmann, Laurence; Aboura, Azeddine; Quimsiyeh, Mazin; Menezo, Yves; Tachdjian, Gerard; Benkhalifa, Moncef


    In routine Assisted Reproductive Technology (ART) men with severe oligozoospermia or azoospermia should be informed about the risk of de novo congenital or chromosomal abnormalities in ICSI program. Also the benefits of preimplantation or prenatal genetic diagnosis practice need to be explained to the couple. From a routine ICSI attempt, using ejaculated sperm from male with severe oligozoospermia and having normal karyotype, a 30 years old pregnant woman was referred to prenatal diagnosis in the 17th week for bichorionic biamniotic twin gestation. Amniocentesis was performed because of the detection of an increased foetal nuchal translucency for one of the fetus by the sonographic examination during the 12th week of gestation (WG). Chromosome and DNA studies of the fetus were realized on cultured amniocytes Conventional, molecular cytogenetic and microarray CGH experiments allowed us to conclude that the fetus had a de novo pericentromeric inversion associated with a duplication of the 9p22.1-p24 chromosomal region, 46,XY,invdup(9)(p22.1p24) [arrCGH 9p22.1p24 (RP11-130C19 --> RP11-87O1)x3]. As containing the critical 9p22 region, our case is in coincidence with the general phenotype features of the partial trisomy 9p syndrome with major growth retardation, microcephaly and microretrognathia. This de novo complex chromosome rearrangement illustrates the possible risk of chromosome or gene defects in ICSI program and the contribution of array-CGH for mapping rapidly de novo chromosomal imbalance.

  18. De novo complex intra chromosomal rearrangement after ICSI: characterisation by BACs micro array-CGH

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    Quimsiyeh Mazin


    Full Text Available Abstract Background In routine Assisted Reproductive Technology (ART men with severe oligozoospermia or azoospermia should be informed about the risk of de novo congenital or chromosomal abnormalities in ICSI program. Also the benefits of preimplantation or prenatal genetic diagnosis practice need to be explained to the couple. Methods From a routine ICSI attempt, using ejaculated sperm from male with severe oligozoospermia and having normal karyotype, a 30 years old pregnant woman was referred to prenatal diagnosis in the 17th week for bichorionic biamniotic twin gestation. Amniocentesis was performed because of the detection of an increased foetal nuchal translucency for one of the fetus by the sonographic examination during the 12th week of gestation (WG. Chromosome and DNA studies of the fetus were realized on cultured amniocytes Results Conventional, molecular cytogenetic and microarray CGH experiments allowed us to conclude that the fetus had a de novo pericentromeric inversion associated with a duplication of the 9p22.1-p24 chromosomal region, 46,XY,invdup(9(p22.1p24 [arrCGH 9p22.1p24 (RP11-130C19 → RP11-87O1x3]. As containing the critical 9p22 region, our case is in coincidence with the general phenotype features of the partial trisomy 9p syndrome with major growth retardation, microcephaly and microretrognathia. Conclusion This de novo complex chromosome rearrangement illustrates the possible risk of chromosome or gene defects in ICSI program and the contribution of array-CGH for mapping rapidly de novo chromosomal imbalance.

  19. Multiple Break-Points Detection in Array CGH Data via the Cross-Entropy Method. (United States)

    Priyadarshana, W J R M; Sofronov, Georgy


    Array comparative genome hybridization (aCGH) is a widely used methodology to detect copy number variations of a genome in high resolution. Knowing the number of break-points and their corresponding locations in genomic sequences serves different biological needs. Primarily, it helps to identify disease-causing genes that have functional importance in characterizing genome wide diseases. For human autosomes the normal copy number is two, whereas at the sites of oncogenes it increases (gain of DNA) and at the tumour suppressor genes it decreases (loss of DNA). The majority of the current detection methods are deterministic in their set-up and use dynamic programming or different smoothing techniques to obtain the estimates of copy number variations. These approaches limit the search space of the problem due to different assumptions considered in the methods and do not represent the true nature of the uncertainty associated with the unknown break-points in genomic sequences. We propose the Cross-Entropy method, which is a model-based stochastic optimization technique as an exact search method, to estimate both the number and locations of the break-points in aCGH data. We model the continuous scale log-ratio data obtained by the aCGH technique as a multiple break-point problem. The proposed methodology is compared with well established publicly available methods using both artificially generated data and real data. Results show that the proposed procedure is an effective way of estimating number and especially the locations of break-points with high level of precision. Availability: The methods described in this article are implemented in the new R package breakpoint and it is available from the Comprehensive R Archive Network at

  20. Genomic analysis of clonal eosinophils by CGH arrays reveals new genetic regions involved in chronic eosinophilia. (United States)

    Arefi, Maryam; Robledo, Cristina; Peñarrubia, María J; García de Coca, Alfonso; Cordero, Miguel; Hernández-Rivas, Jesús M; García, Juan Luis


    To assess the presence of genetic imbalances in patients with myeloproliferative neoplasms (MPNs), 38 patients with chronic eosinophilia were studied by array comparative genomic hybridization (aCGH): seven had chronic myelogenous leukaemia (CML), BCR-ABL1 positive, nine patients had myeloproliferative neoplasia Ph- (MPN-Ph-), three had a myeloid neoplasm associated with a PDGFRA rearrangement, and the remaining two cases were Lymphoproliferative T neoplasms associated with eosinophilia. In addition, 17 patients had a secondary eosinophilia and were used as controls. Eosinophilic enrichment was carried out in all cases. Genomic imbalances were found in 76% of all MPN patients. Losses on 20q were the most frequent genetic abnormality in MPNs (32%), affected the three types of MPN studied. This study also found losses at 11q13.3 in 26% of patients with MPN-Ph- and in 19p13.11 in two of the three patients with an MPN associated with a PDGFRA rearrangement. In addition, 29% of patients with CML had losses on 8q24. In summary, aCGH revealed clonality in eosinophils in most MPNs, suggesting that it could be a useful technique for defining clonality in these diseases. The presence of genetic losses in new regions could provide new insights into the knowledge of these MPN associated with eosinophilia. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. The development of a mini-array for estimating the disease state of gastric adenocarcinoma by array CGH

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    Oga Atsunori


    Full Text Available Abstract Background The treatment strategy usually depends on the disease state in the individual patient. However, it is difficult to estimate the disease state before treatment in many patients. The purpose of this study was to develop a BAC (bacterial artificial chromosome mini-array allowing for the estimation of node metastasis, liver metastasis, peritoneal dissemination and the depth of tumor invasion in gastric cancers. Methods Initially, the DNA copy number aberrations (DCNAs were analyzed by array-based comparative genomic hybridization (aCGH in 83 gastric adenocarcinomas as a training-sample set. Next, two independent analytical methods were applied to the aCGH data to identify the BAC clones with DNA copy number aberrations that were linked with the disease states. One of the methods, a decision-tree model classifier, identified 6, 4, 4, 4, and 7 clones for estimating lymph node metastasis, liver metastasis, peritoneal dissemination, depth of tumor invasion, and histological type, respectively. In the other method, a clone-by-clone comparison of the frequency of the DNA copy number aberrations selected 26 clones to estimate the disease states. Results By spotting these 50 clones together with 26 frequently or rarely involved clones and 62 reference clones, a mini-array was made to estimate the above parameters, and the diagnostic performance of the mini-array was evaluated for an independent set of 30 gastric cancers (blinded – sample set. In comparison to the clinicopathological features, the overall accuracy was 66.7% for node metastasis, 86.7% for liver metastasis, 86.7% for peritoneal dissemination, and 96.7% for depth of tumor invasion. The intratumoral heterogeneity barely affected the diagnostic performance of the mini-array. Conclusion These results suggest that the mini-array makes it possible to determine an optimal treatment for each of the patients with gastric adenocarcinoma.

  2. CGHregions: Dimension Reduction for Array CGH Data with Minimal Information Loss

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    Mark A. van de Wiel


    Full Text Available An algorithm to reduce multi-sample array CGH data from thousands of clones to tens or hundreds of clone regions is introduced. This reduction of the data is performed such that little information is lost, which is possible due to the high dependencies between neighboring clones. The algorithm is explained using a small example. The potential beneficial effects of the algorithm for downstream analysis are illustrated by re-analysis of previously published colorectal cancer data. Using multiple testing corrections suitable for these data, we provide statistical evidence for genomic differences on several clone regions between MSI+ and CIN+ tumors. The algorithm, named CGHregions, is available as an easy-to-use script in R.

  3. Clinical applications of BAC array-CGH to the study of diffuse large B-cell lymphomas. (United States)

    Robledo, Cristina; García, Juan Luis; Hernández, Jesús M


    BAC array-CGH is a powerful method to identify DNA copy number changes (gains, amplifications and deletions) on a genome-wide scale, and to map these changes to genomic sequence. It is based on the analysis of genomic DNA isolated from test and reference cell populations, the differential labelling with fluorescent dyes and the co-hybridization with a genomic array. BAC array-CGH has proven to be a specific, sensitive, and reliable technique, with considerable advantages compared to other methods used for the analysis of DNA copy number changes. The application of genome scanning technologies and the recent advances in bioinformatics tools that enable us to perform a robust and highly sensitive analysis of array-CGH data, useful not only for genome scanning of tumor cells but also in the identification of novel cancer related genes, oncogenes and suppressor genes. Cytogenetic analysis provides essential information for diagnosis and prognosis in patients with hematologic malignancies such as lymphomas. However, the chromosomal interpretation in non-Hodgkin lymphoma (NHL) is sometimes inconclusive. Copy number aberrations identified by BAC array-CGH analyses could be a complementary methodology to chromosomal analysis. In NHL the genomic imbalances might have a prognostic rather than a diagnostic value. In fact, the diagnosis of NHL is based on pathological and molecular cytogenetics data. Furthermore genetic variations and their association with specific types of lymphoma development, and elucidation of the variable genetic pathways leading to lymphoma development, are important directions for future cancer research. Array-CGH, along with FISH and PCR, will be used for routine diagnostic purposes in near future.

  4. Parsimonious higher-order hidden Markov models for improved array-CGH analysis with applications to Arabidopsis thaliana.

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    Michael Seifert


    Full Text Available Array-based comparative genomic hybridization (Array-CGH is an important technology in molecular biology for the detection of DNA copy number polymorphisms between closely related genomes. Hidden Markov Models (HMMs are popular tools for the analysis of Array-CGH data, but current methods are only based on first-order HMMs having constrained abilities to model spatial dependencies between measurements of closely adjacent chromosomal regions. Here, we develop parsimonious higher-order HMMs enabling the interpolation between a mixture model ignoring spatial dependencies and a higher-order HMM exhaustively modeling spatial dependencies. We apply parsimonious higher-order HMMs to the analysis of Array-CGH data of the accessions C24 and Col-0 of the model plant Arabidopsis thaliana. We compare these models against first-order HMMs and other existing methods using a reference of known deletions and sequence deviations. We find that parsimonious higher-order HMMs clearly improve the identification of these polymorphisms. Moreover, we perform a functional analysis of identified polymorphisms revealing novel details of genomic differences between C24 and Col-0. Additional model evaluations are done on widely considered Array-CGH data of human cell lines indicating that parsimonious HMMs are also well-suited for the analysis of non-plant specific data. All these results indicate that parsimonious higher-order HMMs are useful for Array-CGH analyses. An implementation of parsimonious higher-order HMMs is available as part of the open source Java library Jstacs (

  5. Parsimonious higher-order hidden Markov models for improved array-CGH analysis with applications to Arabidopsis thaliana. (United States)

    Seifert, Michael; Gohr, André; Strickert, Marc; Grosse, Ivo


    Array-based comparative genomic hybridization (Array-CGH) is an important technology in molecular biology for the detection of DNA copy number polymorphisms between closely related genomes. Hidden Markov Models (HMMs) are popular tools for the analysis of Array-CGH data, but current methods are only based on first-order HMMs having constrained abilities to model spatial dependencies between measurements of closely adjacent chromosomal regions. Here, we develop parsimonious higher-order HMMs enabling the interpolation between a mixture model ignoring spatial dependencies and a higher-order HMM exhaustively modeling spatial dependencies. We apply parsimonious higher-order HMMs to the analysis of Array-CGH data of the accessions C24 and Col-0 of the model plant Arabidopsis thaliana. We compare these models against first-order HMMs and other existing methods using a reference of known deletions and sequence deviations. We find that parsimonious higher-order HMMs clearly improve the identification of these polymorphisms. Moreover, we perform a functional analysis of identified polymorphisms revealing novel details of genomic differences between C24 and Col-0. Additional model evaluations are done on widely considered Array-CGH data of human cell lines indicating that parsimonious HMMs are also well-suited for the analysis of non-plant specific data. All these results indicate that parsimonious higher-order HMMs are useful for Array-CGH analyses. An implementation of parsimonious higher-order HMMs is available as part of the open source Java library Jstacs (

  6. CGH and SNP array using DNA extracted from fixed cytogenetic preparations and long-term refrigerated bone marrow specimens

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    MacKinnon Ruth N


    Full Text Available Abstract Background The analysis of nucleic acids is limited by the availability of archival specimens and the quality and amount of the extracted material. Archived cytogenetic preparations are stored in many laboratories and are a potential source of total genomic DNA for array karyotyping and other applications. Array CGH using DNA from fixed cytogenetic preparations has been described, but it is not known whether it can be used for SNP arrays. Diagnostic bone marrow specimens taken during the assessment of hematological malignancies are also a potential source of DNA, but it is generally assumed that DNA must be extracted, or the specimen frozen, within a day or two of collection, to obtain DNA suitable for further analysis. We have assessed DNA extracted from these materials for both SNP array and array CGH. Results We show that both SNP array and array CGH can be performed on genomic DNA extracted from cytogenetic specimens stored in Carnoy's fixative, and from bone marrow which has been stored unfrozen, at 4°C, for at least 36 days. We describe a procedure for extracting a usable concentration of total genomic DNA from cytogenetic suspensions of low cellularity. Conclusions The ability to use these archival specimens for DNA-based analysis increases the potential for retrospective genetic analysis of clinical specimens. Fixed cytogenetic preparations and long-term refrigerated bone marrow both provide DNA suitable for array karyotyping, and may be suitable for a wider range of analytical procedures.

  7. Mapping genomic library clones using oligonucleotide arrays

    Energy Technology Data Exchange (ETDEWEB)

    Sapolsky, R.J.; Lipshutz, R.J. [Affymetrix, Santa Clara, CA (United States)


    We have developed a high-density DNA probe array and accompanying biochemical and informatic methods to order clones from genomic libraries. This approach involves a series of enzymatic steps for capturing a set of short dispersed sequence markers scattered throughout a high-molecular-weight DNA. By this process, all the ambiguous sequences lying adjacent to a given Type IIS restriction site are ligated between two DNA adaptors. These markers, once amplified and labeled by PCR, can be hybridized and detected on a high-density olligonucleotide array bearing probes complementary to all possible markers. The array is synthesized using light-directed combinatorial chemistry. For each clone in a genomic library, a characteristic set of sequence markers can be determined. On the basis of the similarity between the marker sets for each pair of clones, their relative overlap can be measured. The library can be sequentially ordered into a contig map using this overlap information. This new methodology does not require gel-based methods or prior sequence information and involves manipulations that should allow for easy adaptation to automated processing and data collection. 28 refs., 9 figs., 2 tabs.

  8. DNA copy number changes in high-grade malignant peripheral nerve sheath tumors by array CGH

    Directory of Open Access Journals (Sweden)

    Bjerkehagen Bodil


    Full Text Available Abstract Background Malignant peripheral nerve sheath tumors (MPNSTs are rare and highly aggressive soft tissue tumors showing complex chromosomal aberrations. In order to identify recurrent chromosomal regions of gain and loss, and thereby novel gene targets of potential importance for MPNST development and/or progression, we have analyzed DNA copy number changes in seven high-grade MPNSTs using microarray-based comparative genomic hybridization (array CGH. Results Considerable more gains than losses were observed, and the most frequent minimal recurrent regions of gain included 1q24.1-q24.2, 1q24.3-q25.1, 8p23.1-p12, 9q34.11-q34.13 and 17q23.2-q25.3, all gained in five of seven samples. The 17q23.2-q25.3 region was gained in all five patients with poor outcome and not in the two patients with disease-free survival. cDNA microarray analysis and quantitative real-time reverse transcription PCR were used to investigate expression of genes located within these regions. The gene lysyl oxidase-like 2 (LOXL2 was identified as a candidate target for the 8p23.1-p12 gain. Within 17q, the genes topoisomerase II-α (TOP2A, ets variant gene 4 (E1A enhancer binding protein, E1AF (ETV4 and baculoviral IAP repeat-containing 5 (survivin (BIRC5 showed increased expression in all samples compared to two benign tumors. Increased expression of these genes has previously been associated with poor survival in other malignancies, and for TOP2A, in MPNSTs as well. In addition, we have analyzed the expression of five micro RNAs located within the 17q23.2-q25.3 region, but none of them showed high expression levels compared to the benign tumors. Conclusion Our study shows the potential of using DNA copy number changes obtained by array CGH to predict the prognosis of MPNST patients. Although no clear correlations between the expression level and patient outcome were observed, the genes TOP2A, ETV4 and BIRC5 are interesting candidate targets for the 17q gain associated

  9. Identification of novel genomic aberrations in AML-M5 in a level of array CGH.

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    Rui Zhang

    Full Text Available To assess the possible existence of unbalanced chromosomal abnormalities and delineate the characterization of copy number alterations (CNAs of acute myeloid leukemia-M5 (AML-M5, R-banding karyotype, oligonucelotide array CGH and FISH were performed in 24 patients with AML-M5. A total of 117 CNAs with size ranging from 0.004 to 146.263 Mb was recognized in 12 of 24 cases, involving all chromosomes other than chromosome 1, 4, X and Y. Cryptic CNAs with size less than 5 Mb accounted for 59.8% of all the CNAs. 12 recurrent chromosomal alterations were mapped. Seven out of them were described in the previous AML studies and five were new candidate AML-M5 associated CNAs, including gains of 3q26.2-qter and 13q31.3 as well as losses of 2q24.2, 8p12 and 14q32. Amplication of 3q26.2-qter was the sole large recurrent chromosomal anomaly and the pathogenic mechanism in AML-M5 was possibly different from the classical recurrent 3q21q26 abnormality in AML. As a tumor suppressor gene, FOXN3, was singled out from the small recurrent CNA of 14q32, however, it is proved that deletion of FOXN3 is a common marker of myeloid leukemia rather than a specific marker for AML-M5 subtype. Moreover, the concurrent amplication of MLL and deletion of CDKN2A were noted and it might be associated with AML-M5. The number of CNA did not show a significant association with clinico-biological parameters and CR number of the 22 patients received chemotherapy. This study provided the evidence that array CGH served as a complementary platform for routine cytogenetic analysis to identify those cryptic alterations in the patients with AML-M5. As a subtype of AML, AML-M5 carries both common recurrent CNAs and unique CNAs, which may harbor novel oncogenes or tumor suppressor genes. Clarifying the role of these genes will contribute to the understanding of leukemogenic network of AML-M5.

  10. Aneuploidy analysis of non-pronuclear embryos from IVF with use of array CGH: a case report. (United States)

    Lixin, Deng; Zhifeng, Xiang; Cong, He; Jinzhou, Zhang; Hongbin, Xie


    By using array comparative genomic hybridization (array CGH), to analyze the aneuploidy of the single blastomeres from non-pronuclear embryos on cleavage-stage in IVF cycle. Four non-pronuclear embryos were got from an IVF cycle, and the each single cell was biopsied from the four cleavage-stage embryos on the third day after the insemination which was investigated by using array CGH. After the biopsy, all the embryos continued to cleave, and lately entered the morula stage on the fifth day, just one embryo 3 was developed to early blastocyst stage on the sixth day. The four blastomere 24 chromosomes showed one X monomer and three normal XY diploids; the autosome chromosomes of blastomeres were abnormally gained or lost at different chromosome from four embryos, such as Embryo 1 : 49,X (-1, -5, -11, -19, -20, -21, -Y, +3, +6, +7, +8, +10, +13, +14, +16, +17, +18); Embryo 2 : 44,XY (-12, -15); Embryo 3: 47,XY (-3, -8, -9, -21, +7, +17, +18, +19, +20); Embryo 4 : 54,XY (+4, +7, +10, +12, +13, +16, +17, +22). With the use of the array CGH, the aneuploidy analysis could review the abnormal chromosomes of single blastomere from the non-pronuclear embryos, which can harbor the risk of abnormal sex chromosome and autosome chromosomes.

  11. Microarray based comparative genomic hybridisation (array-CGH) detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features

    National Research Council Canada - National Science Library

    Shaw-Smith, C; Redon, R; Rickman, L; Rio, M; Willatt, L; Fiegler, H; Firth, H; Sanlaville, D; Winter, R; Colleaux, L; Bobrow, M; Carter, N P


    ...). The presence of subtle DNA copy number changes was investigated by array-CGH in 50 patients with learning disability and dysmorphism, employing a DNA microarray constructed from large insert clones...

  12. High-resolution copy number profiling by array CGH using DNA isolated from formalin-fixed, paraffin-embedded tissues. (United States)

    van Essen, Hendrik F; Ylstra, Bauke


    We describe protocols to acquire high-quality DNA from formalin-fixed, paraffin-embedded (FFPE) tissues for the use in array comparative genome hybridization (CGH). Formalin fixation combined with paraffin embedding is routine procedure for solid malignancies in the diagnostic practice of the pathologist. As a consequence, large archives of FFPE tissues are available in pathology institutes across the globe. This archival material is for many research questions an invaluable resource, with long-term clinical follow-up and survival data available. FFPE is, thus, highly attractive for large genomics studies, including experiments requiring samples for test/learning and validation. Most larger array CGH studies have, therefore, made use of FFPE material and show that CNAs have tumor- and tissue-specific traits (Chin et al. Cancer Cell 10: 529-541, 2006; Fridlyand et al. BMC Cancer 6: 96, 2006; Weiss et al. Oncogene 22: 1872-1879, 2003; Jong et al. Oncogene 26: 1499-1506, 2007). The protocols described are tailored to array CGH of FFPE solid malignancies: from sectioning FFPE blocks to specific cynosures for pathological revisions of sections, DNA isolation, quality testing, and amplification. The protocols are technical in character and elaborate up to the labeling of isolated DNA while further processes and interpretation and data analysis are beyond the scope.

  13. Discovery of common Asian copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing. (United States)

    Park, Hansoo; Kim, Jong-Il; Ju, Young Seok; Gokcumen, Omer; Mills, Ryan E; Kim, Sheehyun; Lee, Seungbok; Suh, Dongwhan; Hong, Dongwan; Kang, Hyunseok Peter; Yoo, Yun Joo; Shin, Jong-Yeon; Kim, Hyun-Jin; Yavartanoo, Maryam; Chang, Young Wha; Ha, Jung-Sook; Chong, Wilson; Hwang, Ga-Ram; Darvishi, Katayoon; Kim, Hyeran; Yang, Song Ju; Yang, Kap-Seok; Kim, Hyungtae; Hurles, Matthew E; Scherer, Stephen W; Carter, Nigel P; Tyler-Smith, Chris; Lee, Charles; Seo, Jeong-Sun


    Copy number variants (CNVs) account for the majority of human genomic diversity in terms of base coverage. Here, we have developed and applied a new method to combine high-resolution array comparative genomic hybridization (CGH) data with whole-genome DNA sequencing data to obtain a comprehensive catalog of common CNVs in Asian individuals. The genomes of 30 individuals from three Asian populations (Korean, Chinese and Japanese) were interrogated with an ultra-high-resolution array CGH platform containing 24 million probes. Whole-genome sequencing data from a reference genome (NA10851, with 28.3x coverage) and two Asian genomes (AK1, with 27.8x coverage and AK2, with 32.0x coverage) were used to transform the relative copy number information obtained from array CGH experiments into absolute copy number values. We discovered 5,177 CNVs, of which 3,547 were putative Asian-specific CNVs. These common CNVs in Asian populations will be a useful resource for subsequent genetic studies in these populations, and the new method of calling absolute CNVs will be essential for applying CNV data to personalized medicine.

  14. Formalin-fixed paraffin-embedded clinical tissues show spurious copy number changes in array-CGH profiles. (United States)

    Mc Sherry, E A; Mc Goldrick, A; Kay, E W; Hopkins, A M; Gallagher, W M; Dervan, P A


    Formalin-fixed paraffin-embedded (FFPE) archival clinical specimens are invaluable in discovery of prognostic and therapeutic targets for diseases such as cancer. However, the suitability of FFPE-derived genetic material for array-based comparative genomic hybridization (array-CGH) studies is underexplored. In this study, genetic profiles of matched FFPE and fresh-frozen specimens were examined to investigate DNA integrity differences between these sample types and determine the impact this may have on genetic profiles. Genomic DNA was extracted from three patient-matched FFPE and fresh-frozen clinical tissue samples. T47D breast cancer control cells were also grown in culture and processed to yield a fresh T47D sample, a fresh-frozen T47D sample and a FFPE T47D sample. DNA was extracted from all the samples; array-CGH conducted and genetic profiles of matched samples were then compared. A loss of high molecular weight DNA was observed in the FFPE clinical tissues and FFPE T47D samples. A dramatic increase in absolute number of genetic alterations was observed in all FFPE tissues relative to matched fresh-frozen counterparts. In future, alternative fixation and tissue-processing procedures, and/or new DNA extraction and CGH profiling protocols, may be implemented, enabling identification of changes involved in disease progression using stored clinical specimens.

  15. Typing of enteroviruses by use of microwell oligonucleotide arrays. (United States)

    Susi, P; Hattara, L; Waris, M; Luoma-Aho, T; Siitari, H; Hyypiä, T; Saviranta, P


    We have developed a straightforward assay for the rapid typing of enteroviruses using oligonucleotide arrays in microtiter wells. The viral nucleic acids are concomitantly amplified and labeled during reverse transcription-PCR, and unpurified PCR products are used for hybridization. DNA strands are separated by alkaline denaturation, and hybridization is started by neutralization. The microarray hybridization reactions and the subsequent washes are performed in standard 96-well microtiter plates, which makes the method easily adaptable to high-throughput analysis. We describe here the assay principle and its potential in clinical laboratory use by correctly identifying 10 different enterovirus reference strains. Furthermore, we explore the detection of unknown sequence variants using serotype consensus oligonucleotide probes. With just two consensus probes for the coxsackievirus A9 (CVA9) serotype, we detected 23 out of 25 highly diverse CVA9 isolates. Overall, the assay involves several features aiming at ease of performance, robustness, and applicability to large-scale studies.

  16. Oligonucleotide array outperforms SNP array on formalin-fixed paraffin-embedded clinical samples. (United States)

    Nasri, Soroush; Anjomshoaa, Ahmad; Song, Sarah; Guilford, Parry; McNoe, Les; Black, Michael; Phillips, Vicky; Reeve, Anthony; Humar, Bostjan


    Compromised quality of formalin-fixed paraffin-embedded (FFPE)-derived DNA has compounded the use of archival specimens for array-based genomic studies. Recent technological advances have led to first successes in this field; however, there is currently no general agreement on the most suitable platform for the array-based analysis of FFPE DNA. In this study, FFPE and matched fresh-frozen (FF) specimens were separately analyzed with Affymetrix single nucleotide polymorphism (SNP) 6.0 and Agilent 4x44K oligonucleotide arrays to compare the genomic profiles from the two tissue sources and to assess the relative performance of the two platforms on FFPE material. Genomic DNA was extracted from matched FFPE-FF pairs of normal intestinal epithelium from four patients and were applied to the SNP and oligonucleotide platforms according to the manufacturer-recommended protocols. On the Affymetrix platform, a substantial increase in apparent copy number alterations was observed in all FFPE tissues relative to their matched FF counterparts. In contrast, FFPE and matched FF genomic profiles obtained via the Agilent platform were very similar. Both the SNP and the oligonucleotide platform performed comparably on FF material. This study demonstrates that Agilent oligonucleotide array comparative genomic hybridization generates reliable results from FFPE extracted DNA, whereas the Affymetrix SNP-based array seems less suitable for the analysis of FFPE material.

  17. Combining gene expression data from different generations of oligonucleotide arrays

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    Kong Sek


    Full Text Available Abstract Background One of the important challenges in microarray analysis is to take full advantage of previously accumulated data, both from one's own laboratory and from public repositories. Through a comparative analysis on a variety of datasets, a more comprehensive view of the underlying mechanism or structure can be obtained. However, as we discover in this work, continual changes in genomic sequence annotations and probe design criteria make it difficult to compare gene expression data even from different generations of the same microarray platform. Results We first describe the extent of discordance between the results derived from two generations of Affymetrix oligonucleotide arrays, as revealed in cluster analysis and in identification of differentially expressed genes. We then propose a method for increasing comparability. The dataset we use consists of a set of 14 human muscle biopsy samples from patients with inflammatory myopathies that were hybridized on both HG-U95Av2 and HG-U133A human arrays. We find that the use of the probe set matching table for comparative analysis provided by Affymetrix produces better results than matching by UniGene or LocusLink identifiers but still remains inadequate. Rescaling of expression values for each gene across samples and data filtering by expression values enhance comparability but only for few specific analyses. As a generic method for improving comparability, we select a subset of probes with overlapping sequence segments in the two array types and recalculate expression values based only on the selected probes. We show that this filtering of probes significantly improves the comparability while retaining a sufficient number of probe sets for further analysis. Conclusions Compatibility between high-density oligonucleotide arrays is significantly affected by probe-level sequence information. With a careful filtering of the probes based on their sequence overlaps, data from different

  18. [Accidental finding of a cri du chat syndrome in an adult patient by means of array-CGH]. (United States)

    Ferreirós-Martínez, Raquel; López-Manzanares, Lydia; Alonso-Cerezo, Concepción


    Introduccion. El sindrome cri du chat (SCDC) tiene su origen en una delecion parcial o total del brazo corto del cromosoma 5, y es uno de los sindromes de delecion cromosomica mas frecuentes en humanos. La mayoria de los pacientes se diagnostica entre el primer mes y el primer año de vida, si bien aqui se describe el hallazgo de un SCDC en una mujer con sospecha de ataxia espinocerebelar y antecedentes familiares de trastorno bipolar y ataxia, con especial atencion a las caracteristicas clinicas y las tecnicas diagnosticas que permitieron su identificacion. Caso clinico. Mujer de 46 años que presentaba una inteligencia limite, intervenida a los 43 años de faquectomia bilateral. El inicio de la sintomatologia fue durante la infancia, e incluia hipoacusia, ataxia, disartria, disfagia, depresion, deterioro cognitivo y trastorno bipolar. La exploracion fisica revelo microcefalia, micrognatia, pies equinos y ataxia. Se realizo cariotipo y array-CGH en sangre periferica. La paciente presentaba una traslocacion que involucraba los cromosomas 5 y 15, y una inversion del cromosoma 9: 45,XX,inv9(p11q13);t(5,15)(p15.33;q11.2). El array-CGH mostro una delecion de 2,91 Mb en 5p15.33, formula genomica arr 5p15.33 (151537-3057771)x1, que involucraba 20 genes, incluyendo el gen TERT. Conclusiones. La delecion de multiples genes confirmo el diagnostico de SCDC y es la responsable del fenotipo de la paciente. Se pone de manifiesto la importancia de utilizar tecnicas adecuadas de diagnostico (array-CGH, cariotipo en sangre periferica) y la correcta eleccion de estas.

  19. Phenotype and 244k array-CGH characterization of chromosome 13q deletions: an update of the phenotypic map of 13q21.1-qter

    DEFF Research Database (Denmark)

    Kirchhoff, Maria; Bisgaard, Anne-Marie; Stoeva, Radka


    Partial deletions of the long arm of chromosome 13 lead to variable phenotypes dependant on the size and position of the deleted region. In order to update the phenotypic map of chromosome 13q21.1-qter deletions, we applied 244k Agilent oligonucleotide-based array-CGH to determine the exact.......1), lung hypoplasia (13q31.3-13q33.1), and thumb a-/hypoplasia (13q31.3-q33.1 and 13q33.3-q34). Based on observations of this study and previous reports we suggest a new entity, "distal limb anomalies association," linked to 13q31.3q33.1 segment. Most of the individuals with deletion of any part of 13q21......-genotype correlation on chromosome 13. In contrast to previous reports of carriers of 13q32 band deletions as the most seriously affected patients, we present two such individuals with long-term survival, 28 and 2.5 years....

  20. [Middle ear salivary gland choristoma related to branchio-oto-renal syndrome diagnosed by array-CGH]. (United States)

    Amrhein, P; Sittel, C; Spaich, C; Kohlhase, J; Boppert, R; Kohlhof, P; Koitschev, A


    Branchio-oto-renal (BOR) syndrome is characterized by ear malformations associated with sensorineural or mixed hearing loss. In addition, preauricular tags, preauricular pits, branchial cleft fistulas and cysts, as well as renal dysplasia are seen. A genetic mutation on chromosome 8, either autosomal dominantly inherited or occuring as a spontaneous mutation, is the cause in the majority of cases. Using array-based comparative genomic hybridization (CGH), it is possible to detect even the smallest genetic changes. Salivary gland choristoma in the middle ear is very rare. Surgical removal and histological clarification are required.

  1. Characterization of genomic alterations in radiation-associated breast cancer among childhood cancer survivors, using comparative genomic hybridization (CGH arrays.

    Directory of Open Access Journals (Sweden)

    Xiaohong R Yang

    Full Text Available Ionizing radiation is an established risk factor for breast cancer. Epidemiologic studies of radiation-exposed cohorts have been primarily descriptive; molecular events responsible for the development of radiation-associated breast cancer have not been elucidated. In this study, we used array comparative genomic hybridization (array-CGH to characterize genome-wide copy number changes in breast tumors collected in the Childhood Cancer Survivor Study (CCSS. Array-CGH data were obtained from 32 cases who developed a second primary breast cancer following chest irradiation at early ages for the treatment of their first cancers, mostly Hodgkin lymphoma. The majority of these cases developed breast cancer before age 45 (91%, n = 29, had invasive ductal tumors (81%, n = 26, estrogen receptor (ER-positive staining (68%, n = 19 out of 28, and high proliferation as indicated by high Ki-67 staining (77%, n = 17 out of 22. Genomic regions with low-copy number gains and losses and high-level amplifications were similar to what has been reported in sporadic breast tumors, however, the frequency of amplifications of the 17q12 region containing human epidermal growth factor receptor 2 (HER2 was much higher among CCSS cases (38%, n = 12. Our findings suggest that second primary breast cancers in CCSS were enriched for an "amplifier" genomic subgroup with highly proliferative breast tumors. Future investigation in a larger irradiated cohort will be needed to confirm our findings.

  2. Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays

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    Khan Shehnaz


    Full Text Available Abstract Background Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray. Results Significantly (p M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p Conclusions This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.

  3. Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays

    Directory of Open Access Journals (Sweden)

    Malloff Chad A


    Full Text Available Abstract Background The recent development of array based comparative genomic hybridization (CGH technology provides improved resolution for detection of genomic DNA copy number alterations. In array CGH, generating spotting solution is a multi-step process where bacterial artificial chromosome (BAC clones are converted to replenishable PCR amplified fragments pools (AFP for use as spotting solution in a microarray format on glass substrate. With completion of the human and mouse genome sequencing, large BAC clone sets providing complete genome coverage are available for construction of whole genome BAC arrays. Currently, Southern hybridization, fluorescent in-situ hybridization (FISH, and BAC end sequencing methods are commonly used to identify the initial BAC clone but not the end product used for spotting arrays. The AFP sequencing technique described in this study is a novel method designed to verify the identity of array spotting solution in a high throughput manner. Results We show here that Southern hybridization, FISH, and AFP sequencing can be used to verify the identity of final spotting solutions using less than 10% of the AFP product. Single pass AFP sequencing identified over half of the 960 AFPs analyzed. Moreover, using two vector primers approximately 90% of the AFP spotting solutions can be identified. Conclusions In this feasibility study we demonstrate that current methods for identifying initial BAC clones can be adapted to verify the identity of AFP spotting solutions used in printing arrays. Of these methods, AFP sequencing proves to be the most efficient for large scale identification of spotting solution in a high throughput manner.

  4. BAC CGH-array identified specific small-scale genomic imbalances in diploid DMBA-induced rat mammary tumors

    Directory of Open Access Journals (Sweden)

    Samuelson Emma


    Full Text Available Abstract Background Development of breast cancer is a multistage process influenced by hormonal and environmental factors as well as by genetic background. The search for genes underlying this malignancy has recently been highly productive, but the etiology behind this complex disease is still not understood. In studies using animal cancer models, heterogeneity of the genetic background and environmental factors is reduced and thus analysis and identification of genetic aberrations in tumors may become easier. To identify chromosomal regions potentially involved in the initiation and progression of mammary cancer, in the present work we subjected a subset of experimental mammary tumors to cytogenetic and molecular genetic analysis. Methods Mammary tumors were induced with DMBA (7,12-dimethylbenz[a]anthrazene in female rats from the susceptible SPRD-Cu3 strain and from crosses and backcrosses between this strain and the resistant WKY strain. We first produced a general overview of chromosomal aberrations in the tumors using conventional kartyotyping (G-banding and Comparative Genome Hybridization (CGH analyses. Particular chromosomal changes were then analyzed in more details using an in-house developed BAC (bacterial artificial chromosome CGH-array platform. Results Tumors appeared to be diploid by conventional karyotyping, however several sub-microscopic chromosome gains or losses in the tumor material were identified by BAC CGH-array analysis. An oncogenetic tree analysis based on the BAC CGH-array data suggested gain of rat chromosome (RNO band 12q11, loss of RNO5q32 or RNO6q21 as the earliest events in the development of these mammary tumors. Conclusions Some of the identified changes appear to be more specific for DMBA-induced mammary tumors and some are similar to those previously reported in ACI rat model for estradiol-induced mammary tumors. The later group of changes is more interesting, since they may represent anomalies that involve

  5. A recurrent copy number variation of the NEB triplicate region: only revealed by the targeted nemaline myopathy CGH array. (United States)

    Kiiski, Kirsi; Lehtokari, Vilma-Lotta; Löytynoja, Ari; Ahlstén, Liina; Laitila, Jenni; Wallgren-Pettersson, Carina; Pelin, Katarina


    Recently, new large variants have been identified in the nebulin gene (NEB) causing nemaline myopathy (NM). NM constitutes a heterogeneous group of disorders among the congenital myopathies, and disease-causing variants in NEB are a main cause of the recessively inherited form of NM. NEB consists of 183 exons and it includes homologous sequences such as a 32-kb triplicate region (TRI), where eight exons are repeated three times (exons 82-89, 90-97, 98-105). In human, the normal copy number of NEB TRI is six (three copies in each allele). Recently, we described a custom NM-CGH microarray designed to detect copy number variations (CNVs) in the known NM genes. The array has now been updated to include all the currently known 10 NM genes. The NM-CGH array is superior in detecting CNVs, especially of the NEB TRI, that is not included in the exome capture kits. To date, we have studied 266 samples from 196 NM families using the NM-CGH microarray, and identified a novel recurrent NEB TRI variation in 13% (26/196) of the families and in 10% of the controls (6/60). An analysis of the breakpoints revealed adjacent repeat elements, which are known to predispose for rearrangements such as CNVs. The control CNV samples deviate only one copy from the normal six copies, whereas the NM samples include CNVs of up to four additional copies. Based on this study, NEB seems to tolerate deviations of one TRI copy, whereas addition of two or more copies might be pathogenic.

  6. Efficient oligonucleotide probe selection for pan-genomic tiling arrays

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    Zhang Wei


    Full Text Available Abstract Background Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome. Results This paper presents a new probe selection algorithm (PanArray that can tile multiple whole genomes using a minimal number of probes. Unlike arrays built on clustered gene families, PanArray uses an unbiased, probe-centric approach that does not rely on annotations, gene clustering, or multi-alignments. Instead, probes are evenly tiled across all sequences of the pan-genome at a consistent level of coverage. To minimize the required number of probes, probes conserved across multiple strains in the pan-genome are selected first, and additional probes are used only where necessary to span polymorphic regions of the genome. The viability of the algorithm is demonstrated by array designs for seven different bacterial pan-genomes and, in particular, the design of a 385,000 probe array that fully tiles the genomes of 20 different Listeria monocytogenes strains with overlapping probes at greater than twofold coverage. Conclusion PanArray is an oligonucleotide probe selection algorithm for tiling multiple genome sequences using a minimal number of probes. It is capable of fully tiling all genomes of a species on

  7. Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains. (United States)

    Dumas, Laura; Dickens, C Michael; Anderson, Nathan; Davis, Jonathan; Bennett, Beth; Radcliffe, Richard A; Sikela, James M


    It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to

  8. A 91 kb microdeletion at Xq26.2 involving the GPC3 gene in a female fetus with Simpson-Golabi-Behmel syndrome detected by prenatal arrayCGH

    DEFF Research Database (Denmark)

    Ramsing, Mette; Becher, Naja Helene; Christensen, Rikke

    A 91 kb microdeletion at Xq26.2 involving the GPC3 gene in a female fetus with Simpson-Golabi-Behmel syndrome detected by prenatal arrayCGH......A 91 kb microdeletion at Xq26.2 involving the GPC3 gene in a female fetus with Simpson-Golabi-Behmel syndrome detected by prenatal arrayCGH...

  9. Genetic characterization of dogs via chromosomal analysis and array-based comparative genomic hybridization (aCGH). (United States)

    Müller, M H; Reimann-Berg, N; Bullerdiek, J; Murua Escobar, H


    The results of cytogenetic and molecular cytogenetic investigations revealed similarities in genetic background and biological behaviour between tumours and genetic diseases of humans and dogs. These findings classify the dog a good and accepted model for human cancers such as osteosarcomas, mammary carcinomas, oral melanomas and others. With the appearance of new studies and advances in canine genome sequencing, the number of known homologies in diseases between these species raised and still is expected to increase. In this context, array-based comparative genomic hybridization (aCGH) provides a novel tool to rapidly characterize numerical aberrations in canine tumours or to detect copy number aberrations between different breeds. As it is possible to spot probes covering the whole genome on each chip to discover copy number aberrations of all chromosomes simultaneously, this method is time-saving and cost-effective - considering the relation of costs and the amount of data obtained. Complemented with traditional methods like karyotyping and fluorescence in situ hybridization (FISH) analyses, the aCGH is able to provide new insights into the underlying causes of canine carcinogenesis.

  10. Array CGH on unstimulated blood does not detect all cases of Pallister-Killian syndrome: a skin biopsy should remain the diagnostic gold standard. (United States)

    Hodge, Jennelle C; Hulshizer, Rachael L; Seger, Pam; St Antoine, Angelique; Bair, Jennifer; Kirmani, Salman


    A child whose features are consistent with Pallister-Killian syndrome (PKS) did not have detectable tetrasomy 12p due to an additional isochromosome 12p in an unstimulated blood specimen by interphase FISH or array CGH analysis. The diagnosis of PKS was made through these methods solely in a skin biopsy specimen. To determine the sensitivity of our array CGH platform to tetrasomy 12p mosaicism, dilutions of DNA from both the child's skin fibroblasts and a PKS cell line were analyzed. Tetrasomy 12p at 10% mosaicism was identifiable but 5% was below the limit of detection. This result suggests through extrapolation that the tetrasomy 12p is present in <10% of cells in our patient's blood, confirming the tissue-limited mosaicism of PKS. Multiple recent studies show that array CGH provides greater sensitivity than chromosome analysis to detect mosaic abnormalities including that of tetrasomy 12p in blood specimens. However, our case demonstrates that the biology of PKS precludes the exclusive use of array CGH on blood for diagnosis. A tissue sample should continue to be the diagnostic gold standard for PKS.

  11. Genome-wide microarray expression and genomic alterations by array-CGH analysis in neuroblastoma stem-like cells.

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    Raquel Ordóñez

    Full Text Available Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC, a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture. Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells.

  12. Novel Genomic Aberrations in Testicular Germ Cell Tumors by Array-CGH, and Associated Gene Expression Changes

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    Rolf I. Skotheim


    Full Text Available Introduction: Testicular germ cell tumors of adolescent and young adult men (TGCTs generally have near triploid and complex karyotypes. The actual genes driving the tumorigenesis remain essentially to be identified. Materials and Methods: To determine the detailed DNA copy number changes, and investigate their impact on gene expression levels, we performed an integrated microarray profiling of TGCT genomes and transcriptomes. We analyzed 17 TGCTs, three precursor lesions, and the embryonal carcinoma cell lines, NTERA2 and 2102Ep, by comparative genomic hybridization microarrays (array-CGH, and integrated the data with transcriptome profiles of the same samples. Results: The gain of chromosome arm 12p was, as expected, the most common aberration, and we found CCND2, CD9, GAPD, GDF3, NANOG, and TEAD4 to be the therein most highly over-expressed genes. Additional frequent genomic aberrations revealed some shorter chromosomal segments, which are novel to TGCT, as well as known aberrations for which we here refined boundaries. These include gains from 7p15.2 and 21q22.2, and losses of 4p16.3 and 22q13.3. Integration of DNA copy number information to gene expression profiles identified that BRCC3, FOS, MLLT11, NES, and RAC1 may act as novel oncogenes in TGCT. Similarly, DDX26, ERCC5, FZD4, NME4, OPTN, and RB1 were both lost and under-expressed genes, and are thus putative TGCT suppressor genes. Conclusion: This first genome-wide integrated array-CGH and gene expression profiling of TGCT provides novel insights into the genome biology underlying testicular tumorigenesis.

  13. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study

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    Yang Zhihong


    Full Text Available Abstract Background Single embryo transfer (SET remains underutilized as a strategy to reduce multiple gestation risk in IVF, and its overall lower pregnancy rate underscores the need for improved techniques to select one embryo for fresh transfer. This study explored use of comprehensive chromosomal screening by array CGH (aCGH to provide this advantage and improve pregnancy rate from SET. Methods First-time IVF patients with a good prognosis (age Results For patients in Group A (n = 55, 425 blastocysts were biopsied and analyzed via aCGH (7.7 blastocysts/patient. Aneuploidy was detected in 191/425 (44.9% of blastocysts in this group. For patients in Group B (n = 48, 389 blastocysts were microscopically examined (8.1 blastocysts/patient. Clinical pregnancy rate was significantly higher in the morphology + aCGH group compared to the morphology-only group (70.9 and 45.8%, respectively; p = 0.017; ongoing pregnancy rate for Groups A and B were 69.1 vs. 41.7%, respectively (p = 0.009. There were no twin pregnancies. Conclusion Although aCGH followed by frozen embryo transfer has been used to screen at risk embryos (e.g., known parental chromosomal translocation or history of recurrent pregnancy loss, this is the first description of aCGH fully integrated with a clinical IVF program to select single blastocysts for fresh SET in good prognosis patients. The observed aneuploidy rate (44.9% among biopsied blastocysts highlights the inherent imprecision of SET when conventional morphology is used alone. Embryos randomized to the aCGH group implanted with greater efficiency, resulted in clinical pregnancy more often, and yielded a lower miscarriage rate than those selected without aCGH. Additional studies are needed to verify our pilot data and confirm a role for on-site, rapid aCGH for IVF patients contemplating fresh SET.

  14. Significance of genome-wide analysis of copy number alterations and UPD in myelodysplastic syndromes using combined CGH - SNP arrays. (United States)

    Ahmad, Ausaf; Iqbal, M Anwar


    Genetic information is an extremely valuable data source in characterizing the personal nature of cancer. Chromosome instability is a hallmark of most cancer cells. Chromosomal abnormalities are correlated with poor prognosis, disease classification, risk stratification, and treatment selection. Copy number alterations (CNAs) are an important molecular signature in cancer initiation, development, and progression. Recent application of whole-genome tools to characterize normal and cancer genomes provides the powerful molecular cytogenetic means to enumerate the multiple somatic, genetic and epigenetic alterations that occur in cancer. Combined array comparative genomic hybridization (aCGH) with single nucleotide polymorphism (SNP) array is a useful technique allowing detection of CNAs and loss of heterozygosity (LOH) or uni-parental disomy (UPD) together in a single experiment. It also provides allelic information on deletions, duplications, and amplifications. UPD can result in an abnormal phenotype when the chromosomes involved are imprinted. Myelodysplastic syndromes (MDS) are the most common clonal stem cell hematologic malignancy characterized by ineffective hematopoiesis, which leads to rapid progression into acute myeloid leukemia. UPD that occurs without concurrent changes in the gene copy number is a common chromosomal defect in hematologic malignancies, especially in MDS. Approximately 40-50% of MDS patients do not have karyotypic abnormalities that are detectable using classical metaphase cytogenetic techniques (MC) because of inherent limitations of MC, low resolution and the requirement of having dividing cells. In this review, we highlight advances in the clinical application of microarray technology in MDS and discuss the clinical potential of microarray.

  15. Array-CGH and clinical characterization in a patient with subtelomeric 6p deletion without ocular dysgenesis. (United States)

    Piccione, Maria; Antona, R; Salzano, E; Cavani, S; Malacarne, M; Morreale Bubella, R; Pierluigi, M; Viaggi, C D; Corsello, Giovanni


    Subtelomeric terminal 6p deletion has been recognized as a clinically identifiable syndrome including facial dysmorphism, malformation of the anterior eye chamber, hearing loss, heart defects, and developmental delay. Genotype-phenotype correlations of previously published patients have strongly suggested anterior eye segment anomalies as one of the major malformations of the syndrome if the critical 6p25 region contains the FOXC 1 gene. In addition, the presence in this region of one or more genes involved in hearing loss has been hypothesized. We report a patient with a 47,XYY karyotype and submicroscopic terminal 6p deletion. Further characterization of the deletion with array comparative genome hybridization also revealed a cryptic microduplication on chromosome 19. The patient showed dysmorphic features, neuromotor retardation, and profound language impairment, in absence of hearing loss and structural eye anomalies. As far as we know this is the first reported terminal 6p25.1 deletion case without eye dysgenesis precisely characterized by array-CGH. Our result suggests that the genes in this region may not be obvious candidates for hearing loss and demonstrate the need for further elucidation of the function of the genes involved in eye developmental processes.

  16. Screening of 50 cypriot patients with autism spectrum disorders or autistic features using 400K custom array-CGH. (United States)

    Kousoulidou, Ludmila; Moutafi, Maria; Nicolaides, Paola; Hadjiloizou, Stavros; Christofi, Christos; Paradesiotou, Anna; Anastasiadou, Violetta; Sismani, Carolina; Patsalis, Philippos C


    Autism spectrum disorders (ASDs) comprise a distinct entity of neurodevelopmental disorders with a strong genetic component. Despite the identification of several candidate genes and causative genomic copy number variations (CNVs), the majority of ASD cases still remain unresolved. We have applied microarray-based comparative genomic hybridization (array-CGH) using Agilent 400K custom array in the first Cyprus population screening for identification of ASD-associated CNVs. A cohort of 50 ASD patients (G1), their parents (G2), 50 ethnically matched normal controls (G3), and 80 normal individuals having children with various developmental and neurological conditions (G4) were tested. As a result, 14 patients were found to carry 20 potentially causative aberrations, two of which were de novo. Comparison of the four population groups revealed an increased rate of rare disease-associated variants in normal parents of children with autism. The above data provided additional evidence, supporting the complexity of ASD aetiology in comparison to other developmental disorders involving cognitive impairment. Furthermore, we have demonstrated the rationale of a more targeted approach combining accurate clinical description with high-resolution population-oriented genomic screening for defining the role of CNVs in autism and identifying meaningful associations on the molecular level.

  17. Screening of 50 Cypriot Patients with Autism Spectrum Disorders or Autistic Features Using 400K Custom Array-CGH

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    Ludmila Kousoulidou


    Full Text Available Autism spectrum disorders (ASDs comprise a distinct entity of neurodevelopmental disorders with a strong genetic component. Despite the identification of several candidate genes and causative genomic copy number variations (CNVs, the majority of ASD cases still remain unresolved. We have applied microarray-based comparative genomic hybridization (array-CGH using Agilent 400K custom array in the first Cyprus population screening for identification of ASD-associated CNVs. A cohort of 50 ASD patients (G1, their parents (G2, 50 ethnically matched normal controls (G3, and 80 normal individuals having children with various developmental and neurological conditions (G4 were tested. As a result, 14 patients were found to carry 20 potentially causative aberrations, two of which were de novo. Comparison of the four population groups revealed an increased rate of rare disease-associated variants in normal parents of children with autism. The above data provided additional evidence, supporting the complexity of ASD aetiology in comparison to other developmental disorders involving cognitive impairment. Furthermore, we have demonstrated the rationale of a more targeted approach combining accurate clinical description with high-resolution population-oriented genomic screening for defining the role of CNVs in autism and identifying meaningful associations on the molecular level.

  18. Genome-wide detection of copy number variations among diverse horse breeds by array CGH. (United States)

    Wang, Wei; Wang, Shenyuan; Hou, Chenglin; Xing, Yanping; Cao, Junwei; Wu, Kaifeng; Liu, Chunxia; Zhang, Dong; Zhang, Li; Zhang, Yanru; Zhou, Huanmin


    Recent studies have found that copy number variations (CNVs) are widespread in human and animal genomes. CNVs are a significant source of genetic variation, and have been shown to be associated with phenotypic diversity. However, the effect of CNVs on genetic variation in horses is not well understood. In the present study, CNVs in 6 different breeds of mare horses, Mongolia horse, Abaga horse, Hequ horse and Kazakh horse (all plateau breeds) and Debao pony and Thoroughbred, were determined using aCGH. In total, seven hundred CNVs were identified ranging in size from 6.1 Kb to 0.57 Mb across all autosomes, with an average size of 43.08 Kb and a median size of 15.11 Kb. By merging overlapping CNVs, we found a total of three hundred and fifty-three CNV regions (CNVRs). The length of the CNVRs ranged from 6.1 Kb to 1.45 Mb with average and median sizes of 38.49 Kb and 13.1 Kb. Collectively, 13.59 Mb of copy number variation was identified among the horses investigated and accounted for approximately 0.61% of the horse genome sequence. Five hundred and eighteen annotated genes were affected by CNVs, which corresponded to about 2.26% of all horse genes. Through the gene ontology (GO), genetic pathway analysis and comparison of CNV genes among different breeds, we found evidence that CNVs involving 7 genes may be related to the adaptation to severe environment of these plateau horses. This study is the first report of copy number variations in Chinese horses, which indicates that CNVs are ubiquitous in the horse genome and influence many biological processes of the horse. These results will be helpful not only in mapping the horse whole-genome CNVs, but also to further research for the adaption to the high altitude severe environment for plateau horses.

  19. De Novo Identification of Single Nucleotide Mutations in Caenorhabditis elegans Using Array Comparative Genomic Hybridization (United States)

    Maydan, Jason S.; Okada, H. Mark; Flibotte, Stephane; Edgley, Mark L.; Moerman, Donald G.


    Array comparative genomic hybridization (aCGH) has been used primarily to detect copy-number variants between two genomes. Here we report using aCGH to detect single nucleotide mutations on oligonucleotide microarrays with overlapping 50-mer probes. This technique represents a powerful method for rapidly detecting novel homozygous single nucleotide mutations in any organism with a sequenced reference genome. PMID:19189945

  20. Enhancing genome-wide copy number variation identification by high density array CGH using diverse resources of pig breeds.

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    Jiying Wang

    Full Text Available Copy number variations (CNVs are important forms of genomic variation, and have attracted extensive attentions in humans as well as domestic animals. In the study, using a custom-designed 2.1 M array comparative genomic hybridization (aCGH, genome-wide CNVs were identified among 12 individuals from diverse pig breeds, including one Asian wild population, six Chinese indigenous breeds and two modern commercial breeds (Yorkshire and Landrace, with one individual of the other modern commercial breed, Duroc, as the reference. A total of 1,344 CNV regions (CNVRs were identified, covering 47.79 Mb (∼1.70% of the pig genome. The length of these CNVRs ranged from 3.37 Kb to 1,319.0 Kb with a mean of 35.56 Kb and a median of 11.11 Kb. Compared with similar studies reported, most of the CNVRs (74.18% were firstly identified in present study. In order to confirm these CNVRs, 21 CNVRs were randomly chosen to be validated by quantitative real time PCR (qPCR and a high rate (85.71% of confirmation was obtained. Functional annotation of CNVRs suggested that the identified CNVRs have important function, and may play an important role in phenotypic and production traits difference among various breeds. Our results are essential complementary to the CNV map in the pig genome, which will provide abundant genetic markers to investigate association studies between various phenotypes and CNVs in pigs.

  1. Combined subtractive cDNA cloning and array CGH: an efficient approach for identification of overexpressed genes in DNA amplicons

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    De Paepe Anne


    Full Text Available Abstract Background Activation of proto-oncogenes by DNA amplification is an important mechanism in the development and maintenance of cancer cells. Until recently, identification of the targeted genes relied on labour intensive and time consuming positional cloning methods. In this study, we outline a straightforward and efficient strategy for fast and comprehensive cloning of amplified and overexpressed genes. Results As a proof of principle, we analyzed neuroblastoma cell line IMR-32, with at least two amplification sites along the short arm of chromosome 2. In a first step, overexpressed cDNA clones were isolated using a PCR based subtractive cloning method. Subsequent deposition of these clones on a custom microarray and hybridization with IMR-32 DNA, resulted in the identification of clones that were overexpressed due to gene amplification. Using this approach, amplification of all previously reported amplified genes in this cell line was detected. Furthermore, four additional clones were found to be amplified, including the TEM8 gene on 2p13.3, two anonymous transcripts, and a fusion transcript, resulting from 2p13.3 and 2p24.3 fused sequences. Conclusions The combinatorial strategy of subtractive cDNA cloning and array CGH analysis allows comprehensive amplicon dissection, which opens perspectives for improved identification of hitherto unknown targeted oncogenes in cancer cells.

  2. Improving molecular diagnosis of aniridia and WAGR syndrome using customized targeted array-based CGH (United States)

    Vallespín, Elena; Villaverde, Cristina; Martín-Arenas, Rubén; Vélez-Monsalve, Camilo; Lorda-Sánchez, Isabel; Nevado, Julián; Trujillo-Tiebas, María José; Lapunzina, Pablo; Ayuso, Carmen; Corton, Marta


    Chromosomal deletions at 11p13 are a frequent cause of congenital Aniridia, a rare pan-ocular genetic disease, and of WAGR syndrome, accounting up to 30% of cases. First-tier genetic testing for newborn with aniridia, to detect 11p13 rearrangements, includes Multiplex Ligation-dependent Probe Amplification (MLPA) and karyotyping. However, neither of these approaches allow obtaining a complete picture of the high complexity of chromosomal deletions and breakpoints in aniridia. Here, we report the development and validation of a customized targeted array-based comparative genomic hybridization, so called WAGR-array, for comprehensive high-resolution analysis of CNV in the WAGR locus. Our approach increased the detection rate in a Spanish cohort of 38 patients with aniridia, WAGR syndrome and other related ocular malformations, allowing to characterize four undiagnosed aniridia cases, and to confirm MLPA findings in four additional patients. For all patients, breakpoints were accurately established and a contiguous deletion syndrome, involving a large number of genes, was identified in three patients. Moreover, we identified novel microdeletions affecting 3' PAX6 regulatory regions in three families with isolated aniridia. This tool represents a good strategy for the genetic diagnosis of aniridia and associated syndromes, allowing for a more accurate CNVs detection, as well as a better delineation of breakpoints. Our results underline the clinical importance of performing exhaustive and accurate analysis of chromosomal rearrangements for patients with aniridia, especially newborns and those without defects in PAX6 after diagnostic screening. PMID:28231309

  3. A male newborn with VACTERL association and Fanconi anemia with a FANCB deletion detected by array comparative genomic hybridization (aCGH). (United States)

    Umaña, Luis A; Magoulas, Pilar; Bi, Weimin; Bacino, Carlos A


    We report on a male newborn with multiple congenital abnormalities consistent with the diagnosis of VACTERL association (vertebral, anal, cardiac, tracheo-esophageal fistula, renal, and limb anomalies), who had Fanconi anemia (complementation group B) recognized by the detection of a deletion in chromosome Xp22.2 using an oligonucleotide array. The diagnosis of Fanconi anemia was confirmed by increased chromosomal breakage abnormalities observed in cultured cells that were treated with cross-linking agents. This is the first report in the literature of Fanconi anemia complementation group B detected by oligonucleotide array testing postnatally.

  4. Strategies in the preparation of DNA oligonucleotide arrays for diagnostic applications. (United States)

    Beaucage, S L


    This report emphasizes the interfacial chemistry that is required to ensure proper attachment of oligonucleotides onto the surface of microarrays. For example, strategies for the covalent attachment of pre-synthesized oligonucleotides to glass slides, gold films, polyacrylamide gel pads, polypyrrole films, and optical fibers are surveyed in an attempt to better define the parameters for optimal formation and detection of DNA hybrids. These parameters include among others, the nature and length of the linkers attaching oligonucleotides to the arrays, and the surface density of oligonucleotides required for unhindered hybridization with DNA targets. Sensitive detection methods such as the use of light-scattering techniques, molecular beacons, surface plasmon resonance, attenuated total internal reflection-FTIR, and the evanescent field excitation of fluorescence from surface-bound fluorophores have been developed to study the kinetics and specificity of hybridization events. Finally, the synthesis of oligonucleotides directly on glass surfaces and polypropylene sheets has been investigated to enable DNA sequencing by hybridization and achieve oligonucleotide densities of ca. 10(6) sequences per cm(2) on DNA chips.

  5. Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species. (United States)

    Chen, Wen; Djama, Zeinab Robleh; Coffey, Michael D; Martin, Frank N; Bilodeau, Guillaume J; Radmer, Lorien; Denton, Geoff; Lévesque, C André


    Most Phytophthora spp. are destructive plant pathogens; therefore, effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of diseases. In the current study, a membrane-based oligonucleotide array was developed that can detect Phytophthora spp. reliably using three DNA regions; namely, the internal transcribed spacer (ITS), the 5' end of cytochrome c oxidase 1 gene (cox1), and the intergenic region between cytochrome c oxidase 2 gene (cox2) and cox1 (cox2-1 spacer). Each sequence data set contained ≈250 sequences representing 98 described and 15 undescribed species of Phytophthora. The array was validated with 143 pure cultures and 35 field samples. Together, nonrejected oligonucleotides from all three markers have the ability to reliably detect 82 described and 8 undescribed Phytophthora spp., including several quarantine or regulated pathogens such as Phytophthora ramorum. Our results showed that a DNA array containing signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for the detection and differentiation of closely related taxon groups. This array has the potential to be used as a routine diagnostic tool for Phytophthora spp. from complex environmental samples without the need for extensive growth of cultures.

  6. Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array

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    Shiu Lin-Yi


    Full Text Available Abstract Background Airborne fungi play an important role in causing allergy and infections in susceptible people. Identification of these fungi, based on morphological characteristics, is time-consuming, expertise-demanding, and could be inaccurate. Methods We developed an oligonucleotide array that could accurately identify 21 important airborne fungi (13 genera that may cause adverse health problems. The method consisted of PCR amplification of the internal transcribed spacer (ITS regions, hybridization of the PCR products to a panel of oligonucleotide probes immobilized on a nylon membrane, and detection of the hybridization signals with alkaline phosphatase-conjugated antibodies. Results A collection of 72 target and 66 nontarget reference strains were analyzed by the array. Both the sensitivity and specificity of the array were 100%, and the detection limit was 10 pg of genomic DNA per assay. Furthermore, 70 fungal isolates recovered from air samples were identified by the array and the identification results were confirmed by sequencing of the ITS and D1/D2 domain of the large-subunit RNA gene. The sensitivity and specificity of the array for identification of the air isolates was 100% (26/26 and 97.7% (43/44, respectively. Conclusions Identification of airborne fungi by the array was cheap and accurate. The current array may contribute to decipher the relationship between airborne fungi and adverse health effect.

  7. [Genomic abnormalities in children with mental retardation and autism: the use of comparative genomic hybridization in situ (HRCGH) and molecular karyotyping with DNA-microchips (array CGH)]. (United States)

    Vorsanova, S G; Iurov, I Iu; Kurinnaia, O S; Voinova, V Iu; Iurov, Iu B


    Genomic abnormalities occur with high frequency in children with mental retardation and autistic spectrum disorders (ADS). Molecular karyotyping using DNA microarrays is a new technology for diagnosis of genomic and chromosomal abnormalities in autism implemented in the fields of biological psychiatry and medical genetics. We carried out a comparative analysis of the frequency and spectrum of genome abnormalities in children with mental retardation and autism of unknown etiology using high-resolution comparative genomic methods for hybridization (HRCGH) and molecular karyotyping (array CGH). In a study of 100 children with autism, learning difficulties and congenital malformations by HRCGH, we identified genomic rearrangements in 46% of cases. Using array CGH we examined 50 children with autism. In 44 cases out of 50 (88%), different genomic abnormalities and genomic variations (CNV - copy number variations) were identified. Unbalanced genomic rearrangements, including deletions and duplications, were found in 23 cases out of 44 (52%). These data suggest that genomic abnormalities which are not detectable by common methods of chromosome analysis are often discovered by molecular cytogenetic techniques in children autism spectrum disorders. In addition, 54 children with idiopathic mental retardation and congenital malformations (31 boys and 23 girls) without autism spectrum disorders were examined using molecular karyotyping and microarray containing an increased number of DNA samples for genomic loci of chromosome X. Deletions and duplications affecting different regions of the chromosome X were detected in 11 out of 54 children (20.4%).

  8. Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array



    Abstract Background Airborne fungi play an important role in causing allergy and infections in susceptible people. Identification of these fungi, based on morphological characteristics, is time-consuming, expertise-demanding, and could be inaccurate. Methods We developed an oligonucleotide array that could accurately identify 21 important airborne fungi (13 genera) that may cause adverse health problems. The method consisted of PCR amplification of the internal transcribed spacer (ITS) region...

  9. Identification of cryptic microaberrations in osteosarcoma by high-definition oligonucleotide array comparative genomic hybridization. (United States)

    Selvarajah, Shamini; Yoshimoto, Maisa; Maire, Georges; Paderova, Jana; Bayani, Jane; Squire, Jeremy A; Zielenska, Maria


    Osteosarcoma (OS) is an aggressive bone tumor characterized by complex abnormal karyotypes and a high level of genomic instability. Using high-resolution array comparative genomic hybridization (aCGH), a novel class of localized copy number variations called microaberrations has been detected. These genomic anomalies typically involve DNA imbalances affecting 700 kb to 1 Mb DNA, and are often associated with some type of genetic syndromes. Because the origin of instability in OS is poorly understood, we used aCGH to determine whether microaberrations were a characteristic of four OS cell lines: U-2 OS, HOS, MG-63, and SAOS-2. TP53 is mutated in SAOS-2, a line in which 17 microaberrations were found. In contrast, U-2 OS, which has a wild-type TP53, had only six such anomalies, the lowest incidence. A 500-kb microaberration within a region of gain at 5p15.33 in SAOS-2 was confirmed by fluorescence in situ hybridization. Significantly, this genomic location is close to the TERT gene, a region of gain in all four cell lines. To our knowledge, this is the first systematic analysis of the incidence of microaberrations in OS. The high levels of these anomalies detected suggest that the instability processes in OS that lead to a highly abnormal karyotypes may also be associated with acquisition of genomic microaberrations.

  10. Chromosomal Aberrations in ETV6/RUNX1-positive Childhood Acute Lymphoblastic Leukemia using 244K Oligonucleotide Array Comparative Genomic Hybridization

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    Zakaria Zubaidah


    Full Text Available Abstract Background Acute lymphoblastic leukemia (ALL is a heterogeneous form of hematological cancer consisting of various subtypes. We are interested to study the genetic aberration in precursor B-cell ALL with specific t(12;21 translocation in childhood ALL patients. A high resolution 244K array-based Comparative Genomic Hybridization (array-CGH was used to study eleven ETV6/RUNX1-positive childhood acute lymphoblastic leukemia (ALL patients. Result 155 chromosomal aberrations (119 losses, 36 gains were reported in the array findings, corresponding to 76.8% deletions and 23.2% amplifications. The ETV6 gene deletion occurred in 4 of the patients, corresponding to 45% of the sample. The most common alterations above 1 Mb were deletion 6q (13%, 12p (12% and 9p (8%, and duplication 4q (6% and Xq (4%. Other genes important in ALL were also identified in this study including RUNX1, CDKN2A, FHIT, and PAX5. The array-CGH technique was able to detect microdeletion as small as 400 bp. Conclusion The results demonstrate the usefulness of high resolution array-CGH as a complementary tool in the investigation of ALL.

  11. BAC array CGH in patients with Velocardiofacial syndrome-like features reveals genomic aberrations on chromosome region 1q21.1

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    Estivill Xavier


    Full Text Available Abstract Background Microdeletion of the chromosome 22q11.2 region is the most common genetic aberration among patients with velocardiofacial syndrome (VCFS but a subset of subjects do not show alterations of this chromosome region. Methods We analyzed 18 patients with VCFS-like features by comparative genomic hybridisation (aCGH array and performed a face-to-face slide hybridization with two different arrays: a whole genome and a chromosome 22-specific BAC array. Putative rearrangements were confirmed by FISH and MLPA assays. Results One patient carried a combination of rearrangements on 1q21.1, consisting in a microduplication of 212 kb and a close microdeletion of 1.15 Mb, previously reported in patients with variable phenotypes, including mental retardation, congenital heart defects (CHD and schizophrenia. While 326 control samples were negative for both 1q21.1 rearrangements, one of 73 patients carried the same 212-kb microduplication, reciprocal to TAR microdeletion syndrome. Also, we detected four copy number variants (CNVs inherited from one parent (a 744-kb duplication on 10q11.22; a 160 kb duplication and deletion on 22q11.21 in two cases; and a gain of 140 kb on 22q13.2, not present in control subjects, raising the potential role of these CNVs in the VCFS-like phenotype. Conclusions Our results confirmed aCGH as a successful strategy in order to characterize additional submicroscopic aberrations in patients with VCF-like features that fail to show alterations in 22q11.2 region. We report a 212-kb microduplication on 1q21.1, detected in two patients, which may contribute to CHD.

  12. chipD: a web tool to design oligonucleotide probes for high-density tiling arrays (United States)

    Dufour, Yann S.; Wesenberg, Gary E.; Tritt, Andrew J.; Glasner, Jeremy D.; Perna, Nicole T.; Mitchell, Julie C.; Donohue, Timothy J.


    chipD is a web server that facilitates design of DNA oligonucleotide probes for high-density tiling arrays, which can be used in a number of genomic applications such as ChIP-chip or gene-expression profiling. The server implements a probe selection algorithm that takes as an input, in addition to the target sequences, a set of parameters that allow probe design to be tailored to specific applications, protocols or the array manufacturer’s requirements. The algorithm optimizes probes to meet three objectives: (i) probes should be specific; (ii) probes should have similar thermodynamic properties; and (iii) the target sequence coverage should be homogeneous and avoid significant gaps. The output provides in a text format, the list of probe sequences with their genomic locations, targeted strands and hybridization characteristics. chipD has been used successfully to design tiling arrays for bacteria and yeast. chipD is available at PMID:20529880

  13. Characteristics of Highly Polymorphic Segmental Copy-Number Variations Observed in Japanese by BAC-Array-CGH (United States)

    Takahashi, Norio; Satoh, Yasunari; Sasaki, Keiko; Shimoichi, Yuko; Sugita, Keiko; Katayama, Hiroaki


    Segmental copy-number variations (CNVs) may contribute to genetic variation in humans. Reports of the existence and characteristics of CNVs in a large Japanese cohort are quite limited. We report the data from a large Japanese population. We conducted population screening for 213 unrelated Japanese individuals using comparative genomic hybridization based on a bacterial artificial chromosome microarray (BAC-aCGH). We summarize the data by focusing on highly polymorphic CNVs in ≥5.0% of the individual, since they may be informative for demonstrating the relationships between genotypes and their phenotypes. We found a total of 680 CNVs at 16 different BAC-regions in the genome. The majority of the polymorphic CNVs presented on BAC-clones that overlapped with regions of segmental duplication, and the majority of the polymorphic CNVs observed in this population had been previously reported in other publications. Some of the CNVs contained genes which might be related to phenotypic heterogeneity among individuals. PMID:21197411

  14. Analysis of oligonucleotide array experiments with repeated measures using mixed models

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    Getchell Thomas V


    Full Text Available Abstract Background Two or more factor mixed factorial experiments are becoming increasingly common in microarray data analysis. In this case study, the two factors are presence (Patients with Alzheimer's disease or absence (Control of the disease, and brain regions including olfactory bulb (OB or cerebellum (CER. In the design considered in this manuscript, OB and CER are repeated measurements from the same subject and, hence, are correlated. It is critical to identify sources of variability in the analysis of oligonucleotide array experiments with repeated measures and correlations among data points have to be considered. In addition, multiple testing problems are more complicated in experiments with multi-level treatments or treatment combinations. Results In this study we adopted a linear mixed model to analyze oligonucleotide array experiments with repeated measures. We first construct a generalized F test to select differentially expressed genes. The Benjamini and Hochberg (BH procedure of controlling false discovery rate (FDR at 5% was applied to the P values of the generalized F test. For those genes with significant generalized F test, we then categorize them based on whether the interaction terms were significant or not at the α-level (αnew = 0.0033 determined by the FDR procedure. Since simple effects may be examined for the genes with significant interaction effect, we adopt the protected Fisher's least significant difference test (LSD procedure at the level of αnew to control the family-wise error rate (FWER for each gene examined. Conclusions A linear mixed model is appropriate for analysis of oligonucleotide array experiments with repeated measures. We constructed a generalized F test to select differentially expressed genes, and then applied a specific sequence of tests to identify factorial effects. This sequence of tests applied was designed to control for gene based FWER.

  15. Analysis of oligonucleotide array experiments with repeated measures using mixed models. (United States)

    Li, Hao; Wood, Constance L; Getchell, Thomas V; Getchell, Marilyn L; Stromberg, Arnold J


    Two or more factor mixed factorial experiments are becoming increasingly common in microarray data analysis. In this case study, the two factors are presence (Patients with Alzheimer's disease) or absence (Control) of the disease, and brain regions including olfactory bulb (OB) or cerebellum (CER). In the design considered in this manuscript, OB and CER are repeated measurements from the same subject and, hence, are correlated. It is critical to identify sources of variability in the analysis of oligonucleotide array experiments with repeated measures and correlations among data points have to be considered. In addition, multiple testing problems are more complicated in experiments with multi-level treatments or treatment combinations. In this study we adopted a linear mixed model to analyze oligonucleotide array experiments with repeated measures. We first construct a generalized F test to select differentially expressed genes. The Benjamini and Hochberg (BH) procedure of controlling false discovery rate (FDR) at 5% was applied to the P values of the generalized F test. For those genes with significant generalized F test, we then categorize them based on whether the interaction terms were significant or not at the alpha-level (alphanew = 0.0033) determined by the FDR procedure. Since simple effects may be examined for the genes with significant interaction effect, we adopt the protected Fisher's least significant difference test (LSD) procedure at the level of alphanew to control the family-wise error rate (FWER) for each gene examined. A linear mixed model is appropriate for analysis of oligonucleotide array experiments with repeated measures. We constructed a generalized F test to select differentially expressed genes, and then applied a specific sequence of tests to identify factorial effects. This sequence of tests applied was designed to control for gene based FWER.

  16. Rapid and specific detection of Lassa virus by reverse transcription-PCR coupled with oligonucleotide array hybridization. (United States)

    Olschläger, Stephan; Günther, Stephan


    To facilitate sequence-specific detection of DNA amplified in a diagnostic reverse transcription (RT)-PCR for Lassa virus, we developed an array featuring 47 oligonucleotide probes for post-PCR hybridization of the amplicons. The array procedure may be performed with low-tech equipment and does not take longer than agarose gel detection.

  17. Design of oligonucleotides for microarrays and perspectives for design of multi-transcriptome arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Wernersson, Rasmus; Knudsen, Steen


    Optimal design of oligonucleotides for microarrays involves tedious and laborious work evaluating potential oligonucleotides relative to a series of parameters. The currently available tools for this purpose are limited in their flexibility and do not present the oligonucleotide designer with an ......Optimal design of oligonucleotides for microarrays involves tedious and laborious work evaluating potential oligonucleotides relative to a series of parameters. The currently available tools for this purpose are limited in their flexibility and do not present the oligonucleotide designer...

  18. Characteristics of Highly Polymorphic Segmental Copy-Number Variations Observed in Japanese by BAC-Array-CGH

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    Norio Takahashi


    Full Text Available Segmental copy-number variations (CNVs may contribute to genetic variation in humans. Reports of the existence and characteristics of CNVs in a large Japanese cohort are quite limited. We report the data from a large Japanese population. We conducted population screening for 213 unrelated Japanese individuals using comparative genomic hybridization based on a bacterial artificial chromosome microarray (BAC-aCGH. We summarize the data by focusing on highly polymorphic CNVs in ≥5.0% of the individual, since they may be informative for demonstrating the relationships between genotypes and their phenotypes. We found a total of 680 CNVs at 16 different BAC-regions in the genome. The majority of the polymorphic CNVs presented on BAC-clones that overlapped with regions of segmental duplication, and the majority of the polymorphic CNVs observed in this population had been previously reported in other publications. Some of the CNVs contained genes which might be related to phenotypic heterogeneity among individuals.

  19. Copy-Number Variations Observed in a Japanese Population by BAC Array CGH: Summary of Relatively Rare CNVs

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    Yasunari Satoh


    Full Text Available Copy-number variations (CNVs may contribute to genetic variation in humans. Reports regarding existence and characteristics of CNVs in a large apparently healthy Japanese cohort are quite limited. We report the data from a screening of 213 unrelated Japanese individuals using comparative genomic hybridization based on a bacterial artificial chromosome microarray (BAC aCGH. In a previous paper, we summarized the data by focusing on highly polymorphic CNVs (in ≥5.0 % of the individuals. However, rare variations have recently received attention from scientists who espouse a hypothesis called “common disease and rare variants.” Here, we report CNVs identified in fewer than 10 individuals in our study population. We found a total of 126 CNVs at 52 different BAC regions in the genome. The CNVs observed at 27 of the 52 BAC-regions were found in only one unrelated individual. The majority of CNVs found in this study were not identified in the Japanese who were examined in the other studies. Family studies were conducted, and the results demonstrated that the CNVs were inherited from one parent in the families.

  20. PanCGH: a genotype-calling algorithm for pangenome CGH data.

    NARCIS (Netherlands)

    Bayjanov, J.; Wels, M.W.W.; Starrenburg, M.; Hylckama Vlieg, J.E. van; Siezen, R.J.; Molenaar, D.


    MOTIVATION: Pangenome arrays contain DNA oligomers targeting several sequenced reference genomes from the same species. In microbiology, these can be employed to investigate the often high genetic variability within a species by comparative genome hybridization (CGH). The biological interpretation o

  1. Tiling array-CGH for the assessment of genomic similarities among synchronous unilateral and bilateral invasive breast cancer tumor pairs

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    Ringnér Markus


    Full Text Available Abstract Background Today, no objective criteria exist to differentiate between individual primary tumors and intra- or intermammary dissemination respectively, in patients diagnosed with two or more synchronous breast cancers. To elucidate whether these tumors most likely arise through clonal expansion, or whether they represent individual primary tumors is of tumor biological interest and may have clinical implications. In this respect, high resolution genomic profiling may provide a more reliable approach than conventional histopathological and tumor biological factors. Methods 32 K tiling microarray-based comparative genomic hybridization (aCGH was used to explore the genomic similarities among synchronous unilateral and bilateral invasive breast cancer tumor pairs, and was compared with histopathological and tumor biological parameters. Results Based on global copy number profiles and unsupervised hierarchical clustering, five of ten (p = 1.9 × 10-5 unilateral tumor pairs displayed similar genomic profiles within the pair, while only one of eight bilateral tumor pairs (p = 0.29 displayed pair-wise genomic similarities. DNA index, histological type and presence of vessel invasion correlated with the genomic analyses. Conclusion Synchronous unilateral tumor pairs are often genomically similar, while synchronous bilateral tumors most often represent individual primary tumors. However, two independent unilateral primary tumors can develop synchronously and contralateral tumor spread can occur. The presence of an intraductal component is not informative when establishing the independence of two tumors, while vessel invasion, the presence of which was found in clustering tumor pairs but not in tumor pairs that did not cluster together, supports the clustering outcome. Our data suggest that genomically similar unilateral tumor pairs may represent a more aggressive disease that requires the addition of more severe treatment modalities, and

  2. Array-CGH reveals recurrent genomic changes in Merkel cell carcinoma including amplification of L-Myc. (United States)

    Paulson, Kelly G; Lemos, Bianca D; Feng, Bin; Jaimes, Natalia; Peñas, Pablo F; Bi, Xiaohui; Maher, Elizabeth; Cohen, Lisa; Leonard, J Helen; Granter, Scott R; Chin, Lynda; Nghiem, Paul


    Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with poorly characterized genetics. We performed high resolution comparative genomic hybridization on 25 MCC specimens using a high-density oligonucleotide microarray. Tumors frequently carried extra copies of chromosomes 1, 3q, 5p, and 6 and lost chromosomes 3p, 4, 5q, 7, 10, and 13. MCC tumors with less genomic aberration were associated with improved survival (P=0.04). Tumors from 13 of 22 MCC patients had detectable Merkel cell polyomavirus DNA, and these tumors had fewer genomic deletions. Three regions of genomic alteration were of particular interest: a deletion of 5q12-21 occurred in 26% of tumors, a deletion of 13q14-21 was recurrent in 26% of tumors and contains the well-characterized tumor suppressor RB1, and a previously unreported focal amplification at 1p34 was present in 39% of tumors and centers on L-Myc (MYCL1). L-Myc is related to the c-Myc proto-oncogene, has transforming activity, and is amplified in the closely related small cell lung cancer. Normal skin showed no L-Myc expression, whereas 4/4 MCC specimens tested expressed L-Myc RNA in relative proportion to the DNA copy number gain. These findings suggest several genes that may contribute to MCC pathogenesis, most notably L-Myc.

  3. Submicroscopic deletions of 11q24-25 in individuals without Jacobsen syndrome: re-examination of the critical region by high-resolution array-CGH

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    VanAllen Margot I


    Full Text Available Abstract Background Jacobsen syndrome is a rare contiguous gene disorder that results from a terminal deletion of the long arm of chromosome 11. It is typically characterized by intellectual disability, a variety of physical anomalies and a distinctive facial appearance. The 11q deletion has traditionally been identified by routine chromosome analysis. Array-based comparative genomic hybridization (array-CGH has offered new opportunities to identify and refine chromosomal abnormalities in regions known to be associated with clinical syndromes. Results Using the 1 Mb BAC array (Spectral Genomics, we screened 70 chromosomally normal children with idiopathic intellectual disability (ID and congenital abnormalities, and identified five cases with submicroscopic abnormalities believed to contribute to their phenotypes. Here, we provide detailed molecular cytogenetic descriptions and clinical presentation of two unrelated subjects with de novo submicroscopic deletions within chromosome bands 11q24-25. In subject 1 the chromosome rearrangement consisted of a 6.18 Mb deletion (from 128.25–134.43 Mb and an adjacent 5.04 Mb duplication (from 123.15–128.19 Mb, while in subject 2, a 4.74 Mb interstitial deletion was found (from 124.29–129.03 Mb. Higher resolution array analysis (385 K Nimblegen was used to refine all breakpoints. Deletions of the 11q24-25 region are known to be associated with Jacobsen syndrome (JBS: OMIM 147791. However, neither of the subjects had the typical features of JBS (trigonocephaly, platelet disorder, heart abnormalities. Both subjects had ID, dysmorphic features and additional phenotypic abnormalities: subject 1 had a kidney abnormality, bilateral preauricular pits, pectus excavatum, mild to moderate conductive hearing loss and behavioral concerns; subject 2 had macrocephaly, an abnormal MRI with delayed myelination, fifth finger shortening and squaring of all fingertips, and sensorineural hearing loss. Conclusion Two

  4. A statistical multiprobe model for analyzing cis and trans genes in genetical genomics experiments with short-oligonucleotide arrays

    NARCIS (Netherlands)

    Alberts, Rudi; Terpstra, Peter; Bystrykh, Leonid V.; Haan, Gerald de; Jansen, Ritsert C.


    Short-oligonucleotide arrays typically contain multiple probes per gene. In genetical genomics applications a statistical model for the individual probe signals can help in separating ‘‘true’’ differential mRNA expression from ‘‘ghost’’ effects caused by polymorphisms, misdesigned probes, and batch

  5. An Xq22.3 duplication detected by comparative genomic hybridization microarray (Array-CGH) defines a new locus (FGS5) for FG syndrome. (United States)

    Jehee, Fernanda Sarquis; Rosenberg, Carla; Krepischi-Santos, Ana Cristina; Kok, Fernando; Knijnenburg, Jeroen; Froyen, Guy; Vianna-Morgante, Angela M; Opitz, John M; Passos-Bueno, Maria Rita


    FG syndrome is an X-linked multiple congenital anomalies (MCA) syndrome. It has been mapped to four distinct loci FGS1-4, through linkage analysis (Xq13, Xp22.3, and Xp11.4-p11.3) and based on the breakpoints of an X chromosome inversion (Xq11:Xq28), but so far no gene has been identified. We describe a boy with FG syndrome who has an inherited duplication at band Xq22.3 detected by comparative genomic hybridization microarray (Array-CGH). These duplication maps outside all four loci described so far for FG syndrome, representing therefore a new locus, which we propose to be called FGS5. MID2, a gene closely related to MID1, which is known to be mutated in Opitz G/BBB syndrome, maps within the duplicated segment of our patient. Since FG and Opitz G/BBB syndromes share many manifestations we considered MID2 a candidate gene for FG syndrome. We also discuss the involvement of other potential genes within the duplicated segment and its relationship with clinical symptoms of our patient, as well as the laboratory abnormalities found in his mother, a carrier of the duplication.

  6. A sensitive method to detect the hepatitis B virus mutations by using solid phase PCR on oligonucleotide array

    Institute of Scientific and Technical Information of China (English)



    A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutations were immobilized on modified glass slides through 5' terminal linker,while the 3' terminal was made to be free. The mutated loci were designed to locate on the last nucleotides of 3' terminal respectively, and the positive control probes lacked the last nucleotide of 3' terminal in comparison with the probes used for detection. Probes fixed on oligonucleotide array were also the solid phase amplification primers. One pair of liquid primers was used to amplify the short template product from whole HBV DNA. Using target DNA as template, the solid primers were extended under the action of Taq DNA polymerase and incorporated with Cy-3dCTP as marker. During the thermal cycling reaction,probes served as solid phase amplification primers and amplification products bound to the oligonucleotide array, which could be visualized by incorporation with fluorescent dyes. After amplification, the oligonucleotide array was washed, performed with laser scanning, and then used for quantitative analysis to obtain the information for mutations. The hybridization results were compared with DNA sequencing. It was demonstrated that in case of sample A, the ratios of fluorescence intensities in wide type and in the muin these two loci. These results correlated to those obtained from DNA sequencing analysis in which the fluorescence intensity ratios in wide type and in the mutated types of 1996G-A and 1901D-A mutations in using solid phase PCR based on oligonucleotide array appears to be a sensitive and promising way to detect mutations with low-density.

  7. Refinement of light-responsive transcript lists using rice oligonucleotide arrays: evaluation of gene-redundancy.

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    Ki-Hong Jung

    Full Text Available Studies of gene function are often hampered by gene-redundancy, especially in organisms with large genomes such as rice (Oryza sativa. We present an approach for using transcriptomics data to focus functional studies and address redundancy. To this end, we have constructed and validated an inexpensive and publicly available rice oligonucleotide near-whole genome array, called the rice NSF45K array. We generated expression profiles for light- vs. dark-grown rice leaf tissue and validated the biological significance of the data by analyzing sources of variation and confirming expression trends with reverse transcription polymerase chain reaction. We examined trends in the data by evaluating enrichment of gene ontology terms at multiple false discovery rate thresholds. To compare data generated with the NSF45K array with published results, we developed publicly available, web-based tools ( The Oligo and EST Anatomy Viewer enables visualization of EST-based expression profiling data for all genes on the array. The Rice Multi-platform Microarray Search Tool facilitates comparison of gene expression profiles across multiple rice microarray platforms. Finally, we incorporated gene expression and biochemical pathway data to reduce the number of candidate gene products putatively participating in the eight steps of the photorespiration pathway from 52 to 10, based on expression levels of putatively functionally redundant genes. We confirmed the efficacy of this method to cope with redundancy by correctly predicting participation in photorespiration of a gene with five paralogs. Applying these methods will accelerate rice functional genomics.

  8. Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays

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    Sodergren Erica


    Full Text Available Abstract Background Syphilis spirochete Treponema pallidum ssp. pallidum remains the enigmatic pathogen, since no virulence factors have been identified and the pathogenesis of the disease is poorly understood. Increasing rates of new syphilis cases per year have been observed recently. Results The genome of the SS14 strain was sequenced to high accuracy by an oligonucleotide array strategy requiring hybridization to only three arrays (Comparative Genome Sequencing, CGS. Gaps in the resulting sequence were filled with targeted dideoxy-terminators (DDT sequencing and the sequence was confirmed by whole genome fingerprinting (WGF. When compared to the Nichols strain, 327 single nucleotide substitutions (224 transitions, 103 transversions, 14 deletions, and 18 insertions were found. On the proteome level, the highest frequency of amino acid-altering substitution polymorphisms was in novel genes, while the lowest was in housekeeping genes, as expected by their evolutionary conservation. Evidence was also found for hypervariable regions and multiple regions showing intrastrain heterogeneity in the T. pallidum chromosome. Conclusion The observed genetic changes do not have influence on the ability of Treponema pallidum to cause syphilitic infection, since both SS14 and Nichols are virulent in rabbit. However, this is the first assessment of the degree of variation between the two syphilis pathogens and paves the way for phylogenetic studies of this fascinating organism.

  9. Identification of novel deletions of 15q11q13 in Angelman syndrome by array-CGH: molecular characterization and genotype-phenotype correlations. (United States)

    Sahoo, Trilochan; Bacino, Carlos A; German, Jennifer R; Shaw, Chad A; Bird, Lynne M; Kimonis, Virginia; Anselm, Irinia; Waisbren, Susan; Beaudet, Arthur L; Peters, Sarika U


    Angelman syndrome (AS) is a neurodevelopmental disorder characterized by mental retardation, absent speech, ataxia, and a happy disposition. Deletions of the 15q11q13 region are found in approximately 70% of AS patients. The deletions are sub-classified into class I and class II based on their sizes of approximately 6.8 and approximately 6.0, respectively, with two different proximal breakpoints and a common distal breakpoint. Utilizing a chromosome 15-specific comparative genomic hybridization genomic microarray (array-CGH), we have identified, determined the deletion sizes, and mapped the breakpoints in a cohort of 44 cases, to relate those breakpoints to the genomic architecture and derive more precise genotype-phenotype correlations. Interestingly four patients of the 44 studied (9.1%) had novel and unusually large deletions, and are reported here. This is the first report of very large deletions of 15q11q13 resulting in AS; the largest deletion being >10.6 Mb. These novel deletions involve three different distal breakpoints, two of which have been earlier shown to be involved in the generation of isodicentric 15q chromosomes (idic15). Additionally, precise determination of the deletion breakpoints reveals the presence of directly oriented low-copy repeats (LCRs) flanking the recurrent and novel breakpoints. The LCRs are adequate in size, orientation, and homology to enable abnormal recombination events leading to deletions and duplications. This genomic organization provides evidence for a common mechanism for the generation of both common and rare deletion types. Larger deletions result in a loss of several genes outside the common Angelman syndrome-Prader-Willi syndrome (AS-PWS) critical interval, and a more severe phenotype.

  10. High-resolution array CGH profiling identifies Na/K transporting ATPase interacting 2 (NKAIN2 as a predisposing candidate gene in neuroblastoma.

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    Paolo Romania

    Full Text Available Neuroblastoma (NB, the most common solid cancer in early childhood, usually occurs sporadically but also its familial occurance is known in 1-2% of NB patients. Germline mutations in the ALK and PHOX2B genes have been found in a subset of familial NBs. However, because some individuals harbouring mutations in these genes do not develop this tumor, additional genetic alterations appear to be required for NB pathogenesis. Herein, we studied an Italian family with three NB patients, two siblings and a first cousin, carrying an ALK germline-activating mutation R1192P, that was inherited from their unaffected mothers and with no mutations in the PHOX2B gene. A comparison between somatic and germline DNA copy number changes in the two affected siblings by a high resolution array-based Comparative Genomic Hybridization (CGH analysis revealed a germline gain at NKAIN2 (Na/K transporting ATPase interacting 2 locus in one of the sibling, that was inherited from the parent who does not carry the ALK mutation. Surprisingly, NKAIN2 was expressed at high levels also in the affected sibling that lacks the genomic gain at this locus, clearly suggesting the existance of other regulatory mechanisms. High levels of NKAIN2 were detected in the MYCN-amplified NB cell lines and in the most aggressive NB lesions as well as in the peripheral blood of a large cohort of NB patients. Consistent with a role of NKAIN2 in NB development, NKAIN2 was down-regulated during all-trans retinoic acid differentiation in two NB cell lines. Taken together, these data indicate a potential role of NKAIN2 gene in NB growth and differentiation.

  11. Evaluation of methods for oligonucleotide array data via quantitative real-time PCR

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    Morris Daryl E


    Full Text Available Abstract Background There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR is generally considered the "gold-standard" assay for measuring gene expression by biologists and is often used to confirm findings from microarray data. Here we use qRT-PCR measurements to validate methods for the components of processing oligo array data: background adjustment, normalization, mismatch adjustment, and probeset summary. An advantage of our approach over spike-in studies is that methods are validated on a real dataset that was collected to address a scientific question. Results We initially identify three of six popular methods that consistently produced the best agreement between oligo array and RT-PCR data for medium- and high-intensity genes. The three methods are generally known as MAS5, gcRMA, and the dChip mismatch mode. For medium- and high-intensity genes, we identified use of data from mismatch probes (as in MAS5 and dChip mismatch and a sequence-based method of background adjustment (as in gcRMA as the most important factors in methods' performances. However, we found poor reliability for methods

  12. Comparative linkage analysis and visualization of high-density oligonucleotide SNP array data

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    Smith Richard JH


    Full Text Available Abstract Background The identification of disease-associated genes using single nucleotide polymorphisms (SNPs has been increasingly reported. In particular, the Affymetrix Mapping 10 K SNP microarray platform uses one PCR primer to amplify the DNA samples and determine the genotype of more than 10,000 SNPs in the human genome. This provides the opportunity for large scale, rapid and cost-effective genotyping assays for linkage analysis. However, the analysis of such datasets is nontrivial because of the large number of markers, and visualizing the linkage scores in the context of genome maps remains less automated using the current linkage analysis software packages. For example, the haplotyping results are commonly represented in the text format. Results Here we report the development of a novel software tool called CompareLinkage for automated formatting of the Affymetrix Mapping 10 K genotype data into the "Linkage" format and the subsequent analysis with multi-point linkage software programs such as Merlin and Allegro. The new software has the ability to visualize the results for all these programs in dChip in the context of genome annotations and cytoband information. In addition we implemented a variant of the Lander-Green algorithm in the dChipLinkage module of dChip software (V1.3 to perform parametric linkage analysis and haplotyping of SNP array data. These functions are integrated with the existing modules of dChip to visualize SNP genotype data together with LOD score curves. We have analyzed three families with recessive and dominant diseases using the new software programs and the comparison results are presented and discussed. Conclusions The CompareLinkage and dChipLinkage software packages are freely available. They provide the visualization tools for high-density oligonucleotide SNP array data, as well as the automated functions for formatting SNP array data for the linkage analysis programs Merlin and Allegro and calling

  13. aCGH-MAS: Analysis of aCGH by means of Multiagent System (United States)

    Benito, Rocío; Bajo, Javier; Rodríguez, Ana Eugenia; Abáigar, María


    There are currently different techniques, such as CGH arrays, to study genetic variations in patients. CGH arrays analyze gains and losses in different regions in the chromosome. Regions with gains or losses in pathologies are important for selecting relevant genes or CNVs (copy-number variations) associated with the variations detected within chromosomes. Information corresponding to mutations, genes, proteins, variations, CNVs, and diseases can be found in different databases and it would be of interest to incorporate information of different sources to extract relevant information. This work proposes a multiagent system to manage the information of aCGH arrays, with the aim of providing an intuitive and extensible system to analyze and interpret the results. The agent roles integrate statistical techniques to select relevant variations and visualization techniques for the interpretation of the final results and to extract relevant information from different sources of information by applying a CBR system. PMID:25874203

  14. Custom CGH array profiling of copy number variations (CNVs on chromosome 6p21.32 (HLA locus in patients with venous malformations associated with multiple sclerosis

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    Salvi Fabrizio


    Full Text Available Abstract Background Multiple sclerosis (MS is a complex disorder thought to result from an interaction between environmental and genetic predisposing factors which have not yet been characterised, although it is known to be associated with the HLA region on 6p21.32. Recently, a picture of chronic cerebrospinal venous insufficiency (CCSVI, consequent to stenosing venous malformation of the main extra-cranial outflow routes (VM, has been described in patients affected with MS, introducing an additional phenotype with possible pathogenic significance. Methods In order to explore the presence of copy number variations (CNVs within the HLA locus, a custom CGH array was designed to cover 7 Mb of the HLA locus region (6,899,999 bp; chr6:29,900,001-36,800,000. Genomic DNA of the 15 patients with CCSVI/VM and MS was hybridised in duplicate. Results In total, 322 CNVs, of which 225 were extragenic and 97 intragenic, were identified in 15 patients. 234 known polymorphic CNVs were detected, the majority of these being situated in non-coding or extragenic regions. The overall number of CNVs (both extra- and intragenic showed a robust and significant correlation with the number of stenosing VMs (Spearman: r = 0.6590, p = 0.0104; linear regression analysis r = 0.6577, p = 0.0106. The region we analysed contains 211 known genes. By using pathway analysis focused on angiogenesis and venous development, MS, and immunity, we tentatively highlight several genes as possible susceptibility factor candidates involved in this peculiar phenotype. Conclusions The CNVs contained in the HLA locus region in patients with the novel phenotype of CCSVI/VM and MS were mapped in detail, demonstrating a significant correlation between the number of known CNVs found in the HLA region and the number of CCSVI-VMs identified in patients. Pathway analysis revealed common routes of interaction of several of the genes involved in angiogenesis and immunity contained within this region

  15. arrayCGHbase: an analysis platform for comparative genomic hybridization microarrays

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    Moreau Yves


    Full Text Available Abstract Background The availability of the human genome sequence as well as the large number of physically accessible oligonucleotides, cDNA, and BAC clones across the entire genome has triggered and accelerated the use of several platforms for analysis of DNA copy number changes, amongst others microarray comparative genomic hybridization (arrayCGH. One of the challenges inherent to this new technology is the management and analysis of large numbers of data points generated in each individual experiment. Results We have developed arrayCGHbase, a comprehensive analysis platform for arrayCGH experiments consisting of a MIAME (Minimal Information About a Microarray Experiment supportive database using MySQL underlying a data mining web tool, to store, analyze, interpret, compare, and visualize arrayCGH results in a uniform and user-friendly format. Following its flexible design, arrayCGHbase is compatible with all existing and forthcoming arrayCGH platforms. Data can be exported in a multitude of formats, including BED files to map copy number information on the genome using the Ensembl or UCSC genome browser. Conclusion ArrayCGHbase is a web based and platform independent arrayCGH data analysis tool, that allows users to access the analysis suite through the internet or a local intranet after installation on a private server. ArrayCGHbase is available at

  16. Error, reproducibility and sensitivity: a pipeline for data processing of Agilent oligonucleotide expression arrays

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    Posch Wilfried


    Full Text Available Abstract Background Expression microarrays are increasingly used to obtain large scale transcriptomic information on a wide range of biological samples. Nevertheless, there is still much debate on the best ways to process data, to design experiments and analyse the output. Furthermore, many of the more sophisticated mathematical approaches to data analysis in the literature remain inaccessible to much of the biological research community. In this study we examine ways of extracting and analysing a large data set obtained using the Agilent long oligonucleotide transcriptomics platform, applied to a set of human macrophage and dendritic cell samples. Results We describe and validate a series of data extraction, transformation and normalisation steps which are implemented via a new R function. Analysis of replicate normalised reference data demonstrate that intrarray variability is small (only around 2% of the mean log signal, while interarray variability from replicate array measurements has a standard deviation (SD of around 0.5 log2 units ( 6% of mean. The common practise of working with ratios of Cy5/Cy3 signal offers little further improvement in terms of reducing error. Comparison to expression data obtained using Arabidopsis samples demonstrates that the large number of genes in each sample showing a low level of transcription reflect the real complexity of the cellular transcriptome. Multidimensional scaling is used to show that the processed data identifies an underlying structure which reflect some of the key biological variables which define the data set. This structure is robust, allowing reliable comparison of samples collected over a number of years and collected by a variety of operators. Conclusions This study outlines a robust and easily implemented pipeline for extracting, transforming normalising and visualising transcriptomic array data from Agilent expression platform. The analysis is used to obtain quantitative estimates of

  17. Characterization of 11p14-p12 deletion in WAGR syndrome by array CGH for identifying genes contributing to mental retardation and autism. (United States)

    Xu, S; Han, J C; Morales, A; Menzie, C M; Williams, K; Fan, Y-S


    WAGR (Wilms tumor, Aniridia, Genitourinary malformations and mental Retardation) syndrome is a rare genomic disorder caused by deletion of the 11p14-p12 chromosome region. The majority of WAGR patients have mental retardation and behavioral problems, and more than 20% of the patients also have features of autism. While the Wilms tumor/genitourinary anomalies and aniridia are caused by deletion of WT1 and PAX6 respectively, the genomic cause of mental retardation and autism in WAGR syndrome remains unknown. Using oligonucleotide arrays, we have characterized the 11p14-p12 deletions in 31 patients and identified all the genes involved in each deletion. The deletions had sizes ranging from 4.9 to 23 Mb that encompass 18-62 genes (40 on average). In addition to WT1 and PAX6, all the patients had deletion of PRRG4 (transmembrane gamma-carboxyglutamic acid protein 4). The majority of them had deletion of BDNF (brain-derived neurotrophic factor) and SLC1A2 [solute carrier family 1 (glial high affinity glutamate transporter) member 2]. Deletion of BDNF and SLC1A2 occurred in patients with autism more frequently than in those without autism. Literature review on the functions of the genes suggests that haploinsufficiency of SLC1A2, PRRG4, and BDNF may contribute to mental retardation and behavioral problems. In particular, BDNF may modulate the risk of autism in WAGR patients as suggested by its link with Rett syndrome as a target of MECP2. We observed that all the de novo deletions occurred in the chromosome 11 inherited from the father in the families genotyped, implying a predisposition for de novo mutations occurring in spermatogenesis and possible involvement of imprinting in cognitive impairment in WAGR patients. Copyright 2008 S. Karger AG, Basel.

  18. Development of a novel ozone- and photo-stable HyPer5 red fluorescent dye for array CGH and microarray gene expression analysis with consistent performance irrespective of environmental conditions

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    Kille Peter


    Full Text Available Abstract Background Array-based comparative genomic hybridization (CGH and gene expression profiling have become vital techniques for identifying molecular defects underlying genetic diseases. Regardless of the microarray platform, cyanine dyes (Cy3 and Cy5 are one of the most widely used fluorescent dye pairs for microarray analysis owing to their brightness and ease of incorporation, enabling high level of assay sensitivity. However, combining both dyes on arrays can become problematic during summer months when ozone levels rise to near 25 parts per billion (ppb. Under such conditions, Cy5 is known to rapidly degrade leading to loss of signal from either "homebrew" or commercial arrays. Cy5 can also suffer disproportionately from dye photobleaching resulting in distortion of (Cy5/Cy3 ratios used in copy number analysis. Our laboratory has been active in fluorescent dye research to find a suitable alternative to Cy5 that is stable to ozone and resistant to photo-bleaching. Here, we report on the development of such a dye, called HyPer5, and describe its' exceptional ozone and photostable properties on microarrays. Results Our results show HyPer5 signal to be stable to high ozone levels. Repeated exposure of mouse arrays hybridized with HyPer5-labeled cDNA to 300 ppb ozone at 5, 10 and 15 minute intervals resulted in no signal loss from the dye. In comparison, Cy5 arrays showed a dramatic 80% decrease in total signal during the same interval. Photobleaching experiments show HyPer5 to be resistant to light induced damage with 3- fold improvement in dye stability over Cy5. In high resolution array CGH experiments, HyPer5 is demonstrated to detect chromosomal aberrations at loci 2p21-16.3 and 15q26.3-26.2 from three patient sample using bacterial artificial chromosome (BAC arrays. The photostability of HyPer5 is further documented by repeat array scanning without loss of detection. Additionally, HyPer5 arrays are shown to preserve sensitivity and

  19. Characterization of genetic rearrangements in esophageal squamous carcinoma cell lines by a combination of M-FISH and array-CGH: further confirmation of some split genomic regions in primary tumors

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    Hao Jia-Jie


    Full Text Available Abstract Background Chromosomal and genomic aberrations are common features of human cancers. However, chromosomal numerical and structural aberrations, breakpoints and disrupted genes have yet to be identified in esophageal squamous cell carcinoma (ESCC. Methods Using multiplex-fluorescence in situ hybridization (M-FISH and oligo array-based comparative hybridization (array-CGH, we identified aberrations and breakpoints in six ESCC cell lines. Furthermore, we detected recurrent breakpoints in primary tumors by dual-color FISH. Results M-FISH and array-CGH results revealed complex numerical and structural aberrations. Frequent gains occurred at 3q26.33-qter, 5p14.1-p11, 7pter-p12.3, 8q24.13-q24.21, 9q31.1-qter, 11p13-p11, 11q11-q13.4, 17q23.3-qter, 18pter-p11, 19 and 20q13.32-qter. Losses were frequent at 18q21.1-qter. Breakpoints that clustered within 1 or 2 Mb were identified, including 9p21.3, 11q13.3-q13.4, 15q25.3 and 3q28. By dual-color FISH, we observed that several recurrent breakpoint regions in cell lines were also present in ESCC tumors. In particular, breakpoints clustered at 11q13.3-q13.4 were identified in 43.3% (58/134 of ESCC tumors. Both 11q13.3-q13.4 splitting and amplification were significantly correlated with lymph node metastasis (LNM (P = 0.004 and 0.022 and advanced stages (P = 0.004 and 0.039. Multivariate logistic regression analysis revealed that only 11q13.3-q13.4 splitting was an independent predictor for LNM (P = 0.026. Conclusions The combination of M-FISH and array-CGH helps produce more accurate karyotypes. Our data provide significant, detailed information for appropriate uses of these ESCC cell lines for cytogenetic and molecular biological studies. The aberrations and breakpoints detected in both the cell lines and primary tumors will contribute to identify affected genes involved in the development and progression of ESCC.

  20. A patient with limb girdle muscular dystrophy carries a TRIM32 deletion, detected by a novel CGH array, in compound heterozygosis with a nonsense mutation. (United States)

    Neri, M; Selvatici, R; Scotton, C; Trabanelli, C; Armaroli, A; De Grandis, D; Levy, N; Gualandi, F; Ferlini, A


    Limb girdle muscular dystrophy 2H is a rare autosomal recessive muscular dystrophy, clinically highly variable, caused by mutations in the TRIM32 gene. Here we describe a 35-years-old who experienced progressive muscle weakness. The muscle biopsy revealed an unspecific pattern of atrophic and hypertrophic fibers; the immunohistochemistry for several proteins was normal. Comparative genomic hybridization (CGH) analysis showed a heterozygous deletion of the entire TRIM32 gene. On the other allele we identified the R316X nonsense mutation. The genetic diagnosis of LGMD2H in this case was reached by using a novel high throughput diagnostic tool.

  1. Screening the Single Nucleotide Polymorphisms in Patients with Internal Carotid Artery Stenosis by Oligonucleotide-Based Custom DNA Array

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    Kenji Nakai


    Full Text Available Early screening of individuals considered to be at risk for severe internal carotid artery (ICA stenosis is an important strategy for preventing ischemic cerebral stroke. The purpose of this study is to screening candidate single nucleotide polymorphisms (SNPs associated with severe ICA stenosis using a newly developed oligonucleotide-based custom DNA array. The subjects consisted of 47 controls and 46 patients with severe ICA stenosis (70% who underwent carotid endarterectomy (CEA. Subjects gave informed consent and we obtained samples of blood and genomic DNA. We studied 8 candidate genes: renin-angiotensin system [angiotensinogen (AGT, angiotensin II receptor type 1 (AGTR1, nitric oxide synthase 3 (NOS3]; growth factor [hepatocyte growth factor (HGF]; transgelin (SM22; cytokine [chemokine receptor 2 (CCR2]; coagulation-fibrinolysis system [5,10-methylenetetrahydrofolate reductase (MTHFR]; and plasminogen activator inhibitor 1 (PAI-1. Genotyping of candidate SNPs was done with a line probe assay (LiPA based on an oligonucleotide-based DNA array. Results: The allele frequency of PAI-1 –1965 delG (odds ratio (OR, 0.3; 95% confidence interval (CI, 0.2–0.6 and MTHFR (OR 1.3, 95% CI, 1.0–1.5 were significantly different between controls and cases with ICA stenosis by Fisher’s exact test. Multiple logistic analysis revealed that diabetes mellitus (DM, SNPs in PAI-1 –1965 delG and MTHFR were an independent risk for ICA stenosis. In conclusion, genetic factors of coagulation-fibrinolysis as well as diabetes mellitus (DM were relevant in ICA stenosis.

  2. Oligonucleotide array discovery of polymorphisms in cultivated tomato (Solanum lycopersicum L.) reveals patterns of SNP variation associated with breeding. (United States)

    Sim, Sung-Chur; Robbins, Matthew D; Chilcott, Charles; Zhu, Tong; Francis, David M


    Cultivated tomato (Solanum lycopersicum L.) has narrow genetic diversity that makes it difficult to identify polymorphisms between elite germplasm. We explored array-based single feature polymorphism (SFP) discovery as a high-throughput approach for marker development in cultivated tomato. Three varieties, FL7600 (fresh-market), OH9242 (processing), and PI114490 (cherry) were used as a source of genomic DNA for hybridization to oligonucleotide arrays. Identification of SFPs was based on outlier detection using regression analysis of normalized hybridization data within a probe set for each gene. A subset of 189 putative SFPs was sequenced for validation. The rate of validation depended on the desired level of significance (alpha) used to define the confidence interval (CI), and ranged from 76% for polymorphisms identified at alpha or= 2 SNPs per locus. We used a subset of validated SNPs for genetic diversity analysis of 92 tomato varieties and accessions. Pairwise estimation of theta (Fst) suggested significant differentiation between collections of fresh-market, processing, vintage, Latin American (landrace), and S. pimpinellifolium accessions. The fresh-market and processing groups displayed high genetic diversity relative to vintage and landrace groups. Furthermore, the patterns of SNP variation indicated that domestication and early breeding practices have led to progressive genetic bottlenecks while modern breeding practices have reintroduced genetic variation into the crop from wild species. Finally, we examined the ratio of non-synonymous (Ka) to synonymous substitutions (Ks) for 20 loci with multiple SNPs (>or= 4 per locus). Six of 20 loci showed ratios of Ka/Ks >or= 0.9. Array-based SFP discovery was an efficient method to identify a large number of molecular markers for genetics and breeding in elite tomato germplasm. Patterns of sequence variation across five major tomato groups provided insight into to the effect of human selection on genetic variation.

  3. How to decide? Different methods of calculating gene expression from short oligonucleotide array data will give different results

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    Voesenek Laurentius ACJ


    Full Text Available Abstract Background Short oligonucleotide arrays for transcript profiling have been available for several years. Generally, raw data from these arrays are analysed with the aid of the Microarray Analysis Suite or GeneChip Operating Software (MAS or GCOS from Affymetrix. Recently, more methods to analyse the raw data have become available. Ideally all these methods should come up with more or less the same results. We set out to evaluate the different methods and include work on our own data set, in order to test which method gives the most reliable results. Results Calculating gene expression with 6 different algorithms (MAS5, dChip PMMM, dChip PM, RMA, GC-RMA and PDNN using the same (Arabidopsis data, results in different calculated gene expression levels. Consequently, depending on the method used, different genes will be identified as differentially regulated. Surprisingly, there was only 27 to 36% overlap between the different methods. Furthermore, 47.5% of the genes/probe sets showed good correlation between the mismatch and perfect match intensities. Conclusion After comparing six algorithms, RMA gave the most reproducible results and showed the highest correlation coefficients with Real Time RT-PCR data on genes identified as differentially expressed by all methods. However, we were not able to verify, by Real Time RT-PCR, the microarray results for most genes that were solely calculated by RMA. Furthermore, we conclude that subtraction of the mismatch intensity from the perfect match intensity results most likely in a significant underestimation for at least 47.5% of the expression values. Not one algorithm produced significant expression values for genes present in quantities below 1 pmol. If the only purpose of the microarray experiment is to find new candidate genes, and too many genes are found, then mutual exclusion of the genes predicted by contrasting methods can be used to narrow down the list of new candidate genes by 64 to 73%.

  4. Detection and precise mapping of germline rearrangements in BRCA1, BRCA2, MSH2, and MLH1 using zoom-in array comparative genomic hybridization (aCGH)

    DEFF Research Database (Denmark)

    Staaf, Johan; Törngren, Therese; Rambech, Eva


    of primers for sequence determination of the breakpoints. The array platform can be streamlined for a particular application, e.g., focusing on breast cancer susceptibility genes, with increased capacity using multiformat design, and represents a valuable new tool and complement for genetic screening...

  5. An array CGH based genomic instability index (G2I is predictive of clinical outcome in breast cancer and reveals a subset of tumors without lymph node involvement but with poor prognosis

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    Bonnet Françoise


    Full Text Available Abstract Background Despite entering complete remission after primary treatment, a substantial proportion of patients with early stage breast cancer will develop metastases. Prediction of such an outcome remains challenging despite the clinical use of several prognostic parameters. Several reports indicate that genomic instability, as reflected in specific chromosomal aneuploidies and variations in DNA content, influences clinical outcome but no precise definition of this parameter has yet been clearly established. Methods To explore the prognostic value of genomic alterations present in primary tumors, we performed a comparative genomic hybridization study on BAC arrays with a panel of breast carcinomas from 45 patients with metastatic relapse and 95 others, matched for age and axillary node involvement, without any recurrence after at least 11 years of follow-up. Array-CGH data was used to establish a two-parameter index representative of the global level of aneusomy by chromosomal arm, and of the number of breakpoints throughout the genome. Results Application of appropriate thresholds allowed us to distinguish three classes of tumors highly associated with metastatic relapse. This index used with the same thresholds on a published set of tumors confirms its prognostic significance with a hazard ratio of 3.24 [95CI: 1.76-5.96] p = 6.7x10-5 for the bad prognostic group with respect to the intermediate group. The high prognostic value of this genomic index is related to its ability to individualize a specific group of breast cancers, mainly luminal type and axillary node negative, showing very high genetic instability and poor outcome. Indirect transcriptomic validation was obtained on independent data sets. Conclusion Accurate evaluation of genetic instability in breast cancers by a genomic instability index (G2I helps individualizing specific tumors with previously unexpected very poor prognosis.

  6. Oligonucleotide array discovery of polymorphisms in cultivated tomato (Solanum lycopersicum L. reveals patterns of SNP variation associated with breeding

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    Zhu Tong


    Full Text Available Abstract Background Cultivated tomato (Solanum lycopersicum L. has narrow genetic diversity that makes it difficult to identify polymorphisms between elite germplasm. We explored array-based single feature polymorphism (SFP discovery as a high-throughput approach for marker development in cultivated tomato. Results Three varieties, FL7600 (fresh-market, OH9242 (processing, and PI114490 (cherry were used as a source of genomic DNA for hybridization to oligonucleotide arrays. Identification of SFPs was based on outlier detection using regression analysis of normalized hybridization data within a probe set for each gene. A subset of 189 putative SFPs was sequenced for validation. The rate of validation depended on the desired level of significance (α used to define the confidence interval (CI, and ranged from 76% for polymorphisms identified at α ≤ 10-6 to 60% for those identified at α ≤ 10-2. Validation percentage reached a plateau between α ≤ 10-4 and α ≤ 10-7, but failure to identify known SFPs (Type II error increased dramatically at α ≤ 10-6. Trough sequence validation, we identified 279 SNPs and 27 InDels in 111 loci. Sixty loci contained ≥ 2 SNPs per locus. We used a subset of validated SNPs for genetic diversity analysis of 92 tomato varieties and accessions. Pairwise estimation of θ (Fst suggested significant differentiation between collections of fresh-market, processing, vintage, Latin American (landrace, and S. pimpinellifolium accessions. The fresh-market and processing groups displayed high genetic diversity relative to vintage and landrace groups. Furthermore, the patterns of SNP variation indicated that domestication and early breeding practices have led to progressive genetic bottlenecks while modern breeding practices have reintroduced genetic variation into the crop from wild species. Finally, we examined the ratio of non-synonymous (Ka to synonymous substitutions (Ks for 20 loci with multiple SNPs (≥ 4 per

  7. Array-CGH and quantitative PCR genetic analysis in a case with bilateral hypoplasia of pulmonary arteries and lungs and simultaneous unilateral renal agenesis. (United States)

    Hussein, Kais; Steinemann, Doris; Scholz, Henrike; Menkhaus, Ralf; Feist, Henning; Kreipe, Hans


    We describe the clinical course and have characterised anatomically and genetically a unique case of a newborn with bilateral hypoplasia of pulmonary arteries, consecutive extremely hypoplastic lung tissue and associated unilateral renal agenesis. Intrauterine oxygenation by the placenta seemed to have allowed normotrophic body maturity but immediately after delivery, in the third trimester, progressive hypoxemia developed and the newborn succumbed to acute respiratory failure. Genetic analysis by array-based comparative genomic hybridisation and quantitative PCR revealed duplication of 1p21, which, however, might not be the disease causing aberration. This case might represent an extreme form of previously reported, rare cases with simultaneous dysorganogenesis of lungs and kidneys.

  8. Empirical evaluation of oligonucleotide probe selection for DNA microarrays.

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    Jennifer G Mulle

    Full Text Available DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs, and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.

  9. Empirical evaluation of oligonucleotide probe selection for DNA microarrays. (United States)

    Mulle, Jennifer G; Patel, Viren C; Warren, Stephen T; Hegde, Madhuri R; Cutler, David J; Zwick, Michael E


    DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.

  10. Array CGH in patients with learning disability (mental retardation) and congenital anomalies: updated systematic review and meta-analysis of 19 studies and 13,926 subjects. (United States)

    Sagoo, Gurdeep S; Butterworth, Adam S; Sanderson, Simon; Shaw-Smith, Charles; Higgins, Julian P T; Burton, Hilary


    Array-based comparative genomic hybridization is being increasingly used in patients with learning disability (mental retardation) and congenital anomalies. In this article, we update our previous meta-analysis evaluating the diagnostic and false-positive yields of this technology. An updated systematic review and meta-analysis was conducted investigating patients with learning disability and congenital anomalies in whom conventional cytogenetic analyses have proven negative. Nineteen studies (13,926 patients) were included of which 12 studies (13,464 patients) were published since our previous analysis. The overall diagnostic yield of causal abnormalities was 10% (95% confidence interval: 8-12%). The overall number needed to test to identify an extra causal abnormality was 10 (95% confidence interval: 8-13). The overall false-positive yield of noncausal abnormalities was 7% (95% confidence interval: 5-10%). This updated meta-analysis provides new evidence to support the use of array-based comparative genomic hybridization in investigating patients with learning disability and congenital anomalies in whom conventional cytogenetic tests have proven negative. However, given that this technology also identifies false positives at a similar rate to causal variants, caution in clinical practice should be advised.

  11. Array CGH characterization of an unbalanced X-autosome translocation associated with Xq27.2-qter deletion, 11q24.3-qter duplication and Xq22.3-q27.1 duplication in a girl with primary amenorrhea and mental retardation. (United States)

    Chen, Chih-Ping; Lin, Shuan-Pei; Chern, Schu-Rern; Kuo, Yu-Ling; Wu, Peih-Shan; Chen, Yu-Ting; Lee, Meng-Shan; Wang, Wayseen


    We present array comparative genomic hybridization (aCGH) characterization of an unbalanced X-autosome translocation with an Xq interstitial segmental duplication in a 16-year-old girl with primary ovarian failure, mental retardation, attention deficit disorder, learning difficulty and facial dysmorphism. aCGH analysis revealed an Xq27.2-q28 deletion, an 11q24.3-q25 duplication, and an inverted duplication of Xq22.3-q27.1. The karyotype was 46,X,der(X)t(X;11)(q27.2;q24.3) dup(X)(q27.1q22.3). We discuss the genotype-phenotype correlation in this case. Our case provides evidence for an association of primary amenorrhea and mental retardation with concomitant unbalanced X-autosome translocation and X chromosome rearrangement.

  12. Genomic profiling of plasmablastic lymphoma using array comparative genomic hybridization (aCGH: revealing significant overlapping genomic lesions with diffuse large B-cell lymphoma

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    Lu Xin-Yan


    Full Text Available Abstract Background Plasmablastic lymphoma (PL is a subtype of diffuse large B-cell lymphoma (DLBCL. Studies have suggested that tumors with PL morphology represent a group of neoplasms with clinopathologic characteristics corresponding to different entities including extramedullary plasmablastic tumors associated with plasma cell myeloma (PCM. The goal of the current study was to evaluate the genetic similarities and differences among PL, DLBCL (AIDS-related and non AIDS-related and PCM using array-based comparative genomic hybridization. Results Examination of genomic data in PL revealed that the most frequent segmental gain (> 40% include: 1p36.11-1p36.33, 1p34.1-1p36.13, 1q21.1-1q23.1, 7q11.2-7q11.23, 11q12-11q13.2 and 22q12.2-22q13.3. This correlated with segmental gains occurring in high frequency in DLBCL (AIDS-related and non AIDS-related cases. There were some segmental gains and some segmental loss that occurred in PL but not in the other types of lymphoma suggesting that these foci may contain genes responsible for the differentiation of this lymphoma. Additionally, some segmental gains and some segmental loss occurred only in PL and AIDS associated DLBCL suggesting that these foci may be associated with HIV infection. Furthermore, some segmental gains and some segmental loss occurred only in PL and PCM suggesting that these lesions may be related to plasmacytic differentiation. Conclusion To the best of our knowledge, the current study represents the first genomic exploration of PL. The genomic aberration pattern of PL appears to be more similar to that of DLBCL (AIDS-related or non AIDS-related than to PCM. Our findings suggest that PL may remain best classified as a subtype of DLBCL at least at the genome level.

  13. Whole genome amplification for CGH analysis: Linker-adapter PCR as the method of choice for difficult and limited samples. (United States)

    Pirker, Christine; Raidl, Maria; Steiner, Elisabeth; Elbling, Leonilla; Holzmann, Klaus; Spiegl-Kreinecker, Sabine; Aubele, Michaela; Grasl-Kraupp, Bettina; Marosi, Christine; Micksche, Michael; Berger, Walter


    Comparative genomic hybridization (CGH) is a powerful method to investigate chromosomal imbalances in tumor cells. However, DNA quantity and quality can be limiting factors for successful CGH analysis. The aim of this study was to investigate the applicability of degenerate oligonucleotide-primed PCR (DOP-PCR) and a recently developed linker-adapter-mediated PCR (LA-PCR) for whole genome amplification for use in CGH, especially for difficult source material. We comparatively analyzed DNA of variable quality derived from different cell/tissue types. Additionally, dilution experiments down to the DNA content of a single cell were performed. FISH and/or classical cytogenetic analyses were used as controls. In the case of high quality DNA samples, both methods were equally suitable for CGH. When analyzing very small amounts of these DNA samples (equivalent to one or a few human diploid cells), DOP-PCR-CGH, but not LA-PCR-CGH, frequently produced false-positive signals (e.g., gains in 1p and 16p, and losses in chromosome 4q). In case of formalin-fixed paraffin-embedded tissues, success rates by LA-PCR-CGH were significantly higher as compared to DOP-PCR-CGH. DNA of minor quality frequently could be analyzed correctly by LA-PCR-CGH, but was prone to give false-positive and/or false-negative results by DOP-PCR-CGH. LA-PCR is superior to DOP-PCR for amplification of DNA for CGH analysis, especially in the case of very limited or partly degraded source material. Copyright 2004 Wiley-Liss, Inc

  14. Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

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    Townsend Henrik J


    Full Text Available Abstract High-density oligonucleotide (oligo arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L. Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available and may be used to facilitate transcriptomic analyses of a wide range of plant and animal

  15. ADaCGH: A parallelized web-based application and R package for the analysis of aCGH data.

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    Ramón Díaz-Uriarte

    Full Text Available BACKGROUND: Copy number alterations (CNAs in genomic DNA have been associated with complex human diseases, including cancer. One of the most common techniques to detect CNAs is array-based comparative genomic hybridization (aCGH. The availability of aCGH platforms and the need for identification of CNAs has resulted in a wealth of methodological studies. METHODOLOGY/PRINCIPAL FINDINGS: ADaCGH is an R package and a web-based application for the analysis of aCGH data. It implements eight methods for detection of CNAs, gains and losses of genomic DNA, including all of the best performing ones from two recent reviews (CBS, GLAD, CGHseg, HMM. For improved speed, we use parallel computing (via MPI. Additional information (GO terms, PubMed citations, KEGG and Reactome pathways is available for individual genes, and for sets of genes with altered copy numbers. CONCLUSIONS/SIGNIFICANCE: ADACGH represents a qualitative increase in the standards of these types of applications: a all of the best performing algorithms are included, not just one or two; b we do not limit ourselves to providing a thin layer of CGI on top of existing BioConductor packages, but instead carefully use parallelization, examining different schemes, and are able to achieve significant decreases in user waiting time (factors up to 45x; c we have added functionality not currently available in some methods, to adapt to recent recommendations (e.g., merging of segmentation results in wavelet-based and CGHseg algorithms; d we incorporate redundancy, fault-tolerance and checkpointing, which are unique among web-based, parallelized applications; e all of the code is available under open source licenses, allowing to build upon, copy, and adapt our code for other software projects.

  16. High resolution microarray comparative genomic hybridisation analysis using spotted oligonucleotides.

    NARCIS (Netherlands)

    Carvalho, B; Ouwerkerk, E; Meijer, G.A.; Ylstra, B.


    BACKGROUND: Currently, comparative genomic hybridisation array (array CGH) is the method of choice for studying genome wide DNA copy number changes. To date, either amplified representations of bacterial artificial chromosomes (BACs)/phage artificial chromosomes (PACs) or cDNAs have been spotted as

  17. Experimental analysis of oligonucleotide microarray design criteria to detect deletions by comparative genomic hybridization

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    Moerman Donald G


    Full Text Available Abstract Background Microarray comparative genomic hybridization (CGH is currently one of the most powerful techniques to measure DNA copy number in large genomes. In humans, microarray CGH is widely used to assess copy number variants in healthy individuals and copy number aberrations associated with various diseases, syndromes and disease susceptibility. In model organisms such as Caenorhabditis elegans (C. elegans the technique has been applied to detect mutations, primarily deletions, in strains of interest. Although various constraints on oligonucleotide properties have been suggested to minimize non-specific hybridization and improve the data quality, there have been few experimental validations for CGH experiments. For genomic regions where strict design filters would limit the coverage it would also be useful to quantify the expected loss in data quality associated with relaxed design criteria. Results We have quantified the effects of filtering various oligonucleotide properties by measuring the resolving power for detecting deletions in the human and C. elegans genomes using NimbleGen microarrays. Approximately twice as many oligonucleotides are typically required to be affected by a deletion in human DNA samples in order to achieve the same statistical confidence as one would observe for a deletion in C. elegans. Surprisingly, the ability to detect deletions strongly depends on the oligonucleotide 15-mer count, which is defined as the sum of the genomic frequency of all the constituent 15-mers within the oligonucleotide. A similarity level above 80% to non-target sequences over the length of the probe produces significant cross-hybridization. We recommend the use of a fairly large melting temperature window of up to 10°C, the elimination of repeat sequences, the elimination of homopolymers longer than 5 nucleotides, and a threshold of -1 kcal/mol on the oligonucleotide self-folding energy. We observed very little difference in data

  18. Molecular Mechanisms of Antisense Oligonucleotides. (United States)

    Crooke, Stanley T


    In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs.

  19. Detection of genomic copy number changes in patients with idiopathic mental retardation by high-resolution X-array-CGH: important role for increased gene dosage of XLMR genes. (United States)

    Froyen, Guy; Van Esch, Hilde; Bauters, Marijke; Hollanders, Karen; Frints, Suzanna G M; Vermeesch, Joris R; Devriendt, Koen; Fryns, Jean-Pierre; Marynen, Peter


    A tiling X-chromosome-specific genomic array with a theoretical resolution of 80 kb was developed to screen patients with idiopathic mental retardation (MR) for submicroscopic copy number differences. Four patients with aberrations previously detected at lower resolution were first analyzed. This facilitated delineation of the location and extent of the aberration at high resolution and subsequently, more precise genotype-phenotype analyses. A cohort of 108 patients was screened, 57 of which were suspected of X-linked mental retardation (XLMR), 26 were probands of brother pairs, and 25 were sporadic cases. A total of 15 copy number changes in 14 patients (13%) were detected, which included two deletions and 13 duplications ranging from 0.1 to 2.7 Mb. The aberrations are associated with the phenotype in five patients (4.6%), based on the following criteria: de novo aberration; involvement of a known or candidate X-linked nonsyndromic(syndromic) MR (MRX(S)) gene; segregation with the disease in the family; absence in control individuals; and skewed X-inactivation in carrier females. These include deletions that contain the MRX(S) genes CDKL5, OPHN1, and CASK, and duplications harboring CDKL5, NXF5, MECP2, and GDI1. In addition, seven imbalances were apparent novel polymorphic regions because they do not fulfill the proposed criteria. Taken together, our data strongly suggest that not only deletions but also duplications on the X chromosome contribute to the phenotype more often than expected, supporting the increased gene dosage mechanism for deregulation of normal cognitive development.

  20. Application of the cghRA framework to the genomic characterization of Diffuse Large B-Cell Lymphoma. (United States)

    Mareschal, Sylvain; Ruminy, Philippe; Alcantara, Marion; Villenet, Céline; Figeac, Martin; Dubois, Sydney; Bertrand, Philippe; Bouzelfen, Abdelilah; Viailly, Pierre-Julien; Penther, Dominique; Tilly, Hervé; Bastard, Christian; Jardin, Fabrice


    Although sequencing-based technologies are becoming the new reference in genome analysis, comparative genomic hybridization arrays (aCGH) still constitute a simple and reliable approach for copy number analysis. The most powerful algorithms to analyse such data have been freely provided by the scientific community for many years, but combining them is a complex scripting task. The cghRA framework combines a user-friendly graphical interface and a powerful objectoriented command-line interface to handle a full aCGH analysis, as is illustrated in an original series of 107 Diffuse Large B-Cell Lymphomas. New algorithms for copy-number calling, polymorphism detection and minimal common region (MCR) prioritization were also developed and validated. While their performances will only be demonstrated with aCGH, these algorithms could actually prove useful to any copy-number analysis, whatever the technique used. R package and source for Linux, MS Windows and MacOS are freely available at . Supplementary data are available at Bioinformatics online.

  1. Molecular testing for familial hypercholesterolaemia-associated mutations in a UK-based cohort: development of an NGS-based method and comparison with multiplex polymerase chain reaction and oligonucleotide arrays. (United States)

    Reiman, Anne; Pandey, Sarojini; Lloyd, Kate L; Dyer, Nigel; Khan, Mike; Crockard, Martin; Latten, Mark J; Watson, Tracey L; Cree, Ian A; Grammatopoulos, Dimitris K


    Background Detection of disease-associated mutations in patients with familial hypercholesterolaemia is crucial for early interventions to reduce risk of cardiovascular disease. Screening for these mutations represents a methodological challenge since more than 1200 different causal mutations in the low-density lipoprotein receptor has been identified. A number of methodological approaches have been developed for screening by clinical diagnostic laboratories. Methods Using primers targeting, the low-density lipoprotein receptor, apolipoprotein B, and proprotein convertase subtilisin/kexin type 9, we developed a novel Ion Torrent-based targeted re-sequencing method. We validated this in a West Midlands-UK small cohort of 58 patients screened in parallel with other mutation-targeting methods, such as multiplex polymerase chain reaction (Elucigene FH20), oligonucleotide arrays (Randox familial hypercholesterolaemia array) or the Illumina next-generation sequencing platform. Results In this small cohort, the next-generation sequencing method achieved excellent analytical performance characteristics and showed 100% and 89% concordance with the Randox array and the Elucigene FH20 assay. Investigation of the discrepant results identified two cases of mutation misclassification of the Elucigene FH20 multiplex polymerase chain reaction assay. A number of novel mutations not previously reported were also identified by the next-generation sequencing method. Conclusions Ion Torrent-based next-generation sequencing can deliver a suitable alternative for the molecular investigation of familial hypercholesterolaemia patients, especially when comprehensive mutation screening for rare or unknown mutations is required.

  2. límites organizacionales del CGH

    Directory of Open Access Journals (Sweden)

    Carlos Chávez Becker


    Full Text Available El presente trabajo se centra en el análisis de los mecanismos que el Consejo General de Huelga empleó para tomar decisiones, su esquema de representación y su forma de operación política con el fin de establecer algunas relaciones significativas entre tales elementos analíticos y los resultados obtenidos por la organización al término del conflicto en febrero de 2000. La principal intención es evaluar al CGH como la organización de los estudiantes en términos de su actuar político a la luz de los costos y beneficios que representó su actividad.

  3. CGH, cDNA and Tissue Microarray Analyses Implicate FGFR2 Amplification in a Small Subset of Breast Tumors

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    Mervi Heiskanen


    Full Text Available Multiple regions of the genome are often amplified during breast cancer development and progression, as evidenced in a number of published studies by comparative genomic hybridization (CGH. However, only relatively few target genes for such amplifications have been identified. Here, we indicate how small‐scale commercially available cDNA and CGH microarray formats combined with the tissue microarray technology enable rapid identification of putative amplification target genes as well as analysis of their clinical significance. According to CGH, the SUM‐52 breast cancer cell line harbors several high‐level DNA amplification sites, including the 10q26 chromosomal region where the fibroblast growth factor receptor 2 (FGFR2 gene has been localized. High level amplification of FGFR2 in SUM‐52 was identified using CGH analysis on a microarray of BAC clones. A cDNA microarray survey of 588 genes showed >40‐fold overexpression of FGFR2. Finally, a tissue microarray based FISH analysis of 750 uncultured primary breast cancers demonstrated in vivo amplification of the FGFR2 gene in about 1% of the tumors. In conclusion, three consecutive microarray (CGH, cDNA and tissue experiments revealed high‐level amplification and overexpression of the FGFR2 in a breast cancer cell line, but only a low frequency of involvement in primary breast tumors. Applied to a genomic scale with larger arrays, this strategy should facilitate identification of the most important target genes for cytogenetic rearrangements, such as DNA amplification sites detected by conventional CGH. Figures on‐4/heiskanen.htm

  4. Evaluation of somatic genomic imbalances in thyroid carcinomas of follicular origin by CGH-based approaches. (United States)

    Baldan, Federica; Mio, Catia; Allegri, Lorenzo; Passon, Nadia; Lepore, Saverio M; Russo, Diego; Damante, Giuseppe


    Application of distinct technologies of cancer genome analysis has provided important information for the molecular characterization of several human neoplasia, including follicular cell-derived thyroid carcinoma. Among them, comparative genomic hybridization (CGH)-based procedures have been extensively applied to evaluate genomic imbalances present in these tumours, obtaining data leading to an increase in the understanding of their complexity and diversity. In this review, after a brief overview of the most commonly used CGH-based technichs, we will describe the major results deriving from the most influential studies in the literature which used this approach to investigate the genomic aberrations of thyroid cancer cells. In most studies a small number of patients have been analyzed. Deletions and duplications at different chromosomal regions were detected in all investigated cohorts. A higher number of genomic imbalances has been detected in anaplastic or poorly differentiated thyroid carcinomas compared to well differentiated ones. Limitations in the interpretation of the results, as well the potential impact in the clinical practice are discussed. Though a quite heterogeneous picture arises from results so far available, CGH array, combined with other methodologies as well as an accurate clinical management, may offer novel opportunities for a better stratification of thyroid cancer patients.

  5. Approximate Dynamic Programming for Fast Denoising of aCGH Data

    CERN Document Server

    Miller, Gary L; Schwartz, Russell; Tsourakakis, Charalampos E


    DNA sequence copy number is the number of copies of DNA at a region of a genome. Identifying genomic regions whose DNA copy number deviates from the normal is a crucial task in understanding cancer evolution. Array-based comparative genomic hybridization (aCGH) is a high-throughput technique for identifying DNA gain or loss. Due to the high level of noise in microarray data, however, interpretation of aCGH output is a difficult and error-prone task. In this paper, we adopt a recent formulation of the denoising aCGH data problem as a regularized least squares problem and propose an approximation algorithm within $\\epsilon$ additive error, where \\epsilon is an arbitrarily small positive constant. Specifically, we show that for n probes, we can approximate the optimal value of our function within additive \\epsilon with an algorithm that runs in $\\tilde{O}(n^{1.5} \\log{(\\frac{U}{\\epsilon}))}$ time, where U is the maximum value over the regularization term and the probes. The basis of our algorithm is the definiti...

  6. Template-Directed Ligation of Peptides to Oligonucleotides (United States)

    Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.


    Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

  7. Programmable CGH on photochromic material using DMD (United States)

    Alata, Romain; Pariani, Giorgio; Zamkotsian, Frederic; Lanzoni, Patrick; Bianco, Andrea; Bertarelli, Chiara


    Computer Generated Holograms (CGHs) are useful for wavefront shaping and complex optics testing, including aspherical and free-form optics. Today, CGHs are recorded directly with a laser or intermediates masks but allows only recording binary CGHs; binary CGHs are efficient but can reconstruct only pixilated images. We propose to use a Digital Micro-mirror Device (DMD) for writing binary CGHs as well as grayscale CGHs, able to reconstruct fulfilled images. DMD is actually studied at LAM, for generating programmable slit masks in multi-object spectrographs. It is composed of 2048x1080 individually controllable micro-mirrors, with a pitch of 13.68 μm. This is a real-time reconfigurable mask, perfect for recording CGHs. A first setup has been developed for hologram recording, where the DMD is enlightened with a collimated beam and illuminates a photosensible plate through an Offner relay, with a magnification of 1:1. Our set up resolution is 2-3 μm, leading to a CGH resolution equal to the DMD micro mirror size. In order to write and erase CGHs during test procedure or on request, we use a photochromic plate called PUR-GD71-50-ST developed at Politecnico di Milano. It is opaque at rest, and becomes transparent when it is illuminated with visible light, between 500 and 700 nm; then it can be erased by a UV flash. We choose to code the CGHs in equally spaced levels, so called stepped CGH. We recorded up to 1000x1000 pixels CGHs with a contrast greater than 50, knowing that the material is able to reach an ultimate contrast of 1000. A second bench has also been developed, dedicated to the reconstruction of the recorded images with a 632.8nm He-Ne laser beam. Very faithful reconstructions have been obtained. Thanks to our recording and reconstruction set-ups, we have been able to successfully record binary and stepped CGHs, and reconstruct them with a high fidelity, revealing the potential of this method for generating programmable/rewritable stepped CGHs on

  8. 2q23.1 microdeletion identified by array comparative genomic hybridisation: an emerging phenotype with Angelman-like features? (United States)

    Jaillard, S; Dubourg, C; Gérard-Blanluet, M; Delahaye, A; Pasquier, L; Dupont, C; Henry, C; Tabet, A-C; Lucas, J; Aboura, A; David, V; Benzacken, B; Odent, S; Pipiras, E


    Genome-wide screening of patients with mental retardation using array comparative genomic hybridisation (CGH) has identified several novel imbalances. With this genotype-first approach, the 2q22.3q23.3 deletion was recently described as a novel microdeletion syndrome. The authors report two unrelated patients with a de novo interstitial deletion mapping in this genomic region and presenting similar "pseudo-Angelman" phenotypes, including severe psychomotor retardation, speech impairment, epilepsy, microcephaly, ataxia, and behavioural disabilities. The microdeletions were identified by array CGH using oligonucleotide and bacterial artificial chromosome (BAC) arrays, and further confirmed by fluorescence in situ hybridisation (FISH) and semi-quantitative polymerase chain reaction (PCR). The boundaries and sizes of the deletions in the two patients were different but an overlapping region of about 250 kb was defined, which mapped to 2q23.1 and included two genes: MBD5 and EPC2. The SIP1 gene associated with the Mowat-Wilson syndrome was not included in the deleted genomic region. Haploinsufficiency of one of the deleted genes (MBD5 or EPC2) could be responsible for the common clinical features observed in the 2q23.1 microdeletion syndrome, and this hypothesis needs further investigation.


    Institute of Scientific and Technical Information of China (English)


    For investigating the possibility of applying degenerate oligonucleotide primer PCR (DOP-PCR) and comparative genomic hybridization (CGH) technique to analyses of genomic genetics in a single cell, the whole genomic DNA of a single cell with XX, XY, XO, XXY, +13 or +21 was amplified by DOP-PCR. Single cell DOP-PCR CGHs with conventional and modified control references, the genomic DNA and a single cell DOP-PCR product from normal male, were carried out respectively. The results showed that the average profile of the fluorescence intensity ratio in CGH with the genomic DNA as reference fluctuates much and that the standard deviation in about 30% haploid is beyond the normal limits. False positive hyper-representation was found to exist in X chromosome while trisomy 13 and 21 were not detected. However, the distributions of the mean and the standard deviation of the ratio in the CGH with DOP-PCR product as reference were quite acceptable. The copy number changes of chromosome X,Y,13 and 21 were revealed. Those results suggested that there is unrandom unequal amplification in a single cell DOP-PCR. Using a single DOP-PCR product as reference can decrease its influence on CGH. Single cell DOP-PCR-CGH and its application in the genetic analyses of preimplantation embryo or fetal cell in maternal blood may be possible.

  10. DNA copy number aberrations in breast cancer by array comparative genomic hybridization

    DEFF Research Database (Denmark)

    Li, J.; Wang, K.; Li, S.;


    Array comparative genomic hybridization (CGH) has been popularly used for analyzing DNA copy number variations in diseases like cancer. In this study, we investigated 82 sporadic samples from 49 breast cancer patients using 1-Mb resolution bacterial artificial chromosome CGH arrays. A number of h...

  11. VizieR Online Data Catalog: Spectroscopy of main-belt Ch/Cgh-type asteroids (Vernazza+, 2016) (United States)

    Vernazza, P.; Marsset, M.; Beck, P.; Binzel, R. P.; Birlan, M.; Cloutis, E. A.; DeMeo, F. E.; Dumas, C.; Hiroi, T.


    We conducted an extensive spectroscopic survey in the near-infrared range of 70 main-belt Ch/Cgh-type asteroids and 4 Ch/Cgh-type families and combined these measurements with available visible wavelength spectra. New data presented here are near-infrared asteroid spectral measurements for Ch- and Cgh-type asteroids from 0.7-2.5μm obtained using SpeX, the low- to medium-resolution near-IR spectrograph and imager on the 3m NASA InfraRed Telescope Facility (IRTF) located on Mauna Kea, HI. Observing runs were conducted remotely primarily from the Observatory of Paris-Meudon, France between 2010 April and 2012 January. The spectrograph SpeX, combined with a 0.8*15arcsec slit, was used in the low-resolution prism mode for acquisition of the spectra in the 0.7-2.5μm wavelength range. In order to monitor the high luminosity and variability of the sky in the near-IR, the telescope was moved along the slit during the acquisition of the data so as to obtain a sequence of spectra located at two different positions (A and B) on the array. In addition, we complemented our data set with additional near-infrared spectra retrieved from the Small Main-Belt Asteroid Spectroscopic Survey (SMASS) database ( Combining these near-infrared measurements with available visible wavelength spectra (Bus, 1999PhDT........50B; Lazzaro et al., 2004Icar..172..179L) allows for the first time an extensive visible and near-infrared (VNIR) spectral database of main-belt Ch and Cgh types with D>45km (78% or 49/63 of all Ch and Cgh types listed in SMASS; see Table1). (1 data file).

  12. Improved analysis of bacterial CGH data beyond the log-ratio paradigm

    Directory of Open Access Journals (Sweden)

    Aakra Ågot


    Full Text Available Abstract Background Existing methods for analyzing bacterial CGH data from two-color arrays are based on log-ratios only, a paradigm inherited from expression studies. We propose an alternative approach, where microarray signals are used in a different way and sequence identity is predicted using a supervised learning approach. Results A data set containing 32 hybridizations of sequenced versus sequenced genomes have been used to test and compare methods. A ROC-analysis has been performed to illustrate the ability to rank probes with respect to Present/Absent calls. Classification into Present and Absent is compared with that of a gaussian mixture model. Conclusion The results indicate our proposed method is an improvement of existing methods with respect to ranking and classification of probes, especially for multi-genome arrays.

  13. Partial biochemical and biological characterization of purified chicken growth hormone (cGH). Isolation of cGH charge variants and evidence that cGH is phosphorylated. (United States)

    Arámburo, C; Carranza, M; Sanchez, R; Perera, G


    Chicken growth hormone (cGH) was purified from frozen pituitary glands obtained from recently sacrificed broilers. Glands were homogenized in a protease inhibitor solution (0.5 mM PMSF, 50 KIU/ml aprotinin, pH 7.2); extract was taken to pH 9.0 with calcium hydroxide and the supernatant was differentially precipitated with 20% (fraction A) and 50% (fraction B) ammonium sulfate. cGH (fraction B-DE-1) was obtained in pure form from fraction B after DEAE-cellulose chromatography at pH 8.6, with a yield of 2.9 mg/g tissue. Three charge variants of cGH (Rf = 0.23, 0.30, and 0.35) could be isolated by electroelution after semipreparative nondenaturing polyacrylamide gel electrophoresis of fraction B-DE-1. These charge variants showed the same apparent molecular weight (26,300 Da) by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. Isoelectric focusing of fraction B-DE-1 revealed two major components (pI = 7.2 and 7.4) and four minor bands (pI = 6.2, 6.7, 7.1, and 7.5). It was found that fraction B-DE-1 contained a significant amount of esterified phosphate (1 nmol PO4/3.5 nmol protein) similar to that reported previously for ovine GH. The functional integrity of the cGH obtained here was characterized by two heterologous and one homologous bioassays. High activity was shown by fraction B-DE-1 in the tibia assay (1.76 UI/mg) and in the liver ornithine decarboxylase assay (sixfold over control), both made in hypophysectomized rats; and it also stimulated lipolysis (138 and 215% at 10 and 100 ng/ml, respectively) on chicken abdominal adipose tissue explants.

  14. The delivery of therapeutic oligonucleotides. (United States)

    Juliano, Rudolph L


    The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology. © The Author 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Development of a human mitochondrial oligonucleotide microarray (h-MitoArray and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency

    Directory of Open Access Journals (Sweden)

    Hansíková Hana


    Full Text Available Abstract Background To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F1Fo ATP synthase deficiency consisting of 2 patients with mt9205ΔTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes. Results Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines – M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known ATP6/ATP8 defect in patients from the M group and indicated several candidate

  16. Antisense oligonucleotides in cancer. (United States)

    Castanotto, Daniela; Stein, Cy A


    Over the past several dozen years, regardless of the substantial effort directed toward developing rational oligonucleotide strategies to silence gene expression, antisense oligonucleotide-based cancer therapy has not been successful. This review focuses on the most likely reasons for this lack of success, and on the barriers that still need to be overcome to make a clinical cancer treatment reality out of the promise of antisense therapy. Considerable progress has been made in the design and delivery of nucleic acid fragments. Chemical modifications have considerably improved oligonucleotide absorption, distribution and metabolism while at the same time reducing toxicity. Nevertheless, the delivery and the cellular uptake of these molecules are still not adequate to provide the desired therapeutic outcome. Recent therapeutic interventional phase III trials of antisense oligodeoxyribonucleotides for a cancer indication will be discussed, in addition to those studies that markedly improve the scientific understanding of the properties of these molecules. We still do not have a marketed antisense oligonucleotide for a cancer indication. This is because critical aspects of the cellular, tumor pharmacology and delivery properties of these agents are still not well understood.

  17. Detection of copy number variation from array intensity and sequencing read depth using a stepwise Bayesian model

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    Gerstein Mark B


    Full Text Available Abstract Background Copy number variants (CNVs have been demonstrated to occur at a high frequency and are now widely believed to make a significant contribution to the phenotypic variation in human populations. Array-based comparative genomic hybridization (array-CGH and newly developed read-depth approach through ultrahigh throughput genomic sequencing both provide rapid, robust, and comprehensive methods to identify CNVs on a whole-genome scale. Results We developed a Bayesian statistical analysis algorithm for the detection of CNVs from both types of genomic data. The algorithm can analyze such data obtained from PCR-based bacterial artificial chromosome arrays, high-density oligonucleotide arrays, and more recently developed high-throughput DNA sequencing. Treating parameters--e.g., the number of CNVs, the position of each CNV, and the data noise level--that define the underlying data generating process as random variables, our approach derives the posterior distribution of the genomic CNV structure given the observed data. Sampling from the posterior distribution using a Markov chain Monte Carlo method, we get not only best estimates for these unknown parameters but also Bayesian credible intervals for the estimates. We illustrate the characteristics of our algorithm by applying it to both synthetic and experimental data sets in comparison to other segmentation algorithms. Conclusions In particular, the synthetic data comparison shows that our method is more sensitive than other approaches at low false positive rates. Furthermore, given its Bayesian origin, our method can also be seen as a technique to refine CNVs identified by fast point-estimate methods and also as a framework to integrate array-CGH and sequencing data with other CNV-related biological knowledge, all through informative priors.

  18. Asterias: A Parallelized Web-based Suite for the Analysis of Expression and aCGH Data

    Directory of Open Access Journals (Sweden)

    Ramón Díaz-Uriarte


    Full Text Available The analysis of expression and CGH arrays plays a central role in the study of complex diseases, especially cancer, including finding markers for early diagnosis and prognosis, choosing an optimal therapy, or increasing our understanding of cancer development and metastasis. Asterias ( is an integrated collection of freely-accessible web tools for the analysis of gene expression and aCGH data. Most of the tools use parallel computing (via MPI and run on a server with 60 CPUs for computation; compared to a desktop or server-based but not parallelized application, parallelization provides speed ups of factors up to 50. Most of our applications allow the user to obtain additional information for user-selected genes (chromosomal location, PubMed ids, Gene Ontology terms, etc. by using clickable links in tables and/or fi gures. Our tools include: normalization of expression and aCGH data (DNMAD; converting between different types of gene/clone and protein identifi ers (IDconverter/IDClight; fi ltering and imputation (preP; finding differentially expressed genes related to patient class and survival data (Pomelo II; searching for models of class prediction (Tnasas; using random forests to search for minimal models for class prediction or for large subsets of genes with predictive capacity (GeneSrF; searching for molecular signatures and predictive genes with survival data (SignS; detecting regions of genomic DNA gain or loss (ADaCGH. The capability to send results between different applications, access to additional functional information, and parallelized computation make our suite unique and exploit features only available to web-based applications.

  19. A web server for mining Comparative Genomic Hybridization (CGH) data (United States)

    Liu, Jun; Ranka, Sanjay; Kahveci, Tamer


    Advances in cytogenetics and molecular biology has established that chromosomal alterations are critical in the pathogenesis of human cancer. Recurrent chromosomal alterations provide cytological and molecular markers for the diagnosis and prognosis of disease. They also facilitate the identification of genes that are important in carcinogenesis, which in the future may help in the development of targeted therapy. A large amount of publicly available cancer genetic data is now available and it is growing. There is a need for public domain tools that allow users to analyze their data and visualize the results. This chapter describes a web based software tool that will allow researchers to analyze and visualize Comparative Genomic Hybridization (CGH) datasets. It employs novel data mining methodologies for clustering and classification of CGH datasets as well as algorithms for identifying important markers (small set of genomic intervals with aberrations) that are potentially cancer signatures. The developed software will help in understanding the relationships between genomic aberrations and cancer types.

  20. Application of Array-Based Comparative Genomic Hybridization to Pediatric Neurologic Diseases



    Purpose Array comparative genomic hybridization (array-CGH) is a technique used to analyze quantitative increase or decrease of chromosomes by competitive DNA hybridization of patients and controls. This study aimed to evaluate the benefits and yield of array-CGH in comparison with conventional karyotyping in pediatric neurology patients. Materials and Methods We included 87 patients from the pediatric neurology clinic with at least one of the following features: developmental delay, mental r...

  1. Exclusion of APC and VHL gene deletions by array-based comparative hybridization in two patients with microscopically visible chromosomal aberrations. (United States)

    Wallerstein, Robert J; Brooks, Susan Sklower; Streck, Deanna L; Kurvathi, Rohini; Toruner, Gokce A


    Karyotyping is a major component of the genetic work-up of patients with dysmorphism. Cytogenetic aberrations close to a known tumor suppressor gene raise important clinical issues because deletion of that tumor suppressor gene can cause genetic predisposition to cancer. We present two cancer-free dysmorphic patients with karyotypes of 46,XX,del(5)(q15q22.3) and 46,XX,del(3)(p25.2~pter). These deletions are close to the APC and VHL genes that confer susceptibility to familial Adenomatous polyposis (OMIM #17510) and von-Hippel-Lindau syndrome (OMIM #193300), respectively. The array-based comparative genomic hybridization (array-CGH) analysis using a custom Agilent 44K oligonucleotide array demonstrated an interstitial 20.7-megabase (Mb) deletion on 5q (chr5: 89,725,638-110,491,345) and a terminal 9.45-Mb deletion on 3p (chr3:pter-9,450,984). According to the March 2006 human reference sequence, the APC gene is located at chr5: 112,101,483-112,209,835 and the VHL gene is located at chr3: 10,158,319-10,168,746. These results indicate that the APC gene is 2,300 kilobases (kb) and the VHL gene is 700 kb away from deleted regions. Southern blot analysis for APC and VHL genes were negative, consistent with array-CGH findings. These results demonstrate the power of array-CCH to assess potential tumor suppressor gene involvement and cancer risk in patients with microscopically visible deletions in areas near tumor suppressors.

  2. An imputation approach for oligonucleotide microarrays.

    Directory of Open Access Journals (Sweden)

    Ming Li

    Full Text Available Oligonucleotide microarrays are commonly adopted for detecting and qualifying the abundance of molecules in biological samples. Analysis of microarray data starts with recording and interpreting hybridization signals from CEL images. However, many CEL images may be blemished by noises from various sources, observed as "bright spots", "dark clouds", and "shadowy circles", etc. It is crucial that these image defects are correctly identified and properly processed. Existing approaches mainly focus on detecting defect areas and removing affected intensities. In this article, we propose to use a mixed effect model for imputing the affected intensities. The proposed imputation procedure is a single-array-based approach which does not require any biological replicate or between-array normalization. We further examine its performance by using Affymetrix high-density SNP arrays. The results show that this imputation procedure significantly reduces genotyping error rates. We also discuss the necessary adjustments for its potential extension to other oligonucleotide microarrays, such as gene expression profiling. The R source code for the implementation of approach is freely available upon request.

  3. Particle-Based Microarrays of Oligonucleotides and Oligopeptides. (United States)

    Nesterov-Mueller, Alexander; Maerkle, Frieder; Hahn, Lothar; Foertsch, Tobias; Schillo, Sebastian; Bykovskaya, Valentina; Sedlmayr, Martyna; Weber, Laura K; Ridder, Barbara; Soehindrijo, Miriam; Muenster, Bastian; Striffler, Jakob; Bischoff, F Ralf; Breitling, Frank; Loeffler, Felix F


    In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches.

  4. Particle-Based Microarrays of Oligonucleotides and Oligopeptides

    Directory of Open Access Journals (Sweden)

    Alexander Nesterov-Mueller


    Full Text Available In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches.

  5. Optimized in situ construction of oligomers on an array surface (United States)

    Tolonen, Andrew C.; Albeanu, Dinu F.; Corbett, Julia F.; Handley, Heather; Henson, Charlotte; Malik, Pratap


    Oligonucleotide arrays are powerful tools to study changes in gene expression for whole genomes. These arrays can be synthesized by adapting photolithographic techniques used in microelectronics. Using this method, oligonucleotides are built base by base directly on the array surface by numerous cycles of photodeprotection and nucleotide addition. In this paper we examine strategies to reduce the number of synthesis cycles required to construct oligonucleotide arrays. By computer modeling oligonucleotide synthesis, we found that the number of required synthesis cycles could be significantly reduced by focusing upon how oligonucleotides are chosen from within genes and upon the order in which nucleotides are deposited on the array. The methods described here could provide a more efficient strategy to produce oligonucleotide arrays. PMID:12384609

  6. Rapid and accurate synthesis of TALE genes from synthetic oligonucleotides. (United States)

    Wang, Fenghua; Zhang, Hefei; Gao, Jingxia; Chen, Fengjiao; Chen, Sijie; Zhang, Cuizhen; Peng, Gang


    Custom synthesis of transcription activator-like effector (TALE) genes has relied upon plasmid libraries of pre-fabricated TALE-repeat monomers or oligomers. Here we describe a novel synthesis method that directly incorporates annealed synthetic oligonucleotides into the TALE-repeat units. Our approach utilizes iterative sets of oligonucleotides and a translational frame check strategy to ensure the high efficiency and accuracy of TALE-gene synthesis. TALE arrays of more than 20 repeats can be constructed, and the majority of the synthesized constructs have perfect sequences. In addition, this novel oligonucleotide-based method can readily accommodate design changes to the TALE repeats. We demonstrated an increased gene targeting efficiency against a genomic site containing a potentially methylated cytosine by incorporating non-conventional repeat variable di-residue (RVD) sequences.

  7. Radiolabeled oligonucleotides for antisense imaging (United States)

    Iyer, Arun K; He, Jiang


    Oligonucleotides radiolabeled with isotopes emitting γ-rays (for SPECT imaging) or positrons (for PET imaging) can be useful for targeting messenger RNA (mRNA) thereby serving as non-invasive imaging tools for detection of gene expression in vivo (antisense imaging). Radiolabeled oligonucleotides may also be used for monitoring their in vivo fate, thereby helping us better understand the barriers to its delivery for antisense targeting. These developments have led to a new area of molecular imaging and targeting, utilizing radiolabeled antisense oligonucleotides. However, the success of antisense imaging relies heavily on overcoming the barriers for its targeted delivery in vivo. Furthermore, the low ability of the radiolabeled antisense oligonucleotide to subsequently internalize into the cell and hybridize with its target mRNA poses additional challenges in realizing its potentials. This review covers the advances in the antisense imaging probe development for PET and SPECT, with an emphasis on radiolabeling strategies, stability, delivery and in vivo targeting. PMID:21822406

  8. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Hansen, Lykke Haastrup; Vester, Birte


    properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper......Although peptide-oligonucleotide conjugates (POCs) are well-known for nucleic acids delivery and therapy, reports on internal attachment of peptides to oligonucleotides are limited in number. To develop a convenient route for preparation of internally labeled POCs with improved biomedical......(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly...

  9. Identification and characterization of a de novo partial trisomy 10p by comparative genomic hybridization (CGH). (United States)

    Benzacken, B; Lapierre, J M; Siffroi, J P; Chalvon, A; Tachdjian, G


    We report the characterization of a de novo unbalanced chromosome rearrangement by comparative genomic hybridization (CGH) in a 15-day-old child with hypotonia and dysmorphia. We describe the combined use of CGH and fluorescence in situ hybridization (FISH) to identify the origin of the additional chromosomal material on the short arm of chromosome 6. Investigation with FISH revealed that the excess material was not derived from chromosome 6. Identification of unknown unbalanced aberrations that could not be identified by traditional cytogenetics procedures is possible by CGH analysis. Visual analysis of digital images from CGH-metaphase spreads revealed a predominantly green signal on the telomeric region of chromosome 10p. After quantitative digital ratio imaging of 10 CGH-metaphase spreads, a region of gain was found in the chromosome band 10p14-pter. The CGH finding was confirmed by FISH analysis, using a whole chromosome 10 paint probe. These results show the usefulness of CGH for a rapid characterization of de novo unbalanced translocation, unidentifiable by karyotype alone.

  10. Design and fabrication of CGH for 600mm diameter SiC primary mirror surface figure testing (United States)

    Pang, Zhihai; Ma, Zhen; Fan, Xuewu; Zou, Gangyi


    Computer-generated hologram (CGH) is an effective way to compensate wavefront aberration in null test of aspheric surfaces and freeform surfaces. Our strategies of CGH design for 600mm diameter SiC primary mirror surface figure testing are presented, and an experiment demonstrating the compensation test results of CGH is reported. We design a CGH including two sections on the same substrate in order to align the CGH to the incident wavefront: main section for compensating wavefront in null test, alignment section for adjusting the relative position between CGH and interferometer. In order to isolate different orders of diffraction, we used power carrier to make different orders of diffraction come to focus at different position along the axis to avoid ghost reflections. We measured the 600mm diameter SiC primary mirror using this CGH, and the surface test result is 0.033λ rms.

  11. Chromosomal aberrations in bladder cancer: fresh versus formalin fixed paraffin embedded tissue and targeted FISH versus wide microarray-based CGH analysis.

    Directory of Open Access Journals (Sweden)

    Elena Panzeri

    Full Text Available Bladder carcinogenesis is believed to follow two alternative pathways driven by the loss of chromosome 9 and the gain of chromosome 7, albeit other nonrandom copy number alterations (CNAs were identified. However, confirmation studies are needed since many aspects of this model remain unclear and considerable heterogeneity among cases has emerged. One of the purposes of this study was to evaluate the performance of a targeted test (UroVysion assay widely used for the detection of Transitional Cell Carcinoma (TCC of the bladder, in two different types of material derived from the same tumor. We compared the results of UroVysion test performed on Freshly Isolated interphasic Nuclei (FIN and on Formalin Fixed Paraffin Embedded (FFPE tissues from 22 TCCs and we didn't find substantial differences. A second goal was to assess the concordance between array-CGH profiles and the targeted chromosomal profiles of UroVysion assay on an additional set of 10 TCCs, in order to evaluate whether UroVysion is an adequately sensitive method for the identification of selected aneuploidies and nonrandom CNAs in TCCs. Our results confirmed the importance of global genomic screening methods, that is array based CGH, to comprehensively determine the genomic profiles of large series of TCCs tumors. However, this technique has yet some limitations, such as not being able to detect low level mosaicism, or not detecting any change in the number of copies for a kind of compensatory effect due to the presence of high cellular heterogeneity. Thus, it is still advisable to use complementary techniques such as array-CGH and FISH, as the former is able to detect alterations at the genome level not excluding any chromosome, but the latter is able to maintain the individual data at the level of single cells, even if it focuses on few genomic regions.

  12. Use of Virtual Medium in Designing of the CGH Wave Front Generator for Aspheric Testing

    Institute of Scientific and Technical Information of China (English)

    KANG Guo-guo; XIE Jing-hui; LIU Yi


    Design method and procedures of computer-generated hologram (CGH) used for aspheric test are introduced in detail.For CGH phase calculation,virtual medium which has zero refractive index at given wavelength is used to model ideal aspheric wavefront.Reflective Fresnel zones located in a ring area concentric to the CGH structure is designed to reduce or eliminate alignment errors.Substrate figure error,pattern distortion,etching and duty cycle variations that influence the reconstructed wavefront are quantitatively analyzed in theory and corresponding error equations are obtained to guide the tolerance distribution during CGH fabricating.A design example is given and the uncertainty of measurement achieves λ/20.

  13. Copy number analysis of the low-copy repeats at the primate NPHP1 locus by array comparative genomic hybridization. (United States)

    Yuan, Bo; Liu, Pengfei; Rogers, Jeffrey; Lupski, James R


    Array comparative genomic hybridization (aCGH) has been widely used to detect copy number variants (CNVs) in both research and clinical settings. A customizable aCGH platform may greatly facilitate copy number analyses in genomic regions with higher-order complexity, such as low-copy repeats (LCRs). Here we present the aCGH analyses focusing on the 45 kb LCRs [1] at the NPHP1 region with diverse copy numbers in humans. Also, the interspecies aCGH analysis comparing human and nonhuman primates revealed dynamic copy number transitions of the human 45 kb LCR orthologues during primate evolution and therefore shed light on the origin of complexity at this locus. The original aCGH data are available at GEO under GSE73962.

  14. Risk assessment models in genetics clinic for array comparative genomic hybridization: Clinical information can be used to predict the likelihood of an abnormal result in patients. (United States)

    Marano, Rachel M; Mercurio, Laura; Kanter, Rebecca; Doyle, Richard; Abuelo, Dianne; Morrow, Eric M; Shur, Natasha


    Array comparative genomic hybridization (aCGH) testing can diagnose chromosomal microdeletions and duplications too small to be detected by conventional cytogenetic techniques. We need to consider which patients are more likely to receive a diagnosis from aCGH testing versus patients that have lower likelihood and may benefit from broader genome wide scanning. We retrospectively reviewed charts of a population of 200 patients, 117 boys and 83 girls, who underwent aCGH testing in Genetics Clinic at Rhode Island hospital between 1 January/2008 and 31 December 2010. Data collected included sex, age at initial clinical presentation, aCGH result, history of seizures, autism, dysmorphic features, global developmental delay/intellectual disability, hypotonia and failure to thrive. aCGH analysis revealed abnormal results in 34 (17%) and variants of unknown significance in 24 (12%). Patients with three or more clinical diagnoses had a 25.0% incidence of abnormal aCGH findings, while patients with two or fewer clinical diagnoses had a 12.5% incidence of abnormal aCGH findings. Currently, we provide families with a range of 10-30% of a diagnosis with aCGH testing. With increased clinical complexity, patients have an increased probability of having an abnormal aCGH result. With this, we can provide individualized risk estimates for each patient.

  15. Thermodynamics of Oligonucleotide Duplex Melting (United States)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.


    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  16. Array comparative genomic hybridization-based characterization of genetic alterations in pulmonary neuroendocrine tumors


    Voortman, Johannes; Lee, Jih-Hsiang; Killian, Jonathan Keith; Suuriniemi, Miia; Wang, Yonghong; Lucchi, Marco; Smith, William I; Meltzer, Paul; Wang, Yisong; Giaccone, Giuseppe


    The goal of this study was to characterize and classify pulmonary neuroendocrine tumors based on array comparative genomic hybridization (aCGH). Using aCGH, we performed karyotype analysis of 33 small cell lung cancer (SCLC) tumors, 13 SCLC cell lines, 19 bronchial carcinoids, and 9 gastrointestinal carcinoids. In contrast to the relatively conserved karyotypes of carcinoid tumors, the karyotypes of SCLC tumors and cell lines were highly aberrant. High copy number (CN) gains were detected in ...

  17. A multi-sample based method for identifying common CNVs in normal human genomic structure using high-resolution aCGH data.

    Directory of Open Access Journals (Sweden)

    Chihyun Park

    Full Text Available BACKGROUND: It is difficult to identify copy number variations (CNV in normal human genomic data due to noise and non-linear relationships between different genomic regions and signal intensity. A high-resolution array comparative genomic hybridization (aCGH containing 42 million probes, which is very large compared to previous arrays, was recently published. Most existing CNV detection algorithms do not work well because of noise associated with the large amount of input data and because most of the current methods were not designed to analyze normal human samples. Normal human genome analysis often requires a joint approach across multiple samples. However, the majority of existing methods can only identify CNVs from a single sample. METHODOLOGY AND PRINCIPAL FINDINGS: We developed a multi-sample-based genomic variations detector (MGVD that uses segmentation to identify common breakpoints across multiple samples and a k-means-based clustering strategy. Unlike previous methods, MGVD simultaneously considers multiple samples with different genomic intensities and identifies CNVs and CNV zones (CNVZs; CNVZ is a more precise measure of the location of a genomic variant than the CNV region (CNVR. CONCLUSIONS AND SIGNIFICANCE: We designed a specialized algorithm to detect common CNVs from extremely high-resolution multi-sample aCGH data. MGVD showed high sensitivity and a low false discovery rate for a simulated data set, and outperformed most current methods when real, high-resolution HapMap datasets were analyzed. MGVD also had the fastest runtime compared to the other algorithms evaluated when actual, high-resolution aCGH data were analyzed. The CNVZs identified by MGVD can be used in association studies for revealing relationships between phenotypes and genomic aberrations. Our algorithm was developed with standard C++ and is available in Linux and MS Windows format in the STL library. It is freely available at:

  18. Improving signal intensities for genes with low-expression on oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Hu Limei


    Full Text Available Abstract Background DNA microarrays using long oligonucleotide probes are widely used to evaluate gene expression in biological samples. These oligonucleotides are pre-synthesized and sequence-optimized to represent specific genes with minimal cross-hybridization to homologous genes. Probe length and concentration are critical factors for signal sensitivity, particularly when genes with various expression levels are being tested. We evaluated the effects of oligonucleotide probe length and concentration on signal intensity measurements of the expression levels of genes in a target sample. Results Selected genes of various expression levels in a single cell line were hybridized to oligonucleotide arrays of four lengths and four concentrations of probes to determine how these critical parameters affected the intensity of the signal representing their expression. We found that oligonucleotides of longer length significantly increased the signals of genes with low-expression in the target. High-expressing gene signals were also boosted but to a lesser degree. Increasing the probe concentration, however, did not linearly increase the signal intensity for either low- or high-expressing genes. Conclusions We conclude that the longer the oligonuclotide probe the better the signal intensities of low expressing genes on oligonucleotide arrays.

  19. Distinct molecular signatures in pediatric infratentorial glioblastomas defined by aCGH. (United States)

    Sharma, S; Free, A; Mei, Y; Peiper, S C; Wang, Z; Cowell, J K


    Glioblastomas (GBM) are rare in children, but reportedly have more varied outcome which suggests differences in tumor etiology compared to typical GBM of adults. To investigate this we performed high resolution array comparative genomic hybridization (aCGH) analysis on three pediatric infratentorial GBM, ages 3.5, 7 and 14 years. Two of these tumors occurred in the brainstem and one in the spinal cord. While histologically typical, one brainstem tumor showed mainly pleomorphic astrocytic cells, whereas the other brainstem and spinal tumors showed a GFAP positive small cell component. Whole chromosomal gains (#1 and #2) and loss (#20) were seen only in the pleomorphic brainstem GBM, which also showed a high level of segmental genomic copy number changes. Segmental loss involving chromosome 8 was seen in all three tumors (Chr8;133039446-136869494, Chr8;pter-3581577, and Chr8;pter-30480019 respectively), whereas loss involving chromosome 16 was seen in only 2 cases with small cell components (Chr16;31827239-qter and Chr16;pter-29754532). Segmental gain of chromosome 7 was shared only between the 2 brainstem cases (Chr7;17187166-qter and Chr7;69824947-qter). Chromosome 17 showed segmental gain of 17q in the backdrop of loss of 17p only in case 1. Segmental gain of chromosome 1q was seen only in case 2. The spinal GBM showed a relatively stable karyotype with a unique loss of Chr19;32848902-qter. None of the frequent losses, gains and amplifications known to occur in adult GBM were identified, suggesting that pediatric infratentorial glioblastomas show a molecular karyotype that was more characteristic of pediatric embryonal tumors than adult GBM.

  20. Association of cGH EcoRV Gene with Production in Tolaki Chicken

    Directory of Open Access Journals (Sweden)

    Muhammad Amrullah Pagala


    Full Text Available cGH (Chicken Growth Hormone gene plays a crucial role in production responses of chicken. The objective of the research was to investigate the association of cGH gene with production in Tolaki chicken. Tolaki chicken is native chickens from Southeast Sulawesi Province of Indonesian. cGH gene was genotyped in 58 Tolaki chicken with PCR-RFLP. PCR was used to amplify genomic DNA for GH gene (399 bp. The amplicon was cutted by EcoRV and produced three genotypes: AA, AG, and GG and two alleles: A and G allele. The study showed the association of GH gene polymorphism with Production traits. GG genotype have better production (daily weight gain and feed conversion than AG genotype in Tolaki chicken, providing evidence that GH gene might be an important candidate gene for production traits.

  1. Matching of array CGH and gene expression microarray features for the purpose of integrative genomic analyses

    Directory of Open Access Journals (Sweden)

    van Wieringen Wessel N


    Full Text Available Abstract Background An increasing number of genomic studies interrogating more than one molecular level is published. Bioinformatics follows biological practice, and recent years have seen a surge in methodology for the integrative analysis of genomic data. Often such analyses require knowledge of which elements of one platform link to those of another. Although important, many integrative analyses do not or insufficiently detail the matching of the platforms. Results We describe, illustrate and discuss six matching procedures. They are implemented in the R-package sigaR (available from Bioconductor. The principles underlying the presented matching procedures are generic, and can be combined to form new matching approaches or be applied to the matching of other platforms. Illustration of the matching procedures on a variety of data sets reveals how the procedures differ in the use of the available data, and may even lead to different results for individual genes. Conclusions Matching of data from multiple genomics platforms is an important preprocessing step for many integrative bioinformatic analysis, for which we present six generic procedures, both old and new. They have been implemented in the R-package sigaR, available from Bioconductor.

  2. 22q13.3 Deletion Syndrome : Clinical and Molecular Analysis Using Array CGH

    NARCIS (Netherlands)

    Dhar, S. U.; del Gaudio, D.; German, J. R.; Peters, S. U.; Ou, Z.; Bader, P. I.; Berg, J. S.; Blazo, M.; Brown, C. W.; Graham, B. H.; Grebe, T. A.; Lalani, S.; Irons, M.; Sparagana, S.; Williams, M.; Phillips, J. A.; Beaudet, A. L.; Stankiewicz, P.; Patel, A.; Cheung, S. W.; Sahoo, T.


    The 22q13.3 deletion syndrome results from loss of terminal segments of varying sizes at 22qter. Few genotype phenotype correlations have been found but all patients have mental retardation and severe delay, or absence of, expressive speech. We carried out clinical and molecular characterization of

  3. Ultrathin oligonucleotide layers for fluorescence-based DNA sensors (United States)

    Furch, M.; Ueberfeld, J.; Hartmann, Andreas; Bock, Daniel; Seeger, Stefan


    Preliminary investigations into the design of an affinity sensor using evanescent wave technology concentrate upon the means of immobilization of the receptor molecules. In this work DNA served as the selective recognition element. The molecular principle of a sequence-selective biosensor for DNA is based on a sandwich-hybridization assay wherein the analyte, a single-stranded (ss)DNA, bound specifically to both an immobilized capture probe and a dye-labeled oligonucleotide in free solution. The efficiency of the capture array depends on the density of highly organized oligonucleotides on the waveguide surface and correlates therefore directly with the specificity and the sensitivity of the sensor. In the present approach using the Langmuir- Blodgett technique cinnamoylbutylether-cellulose monolayers were transferred onto optical fibers or planar waveguides. These films served as matrices for the immobilization of biotinylated oligonucleotides via streptavidin. For the first time streptavidin was immobilized by that manner. The specificity of the streptavidin layer or the following bounded nucleic acid molecules were controlled by an enzyme- linked immunosorbent assay (ELISA). Finally, this application has also shown to be suitable for the detection of Salmonella, which is an important pathogen associated with acute gastroenteritidis and food borne diseases.

  4. Somatic mosaicism detected by exon-targeted, high-resolution aCGH in 10,362 consecutive cases. (United States)

    Pham, Justin; Shaw, Chad; Pursley, Amber; Hixson, Patricia; Sampath, Srirangan; Roney, Erin; Gambin, Tomasz; Kang, Sung-Hae L; Bi, Weimin; Lalani, Seema; Bacino, Carlos; Lupski, James R; Stankiewicz, Pawel; Patel, Ankita; Cheung, Sau-Wai


    Somatic chromosomal mosaicism arising from post-zygotic errors is known to cause several well-defined genetic syndromes as well as contribute to phenotypic variation in diseases. However, somatic mosaicism is often under-diagnosed due to challenges in detection. We evaluated 10,362 patients with a custom-designed, exon-targeted whole-genome oligonucleotide array and detected somatic mosaicism in a total of 57 cases (0.55%). The mosaicism was characterized and confirmed by fluorescence in situ hybridization (FISH) and/or chromosome analysis. Different categories of abnormal cell lines were detected: (1) aneuploidy, including sex chromosome abnormalities and isochromosomes (22 cases), (2) ring or marker chromosomes (12 cases), (3) single deletion/duplication copy number variations (CNVs) (11 cases), (4) multiple deletion/duplication CNVs (5 cases), (5) exonic CNVs (4 cases), and (6) unbalanced translocations (3 cases). Levels of mosaicism calculated based on the array data were in good concordance with those observed by FISH (10-93%). Of the 14 cases evaluated concurrently by chromosome analysis, mosaicism was detected solely by the array in 4 cases (29%). In summary, our exon-targeted array further expands the diagnostic capability of high-resolution array comparative genomic hybridization in detecting mosaicism for cytogenetic abnormalities as well as small CNVs in disease-causing genes.

  5. Somatic mosaicism detected by exon-targeted, high-resolution aCGH in 10 362 consecutive cases (United States)

    Pham, Justin; Shaw, Chad; Pursley, Amber; Hixson, Patricia; Sampath, Srirangan; Roney, Erin; Gambin, Tomasz; Kang, Sung-Hae L; Bi, Weimin; Lalani, Seema; Bacino, Carlos; Lupski, James R; Stankiewicz, Pawel; Patel, Ankita; Cheung, Sau-Wai


    Somatic chromosomal mosaicism arising from post-zygotic errors is known to cause several well-defined genetic syndromes as well as contribute to phenotypic variation in diseases. However, somatic mosaicism is often under-diagnosed due to challenges in detection. We evaluated 10 362 patients with a custom-designed, exon-targeted whole-genome oligonucleotide array and detected somatic mosaicism in a total of 57 cases (0.55%). The mosaicism was characterized and confirmed by fluorescence in situ hybridization (FISH) and/or chromosome analysis. Different categories of abnormal cell lines were detected: (1) aneuploidy, including sex chromosome abnormalities and isochromosomes (22 cases), (2) ring or marker chromosomes (12 cases), (3) single deletion/duplication copy number variations (CNVs) (11 cases), (4) multiple deletion/duplication CNVs (5 cases), (5) exonic CNVs (4 cases), and (6) unbalanced translocations (3 cases). Levels of mosaicism calculated based on the array data were in good concordance with those observed by FISH (10–93%). Of the 14 cases evaluated concurrently by chromosome analysis, mosaicism was detected solely by the array in 4 cases (29%). In summary, our exon-targeted array further expands the diagnostic capability of high-resolution array comparative genomic hybridization in detecting mosaicism for cytogenetic abnormalities as well as small CNVs in disease-causing genes. PMID:24398791

  6. Sex determination of bovine preimplantation embryos by oligonucleotide microarray. (United States)

    Yang, Hua; Zhong, Fagang; Yang, Yonglin; Wang, Xinhua; Liu, Shouren; Zhu, Bin


    The aim has been to set up a rapid and accurate microarray assay using sandwich mode for sex determination of bovine preimplantation embryos. Twelve sequence-specific oligonucleotide capture probes used to discriminate 12 samples were spotted onto the aldehyde-modified glass slides by Arrayer. The 2 recognition probes used to identify coding regions of the sex-determining region of the Y chromosome gene (SRY) and β-casein (CSN2) reference gene were coupled with biotin. The assay was optimized by using genomic DNA extracted from blood samples of known sex individuals. Polymerase chain reaction (PCR) was used to amplify the fragments in the HMG box region of SRY gene and CSN2 gene with sequence-specific primers. The sex of samples was identified by detecting both the SRY and CSN2 genes simultaneously in 2 reaction cells of microarrays, with the male having SRY and CSN2 signals and the female only CSN2. The sex of 20 bovine preimplantation embryos was determined by oligonucleotide microarray. The protocol was run with a blind test that showed a 100% (82/82) specificity and accuracy in sexing of leukocytes. The bovine embryos were transferred into 20 bovine recipients, with a pregnant rate of 40% (8/20). Three calves were born at term, and 5 fetuses were miscarried. Their sexes were fully in accordance with the embryonic sex predetermination predicted by oligonucleotide microarray. This suggests that the oligonucleotide microarray method of SRY gene analysis can be used in early sex prediction of bovine embryos in breeding programs.

  7. Adaptive resolution simulation of oligonucleotides (United States)

    Netz, Paulo A.; Potestio, Raffaello; Kremer, Kurt


    Nucleic acids are characterized by a complex hierarchical structure and a variety of interaction mechanisms with other molecules. These features suggest the need of multiscale simulation methods in order to grasp the relevant physical properties of deoxyribonucleic acid (DNA) and RNA using in silico experiments. Here we report an implementation of a dual-resolution modeling of a DNA oligonucleotide in physiological conditions; in the presented setup only the nucleotide molecule and the solvent and ions in its proximity are described at the atomistic level; in contrast, the water molecules and ions far from the DNA are represented as computationally less expensive coarse-grained particles. Through the analysis of several structural and dynamical parameters, we show that this setup reliably reproduces the physical properties of the DNA molecule as observed in reference atomistic simulations. These results represent a first step towards a realistic multiscale modeling of nucleic acids and provide a quantitatively solid ground for their simulation using dual-resolution methods.

  8. The Chemistry and Biology of Oligonucleotide Conjugates (United States)

    Juliano, R.L.; Ming, Xin; Nakagawa, Osamu


    CONSPECTUS Short DNA or RNA oligonucleotides have tremendous potential as therapeutic agents. Because of their ability to engage in Watson-Crick base pairing they can interact with messenger mRNA or pre-mRNA targets with high selectivity and thus offer the possibility of precise manipulation of gene expression. This possibility has engendered extensive efforts to develop oligonucleotides as drugs, with many candidates already in clinical trials. However, a major impediment to the maturation of oligonucleotide-based therapeutics is the fact that these relatively large and usually highly charged molecules have great difficulty crossing cellular membranes and thus in penetrating to their sites of action in the cytosol or nucleus. In this Account we first summarize some basic aspects of the biology of antisense and siRNA oligonucleotides and then discuss chemical conjugation as an approach to improving the intracellular delivery and therapeutic potential of these agents. Our emphasis will be on the pharmacological ramifications of oligonucleotide conjugates rather than the details of conjugation chemistry. One important approach has been conjugation with ligands designed to bind to particular receptors and thus provide specificity to the interaction of cells with oligonucleotides. Another approach has been to couple antisense or siRNA with agents such as cell penetrating peptides that are designed to provoke escape of the conjugate from intracellular vesicular compartments. Both of these approaches have enjoyed some success. However, there remains much to be learned before oligonucleotide conjugates can find an important place in human therapeutics. PMID:22353142

  9. Stochastic segmentation models for array-based comparative genomic hybridization data analysis. (United States)

    Lai, Tze Leung; Xing, Haipeng; Zhang, Nancy


    Array-based comparative genomic hybridization (array-CGH) is a high throughput, high resolution technique for studying the genetics of cancer. Analysis of array-CGH data typically involves estimation of the underlying chromosome copy numbers from the log fluorescence ratios and segmenting the chromosome into regions with the same copy number at each location. We propose for the analysis of array-CGH data, a new stochastic segmentation model and an associated estimation procedure that has attractive statistical and computational properties. An important benefit of this Bayesian segmentation model is that it yields explicit formulas for posterior means, which can be used to estimate the signal directly without performing segmentation. Other quantities relating to the posterior distribution that are useful for providing confidence assessments of any given segmentation can also be estimated by using our method. We propose an approximation method whose computation time is linear in sequence length which makes our method practically applicable to the new higher density arrays. Simulation studies and applications to real array-CGH data illustrate the advantages of the proposed approach.

  10. Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors.

    NARCIS (Netherlands)

    Veltman, J.A.; Fridlyand, J.; Pejavar, S.; Olshen, A.B.; Korkola, J.E.; Vries, S. de; Carroll, P.; Kuo, W.L.; Pinkel, D.; Albertson, D.; Cordon-Cardo, C.; Jain, A.N.; Waldman, F.M.


    Genome-wide copy number profiles were characterized in 41 primary bladder tumors using array-based comparative genomic hybridization (array CGH). In addition to previously identified alterations in large chromosomal regions, alterations were identified in many small genomic regions, some with high-l

  11. Injection site reactions after subcutaneous oligonucleotide therapy

    NARCIS (Netherlands)

    van Meer, L. (Leonie); M. Moerland (Matthijs); Gallagher, J. (Jolie); M.B.A. van Doorn (Martijn); E.P. Prens (Errol); A.F. Cohen; Rissmann, R. (Robert); J. Burggraaf (Jacobus)


    textabstractOligonucleotides (ONs) are short fragments of nucleic acids, currently being investigated as therapeutic agents. When administered subcutaneously (sc), ONs cause a specific local reaction originating around the injection site, such as erythema, itching, discomfort and pain, including

  12. Direct microcontact printing of oligonucleotides for biochip applications

    Directory of Open Access Journals (Sweden)

    Trévisiol E


    Full Text Available Abstract Background A critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips. Results Contrary to a previous work, we showed that the stamps tailored with an elastomeric poly(dimethylsiloxane material did not require any surface modification to be able to adsorb oligonucleotides or PCR products. The adsorbed DNA molecules are subsequently printed efficiently on a target surface with high sub-micron resolution. Secondly, we showed that successive stamping is characterized by an exponential decay of the amount of transferred DNA molecules to the surface up the 4th print, then followed by a second regime of transfer that was dependent on the contact time and which resulted in reduced quality of the features. Thus, while consecutive stamping was possible, this procedure turned out to be less reproducible and more time consuming than simply re-inking the stamps between each print. Thirdly, we showed that the hybridization signals on arrays made by microcontact printing were 5 to 10-times higher than those made by conventional spotting methods. Finally, we demonstrated the validity of this microcontact printing method in manufacturing oligonucleotides arrays for mutations recognition in a yeast gene. Conclusion The microcontact printing can be considered as a new potential technology platform to pattern DNA microarrays that may have significant advantages over the conventional spotting technologies as it is easy to implement, it uses low cost material to make the stamp, and the arrays made by this technology are 10-times more sensitive in term of hybridization signals than those manufactured by conventional spotting

  13. Genomic characterization of some Iranian children with idiopathic mental retardation using array comparative genomic hybridization

    Directory of Open Access Journals (Sweden)

    Farkhondeh Behjati


    Full Text Available Background: Mental retardation (MR has a prevalence of 1-3% and genetic causes are present in more than 50% of patients. Chromosomal abnormalities are one of the most common genetic causes of MR and are responsible for 4-28% of mental retardation. However, the smallest loss or gain of material visible by standard cytogenetic is about 4 Mb and for smaller abnormalities, molecular cytogenetic techniques such as array comparative genomic hybridization (array CGH should be used. It has been shown that 15-25% of idiopathic MR (IMR has submicroscopic rearrangements detectable by array CGH. In this project, the genomic abnormalities were investigated in 32 MR patients using this technique. Materials and Methods: Patients with IMR with dysmorphism were investigated in this study. Karyotype analysis, fragile X and metabolic tests were first carried out on the patients. The copy number variation was then assessed in a total of 32 patients with normal results for the mentioned tests using whole genome oligo array CGH. Multiple ligation probe amplification was carried out as a confirmation test. Results: In total, 19% of the patients showed genomic abnormalities. This is reduced to 12.5% once the two patients with abnormal karyotypes (upon re-evaluation are removed. Conclusion: The array CGH technique increased the detection rate of genomic imbalances in our patients by 12.5%. It is an accurate and reliable method for the determination of genomic imbalances in patients with IMR and dysmorphism.

  14. Genome-wide mapping of copy number variation in humans: comparative analysis of high resolution array platforms.

    Directory of Open Access Journals (Sweden)

    Rajini R Haraksingh

    Full Text Available Accurate and efficient genome-wide detection of copy number variants (CNVs is essential for understanding human genomic variation, genome-wide CNV association type studies, cytogenetics research and diagnostics, and independent validation of CNVs identified from sequencing based technologies. Numerous, array-based platforms for CNV detection exist utilizing array Comparative Genome Hybridization (aCGH, Single Nucleotide Polymorphism (SNP genotyping or both. We have quantitatively assessed the abilities of twelve leading genome-wide CNV detection platforms to accurately detect Gold Standard sets of CNVs in the genome of HapMap CEU sample NA12878, and found significant differences in performance. The technologies analyzed were the NimbleGen 4.2 M, 2.1 M and 3×720 K Whole Genome and CNV focused arrays, the Agilent 1×1 M CGH and High Resolution and 2×400 K CNV and SNP+CGH arrays, the Illumina Human Omni1Quad array and the Affymetrix SNP 6.0 array. The Gold Standards used were a 1000 Genomes Project sequencing-based set of 3997 validated CNVs and an ultra high-resolution aCGH-based set of 756 validated CNVs. We found that sensitivity, total number, size range and breakpoint resolution of CNV calls were highest for CNV focused arrays. Our results are important for cost effective CNV detection and validation for both basic and clinical applications.

  15. Array-based comparative genomic hybridization is more informative than conventional karyotyping and fluorescence in situ hybridization in the analysis of first-trimester spontaneous abortion

    Directory of Open Access Journals (Sweden)

    Gao Jinsong


    Full Text Available Abstract Background Array-based comparative genomic hybridization (aCGH is a new technique for detecting submicroscopic deletions and duplications, and can overcome many of the limitations associated with classic cytogenetic analysis. However, its clinical use in spontaneous abortion needs comprehensive evaluation. We used aCGH to investigate chromosomal imbalances in 100 spontaneous abortions and compared the results with G-banding karyotyping and fluorescence in situ hybridization (FISH. Inconsistent results were verified by quantitative fluorescence PCR. Results Abnormalities were detected in 61 cases. aCGH achieved the highest detection rate (93.4%, 57/61 compared with traditional karyotyping (77%, 47/61 and FISH analysis (68.9%, 42/61. aCGH identified all chromosome abnormalities reported by traditional karyotyping and interphase FISH analysis, with the exception of four triploids. It also detected three additional aneuploidy cases in 37 specimens with ‘normal’ karyotypes, one mosaicism and 10 abnormalities in 14 specimens that failed to grow in vitro. Conclusions aCGH analysis circumvents many limitations in traditional karyotyping or FISH. The accuracy and efficiency of aCGH in spontaneous abortions highlights its clinical usefulness for the future. As aborted tissues have the potential to be contaminated with maternal cells, the threshold value of detection in aCGH should be lowered to avoid false negatives.

  16. Microdeletion and microduplication analysis of chinese conotruncal defects patients with targeted array comparative genomic hybridization.

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    Xiaohui Gong

    Full Text Available OBJECTIVE: The current study aimed to develop a reliable targeted array comparative genomic hybridization (aCGH to detect microdeletions and microduplications in congenital conotruncal defects (CTDs, especially on 22q11.2 region, and for some other chromosomal aberrations, such as 5p15-5p, 7q11.23 and 4p16.3. METHODS: Twenty-seven patients with CTDs, including 12 pulmonary atresia (PA, 10 double-outlet right ventricle (DORV, 3 transposition of great arteries (TGA, 1 tetralogy of Fallot (TOF and one ventricular septal defect (VSD, were enrolled in this study and screened for pathogenic copy number variations (CNVs, using Agilent 8 x 15K targeted aCGH. Real-time quantitative polymerase chain reaction (qPCR was performed to test the molecular results of targeted aCGH. RESULTS: Four of 27 patients (14.8% had 22q11.2 CNVs, 1 microdeletion and 3 microduplications. qPCR test confirmed the microdeletion and microduplication detected by the targeted aCGH. CONCLUSION: Chromosomal abnormalities were a well-known cause of multiple congenital anomalies (MCA. This aCGH using arrays with high-density coverage in the targeted regions can detect genomic imbalances including 22q11.2 and other 10 kinds CNVs effectively and quickly. This approach has the potential to be applied to detect aneuploidy and common microdeletion/microduplication syndromes on a single microarray.

  17. Visible Genotype Sensor Array

    Directory of Open Access Journals (Sweden)

    Takashi Imai


    Full Text Available A visible sensor array system for simultaneous multiple SNP genotyping has been developed using a new plastic base with specific surface chemistry. Discrimination of SNP alleles is carried out by an allele-specific extension reaction using immobilized oligonucleotide primers. The 3’-ends of oligonucleotide primers are modified with a locked nucleic acid to enhance their efficiency in allelic discrimination. Biotin-dUTPs included in the reaction mixture are selectively incorporated into extending primer sequences and are utilized as tags for alkaline phosphatase-mediated precipitation of colored chemical substrates onto the surface of the plastic base. The visible precipitates allow immediate inspection of typing results by the naked eye and easy recording by a digital camera equipped on a commercial mobile phone. Up to four individuals can be analyzed on a single sensor array and multiple sensor arrays can be handled in a single operation. All of the reactions can be performed within one hour using conventional laboratory instruments. This visible genotype sensor array is suitable for “focused genomics” that follows “comprehensive genomics”.

  18. An oligonucleotide hybridization approach to DNA sequencing. (United States)

    Khrapko, K R; Lysov YuP; Khorlyn, A A; Shick, V V; Florentiev, V L; Mirzabekov, A D


    We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.

  19. Combined array-comparative genomic hybridization and single-nucleotide polymorphism-loss of heterozygosity analysis reveals complex genetic alterations in cervical cancer

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    Kenter Gemma G


    Full Text Available Abstract Background Cervical carcinoma develops as a result of multiple genetic alterations. Different studies investigated genomic alterations in cervical cancer mainly by means of metaphase comparative genomic hybridization (mCGH and microsatellite marker analysis for the detection of loss of heterozygosity (LOH. Currently, high throughput methods such as array comparative genomic hybridization (array CGH, single nucleotide polymorphism array (SNP array and gene expression arrays are available to study genome-wide alterations. Integration of these 3 platforms allows detection of genomic alterations at high resolution and investigation of an association between copy number changes and expression. Results Genome-wide copy number and genotype analysis of 10 cervical cancer cell lines by array CGH and SNP array showed highly complex large-scale alterations. A comparison between array CGH and SNP array revealed that the overall concordance in detection of the same areas with copy number alterations (CNA was above 90%. The use of SNP arrays demonstrated that about 75% of LOH events would not have been found by methods which screen for copy number changes, such as array CGH, since these were LOH events without CNA. Regions frequently targeted by CNA, as determined by array CGH, such as amplification of 5p and 20q, and loss of 8p were confirmed by fluorescent in situ hybridization (FISH. Genome-wide, we did not find a correlation between copy-number and gene expression. At chromosome arm 5p however, 22% of the genes were significantly upregulated in cell lines with amplifications as compared to cell lines without amplifications, as measured by gene expression arrays. For 3 genes, SKP2, ANKH and TRIO, expression differences were confirmed by quantitative real-time PCR (qRT-PCR. Conclusion This study showed that copy number data retrieved from either array CGH or SNP array are comparable and that the integration of genome-wide LOH, copy number and gene

  20. Exome sequencing of germline DNA from non-BRCA1/2 familial breast cancer cases selected on the basis of aCGH tumor profiling.

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    Florentine S Hilbers

    Full Text Available The bulk of familial breast cancer risk (∼70% cannot be explained by mutations in the known predisposition genes, primarily BRCA1 and BRCA2. Underlying genetic heterogeneity in these cases is the probable explanation for the failure of all attempts to identify further high-risk alleles. While exome sequencing of non-BRCA1/2 breast cancer cases is a promising strategy to detect new high-risk genes, rational approaches to the rigorous pre-selection of cases are needed to reduce heterogeneity. We selected six families in which the tumours of multiple cases showed a specific genomic profile on array comparative genomic hybridization (aCGH. Linkage analysis in these families revealed a region on chromosome 4 with a LOD score of 2.49 under homogeneity. We then analysed the germline DNA of two patients from each family using exome sequencing. Initially focusing on the linkage region, no potentially pathogenic variants could be identified in more than one family. Variants outside the linkage region were then analysed, and we detected multiple possibly pathogenic variants in genes that encode DNA integrity maintenance proteins. However, further analysis led to the rejection of all variants due to poor co-segregation or a relatively high allele frequency in a control population. We concluded that using CGH results to focus on a sub-set of families for sequencing analysis did not enable us to identify a common genetic change responsible for the aggregation of breast cancer in these families. Our data also support the emerging view that non-BRCA1/2 hereditary breast cancer families have a very heterogeneous genetic basis.

  1. A review of statistical methods for preprocessing oligonucleotide microarrays. (United States)

    Wu, Zhijin


    Microarrays have become an indispensable tool in biomedical research. This powerful technology not only makes it possible to quantify a large number of nucleic acid molecules simultaneously, but also produces data with many sources of noise. A number of preprocessing steps are therefore necessary to convert the raw data, usually in the form of hybridisation images, to measures of biological meaning that can be used in further statistical analysis. Preprocessing of oligonucleotide arrays includes image processing, background adjustment, data normalisation/transformation and sometimes summarisation when multiple probes are used to target one genomic unit. In this article, we review the issues encountered in each preprocessing step and introduce the statistical models and methods in preprocessing.

  2. Array comparative genomic hybridisation analysis of boys with X linked hypopituitarism identifies a 3.9 Mb duplicated critical region at Xq27 containing SOX3.

    NARCIS (Netherlands)

    Solomon, N.M.; Ross, S.; Morgan, T.; Belsky, J.L.; Hol, F.A.; Karnes, P.; Hopwood, N.J.; Myers, S.E.; Tan, A.; Warne, G.L.; Forrest, S.M.; Thomas, P.Q.


    INTRODUCTION: Array comparative genomic hybridisation (array CGH) is a powerful method that detects alteration of gene copy number with greater resolution and efficiency than traditional methods. However, its ability to detect disease causing duplications in constitutional genomic DNA has not been s

  3. A hybrid Gerchberg-Saxton-like algorithm for DOE and CGH calculation (United States)

    Wang, Haichao; Yue, Weirui; Song, Qiang; Liu, Jingdan; Situ, Guohai


    The Gerchberg-Saxton (GS) algorithm is widely used in various disciplines of modern sciences and technologies where phase retrieval is required. However, this legendary algorithm most likely stagnates after a few iterations. Many efforts have been taken to improve this situation. Here we propose to introduce the strategy of gradient descent and weighting technique to the GS algorithm, and demonstrate it using two examples: design of a diffractive optical element (DOE) to achieve off-axis illumination in lithographic tools, and design of a computer generated hologram (CGH) for holographic display. Both numerical simulation and optical experiments are carried out for demonstration.

  4. Methidium intercalator inserted into synthetic oligonucleotides.

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, E. N.; Smirnov, I. P.; Haff, L. A.; Tishchenko, E. I.; Mirzabekov, A. D.; Florentiev, V. L.; Center for Mechanistic Biology and Biotechnology; Engelhardt Inst. of Molecular Biology; PerSeptive BioSystems Inc.


    A new methidium intercalator phosphoramidite has been synthesized. Methidium incorporation into an oligonucleotide during the synthesis was confirmed by UV and MALDI TOF MS data. UV melting experiments showed enhanced stability of a duplex, containing internal methidium. Methidium phosphoramidite has been synthesized and used for insertion of intercalator into the deoxyoligonucleotides.

  5. Genomic imbalances in 5918 malignant epithelial tumors: an explorative meta-analysis of chromosomal CGH data

    Directory of Open Access Journals (Sweden)

    Baudis Michael


    Full Text Available Abstract Background Chromosomal abnormalities have been associated with most human malignancies, with gains and losses on some genomic regions associated with particular entities. Methods Of the 15429 cases collected for the Progenetix molecular-cytogenetic database, 5918 malignant epithelial neoplasias analyzed by chromosomal Comparative Genomic Hybridization (CGH were selected for further evaluation. For the 22 clinico-pathological entities with more than 50 cases, summary profiles for genomic imbalances were generated from case specific data and analyzed. Results With large variation in overall genomic instability, recurring genomic gains and losses were prominent. Most entities showed frequent gains involving 8q2, while gains on 20q, 1q, 3q, 5p, 7q and 17q were frequent in different entities. Loss "hot spots" included 3p, 4q, 13q, 17p and 18q among others. Related average imbalance patterns were found for clinically distinct entities, e.g. hepatocellular carcinomas (ca. and ductal breast ca., as well as for histologically related entities (squamous cell ca. of different sites. Conclusion Although considerable case-by-case variation of genomic profiles can be found by CGH in epithelial malignancies, a limited set of variously combined chromosomal imbalances may be typical for carcinogenesis. Focus on the respective regions should aid in target gene detection and pathway deduction.

  6. Chemosensitization by antisense oligonucleotides targeting MDM2. (United States)

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo


    The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

  7. Mechanism of antisense oligonucleotide interaction with natural RNAs. (United States)

    Serikov, R; Petyuk, V; Vorobijev, Y; Koval, V; Fedorova, O; Vlassov, V; Zenkova, M


    Oligonucleotides find several numbers of applications: as diagnostic probes, RT and PCR primers and antisense agents due to their ability of forming specific interactions with complementary nucleotide sequences within nucleic acids. These interactions are strongly affected by accessibility of the target sequence in the RNA structure. In the present work the mechanism of invasion of RNA structure by oligonucleotide was investigated using a model system: yeast tRNA(Phe) and oligonucleotides complementary to the 3'-part of this molecule. Kinetics of interaction of oligonucleotides with in vitro transcript of yeast tRNAPhe was studied using stopped-flow technique with fluorescence quenching detection, 5'-DABCYL labeled oligonucleotide was hybridized with 3'-fluorescein labeled tRNA(Phe). The results evidence for a four-step invasion process of the oligonucleotide-RNA complex formation. The process is initiated by formation of transition complexes with nucleotides in the T-loop and ACCA sequence. This complex formation is followed by RNA unfolding and formation of an extended heteroduplex with the oligonucleotide via strand displacement process. Computer modeling of oligonucleotide-tRNA(Phe) interaction revealed potential factors that could favor transition complexes formation and confirmed the proposed mechanism, showing the oligonucleotide to be a molecular "wedge". Our data evidence that oligonucleotide invasion into structured RNA is initiated by loop-single strand interactions, similar to the initial step of the antisense RNA-RNA interactions. The obtained results can be used for choosing efficient oligonucleotide probes.

  8. 1-Mb resolution array-based comparative genomic hybridization using a BAC clone set optimized for cancer gene analysis

    NARCIS (Netherlands)

    Greshock, J; Naylor, TL; Margolin, A; Diskin, S; Cleaver, SH; Futreal, PA; deJong, PJ; Zhao, SY; Liebman, M; Weber, BL


    Array-based comparative genomic hybridization (aCGH) is a recently developed tool for genome-wide determination of DNA copy number alterations. This technology has tremendous potential for disease-gene discovery in cancer and developmental disorders as well as numerous other applications. However, w

  9. Detection and identification of intestinal pathogenic bacteria by hybridization to oligonucleotide microarrays

    Institute of Scientific and Technical Information of China (English)

    Lian-Qun Jin; Jun-Wen Li; Sheng-Qi Wang; Fu-Huan Chao; Xin-Wei Wang; Zheng-Quan Yuan


    AIM: To detect the common intestinal pathogenic bacteria quickly and accurately.METHODS: A rapid (<3 h) experimental procedure was set up based upon the gene chip technology. Target genes were amplified and hybridized by oligonucleotide microarrays.RESULTS: One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 (96.2%) were identified.CONCLUSION: Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus,Staphylococcus aureus, Proteus sp., Bacillus cereus,Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range,and discrimination power of this assay can be continually improved by adding further oligonucleotides to the arrays without any significant increase of complexity or cost.

  10. Dermal/transdermal delivery of small interfering RNA and antisense oligonucleotides- advances and hurdles. (United States)

    Ita, Kevin


    A diverse array of nucleic acids has been studied by several researchers for the management of several diseases. Among these compounds, small interfering RNA and antisense oligonucleotides have attracted considerable attention. Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA while siRNAs, on the other hand, are double-stranded RNA molecules which can hybridize with a specific mRNA sequence and block the translation of numerous genes. One of the main obstacles in the dermal or transdermal delivery of these compounds is their low skin permeability. In this review, various techniques used to enhance the delivery of these molecules into or across the skin are described and in some cases, the correlation between enhanced dermal/transdermal delivery and therapeutic efficacy is highlighted. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. Antisense oligonucleotides in therapy for neurodegenerative disorders. (United States)

    Evers, Melvin M; Toonen, Lodewijk J A; van Roon-Mom, Willeke M C


    Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA. They can not only reduce expression of mutant proteins by breakdown of the targeted transcript, but also restore protein expression or modify proteins through interference with pre-mRNA splicing. There has been a recent revival of interest in the use of antisense oligonucleotides to treat several neurodegenerative disorders using different approaches to prevent disease onset or halt disease progression and the first clinical trials for spinal muscular atrophy and amyotrophic lateral sclerosis showing promising results. For these trials, intrathecal delivery is being used but direct infusion into the brain ventricles and several methods of passing the blood brain barrier after peripheral administration are also under investigation. Copyright © 2015. Published by Elsevier B.V.

  12. 基于微阵列芯片的比较基因组杂交技术在临床实验室产前诊断中的应用%Prenatal diagnosis by array-based comparative genomic hybridization in the clinical laboratory setting

    Institute of Scientific and Technical Information of China (English)

    Amy M. BREMAN; 毕为民; 张秀慧


    Array-based comparative genomic hybridization (array CGH), a method used to detect gains or losses of genetic material, has recently been applied to prenatal diagnosis of genomic imbalance in the clinical laboratory setting. This new and exciting diagnostic tool represents a major technological step forward in cytogenetic testing and addresses many of the limitations of current cytogenetic methods.Conventional chromosome analysis, the current gold standard in prenatal diagnosis, focuses primarily on the detection of common aneuploidies and is limited by its capacity to detect only those copy number changes that are large enough to be microscopically visible (typically 5-6 Mb in size at the 500 band level). In contrast, array CGH analysis simultaneously evaluates regions across the entire genome and al-lows for detection of unbalanced structural and numerical chromosome abnormalities of less than one hun-dred kb. Array CGH analysis also overcomes some of the limitations of chromosome analysis, such as the requirement for cell culture and longer reporting time, by using direct uncultured fetal specimens. With many diagnostic laboratories now embracing this technology, the past year has seen tremendous growth in the use of array CGH analysis for prenatal diagnosis. This review aims to summarize array CGH methodology and its current applications in prenatal diagnosis.

  13. Electrochemical study of hepta–oligonucleotides

    Directory of Open Access Journals (Sweden)

    Zdenka Balcarova


    Full Text Available The study deals with the description and characterization of twohepta–oligonucleotides (DNA and RNA forming special structures.We studied their electrochemical behaviour by means of cyclicvoltammetry (CV and elimination voltammetry with linear scan(EVLS in combination with adsorptive stripping (AdS technique.Differences in electrochemical behaviour of hepta–deoxyribonucleotide and its RNA analog were discussed with regardto their different structures in solutions and their melting points.

  14. Abundant oligonucleotides common to most bacteria.

    Directory of Open Access Journals (Sweden)

    Colin F Davenport

    Full Text Available BACKGROUND: Bacteria show a bias in their genomic oligonucleotide composition far beyond that dictated by G+C content. Patterns of over- and underrepresented oligonucleotides carry a phylogenetic signal and are thus diagnostic for individual species. Patterns of short oligomers have been investigated by multiple groups in large numbers of bacteria genomes. However, global distributions of the most highly overrepresented mid-sized oligomers have not been assessed across all prokaryotes to date. We surveyed overrepresented mid-length oligomers across all prokaryotes and normalised for base composition and embedded oligomers using zero and second order Markov models. PRINCIPAL FINDINGS: Here we report a presumably ancient set of oligomers conserved and overrepresented in nearly all branches of prokaryotic life, including Archaea. These oligomers are either adenine rich homopurines with one to three guanine nucleosides, or homopyridimines with one to four cytosine nucleosides. They do not show a consistent preference for coding or non-coding regions or aggregate in any coding frame, implying a role in DNA structure and as polypeptide binding sites. Structural parameters indicate these oligonucleotides to be an extreme and rigid form of B-DNA prone to forming triple stranded helices under common physiological conditions. Moreover, the narrow minor grooves of these structures are recognised by DNA binding and nucleoid associated proteins such as HU. CONCLUSION: Homopurine and homopyrimidine oligomers exhibit distinct and unusual structural features and are present at high copy number in nearly all prokaryotic lineages. This fact suggests a non-neutral role of these oligonucleotides for bacterial genome organization that has been maintained throughout evolution.

  15. Synthesis and hybridization properties of inverse oligonucleotides.


    Marangoni, M.; Van Aerschot, Arthur; Augustijns, Patrick; Rozenski, Jef; Herdewijn , Piet


    The synthesis of adenine and thymine cyclopentylethyl nucleosides is presented. This novel constrained monomeric building block is very difficult to incorporate into oligonucleotides. It was introduced in 13mer oligodeoxynucleotide sequences at a single position using H-phosphonate chemistry. Phosphoramidite chemistry completely failed in this particular case. The H-phosphonate building blocks were obtained starting from the corresponding phosphoramidites. Stability of duplexes with RNA and D...


    Directory of Open Access Journals (Sweden)

    Bruno Santos Lopes Candido


    Full Text Available The article discusses the evaluation of a Central Hydroelectric Generator (CGH, as an investment. The objective is to project the cash flow of the project and evaluate outcomes to ensure important information for decision-making through technical analysis of investment projects. The work begins with literature on the subject and subsequent case study. To develop this study was carried out data collection on construction and identification of key variables related to energy market in Brazil. Later techniques were used for evaluating investment projects, regarding their financial feasibility. An interview with a business sector, and research and consulting firms. The result was positive and showed that the project is a viable investment thus adding value to shareholders compared to other investment alternatives in the financial market.

  17. The optimizations of CGH generation algorithms based on multiple GPUs for 3D dynamic holographic display (United States)

    Yang, Dan; Liu, Juan; Zhang, Yingxi; Li, Xin; Wang, Yongtian


    Holographic display has been considered as a promising display technology. Currently, low-speed generation of holograms with big holographic data is one of crucial bottlenecks for three dimensional (3D) dynamic holographic display. To solve this problem, the acceleration method computation platform is presented based on look-up table point source method. The computer generated holograms (CGHs) acquisition is sped up by offline file loading and inline calculation optimization, where a pure phase CGH with gigabyte data is encoded to record an object with 10 MB sampling data. Both numerical simulation and optical experiment demonstrate that the CGHs with 1920×1080 resolution by the proposed method can be applied to the 3D objects reconstruction with high quality successfully. It is believed that the CGHs with huge data can be generated by the proposed method with high speed for 3D dynamic holographic display in near future.

  18. Study of improved ray tracing parallel algorithm for CGH of 3D objects on GPU (United States)

    Cong, Bin; Jiang, Xiaoyu; Yao, Jun; Zhao, Kai


    An improved parallel algorithm for holograms of three-dimensional objects was presented. According to the physical characteristics and mathematical properties of the original ray tracing algorithm for computer generated holograms (CGH), using transform approximation and numerical analysis methods, we extract parts of ray tracing algorithm which satisfy parallelization features and implement them on graphics processing unit (GPU). Meanwhile, through proper design of parallel numerical procedure, we did parallel programming to the two-dimensional slices of three-dimensional object with CUDA. According to the experiments, an effective method of dealing with occlusion problem in ray tracing is proposed, as well as generating the holograms of 3D objects with additive property. Our results indicate that the improved algorithm can effectively shorten the computing time. Due to the different sizes of spatial object points and hologram pixels, the speed has increased 20 to 70 times comparing with original ray tracing algorithm.

  19. Correlation between localization, age, and chromosomal imbalances in ependymal tumours as detected by CGH. (United States)

    Jeuken, Judith W M; Sprenger, Sandra H E; Gilhuis, Job; Teepen, Hans L J M; Grotenhuis, Andre J; Wesseling, Pieter


    Ependymal tumours (ETs) are gliomas that arise from the ependymal lining of the cerebral ventricles and from the remnants of the central canal of the spinal cord. Both clinical and genetic studies suggest that distinct genetic subtypes of ETs exist, the subtypes being correlated with patient age and/or tumour site. In the present study, the tumour genome of 20 ETs (15 adult and five paediatric cases) was screened for chromosomal imbalances by comparative genomic hybridization (CGH). The most frequently detected imbalances were -22q (75%), -10q (65%), -21 (50%), -16p (50%), -1p (45%), +4q (45%), -10p (45%), -2q (40%), -6 (40%), -19 (40%), -2p (35%), -3p (35%), and -16q (35%). Comparison of the chromosomal imbalances detected in ETs with those previously reported in oligodendroglial and astrocytic tumours revealed that in this respect ETs show similarities to these other gliomas. By combining these results with those of a recent study of Zheng et al. and Hirose et al., it was found that although ETs from different sites and from adult and paediatric patients show overlap at the CGH level, some chromosomal imbalances occur predominantly in a certain category. In adult patients, spinal ETs relatively often showed +2, +7, +12, and -14q; infratentorial ETs -22; and supratentorial ETs -9. In addition, in posterior fossa ETs, -6 and +9 were much more frequent in adults than in children. It is concluded that the genetic background of ETs is complex and partly determined by tumour site, histopathological subtype, and age of the patient.

  20. Prenatal Diagnosis of a Fetus with de novo Supernumerary Ring Chromosome 16 Characterized by Array Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Pietro Cignini


    Full Text Available A fetus with de novo ring chromosome 16 is presented. At 20 weeks' gestation, ultrasound examination demonstrated bilateral clubfoot, bilateral renal pyelectasis, hypoplasia of the corpus callosum, and transposition of the great vessel. Amniocentesis was performed. Chromosome analysis identified a ring chromosome 16 [47,XY,r(16] and array comparative genomic hybridization (a-CGH demonstrated that the ring included the euchromatic portion 16p11.2. Postmortem examination confirmed prenatal findings. This is the first case of de novo ring chromosome 16 diagnosed prenatally with a new phenotypic pattern and also reinforces the importance of offering amniocentesis with a-CGH if fetal anomalies are detected.

  1. A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides. (United States)

    Magner, Dorota; Biala, Ewa; Lisowiec-Wachnicka, Jolanta; Kierzek, Elzbieta; Kierzek, Ryszard


    Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5-15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.

  2. Analysis of Chinese women with primary ovarian insufficiency by high resolution array-comparative genomic hybridization

    Institute of Scientific and Technical Information of China (English)

    LIAO Can; FU Fang; YANG Xin; SUN Yi-min; LI Dong-zhi


    Background Primary ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea) or premature depletion of ovarian follicles before the age of 40 years. The etiology of primary ovarian insufficiency in human female patients is still unclear. The purpose of this study is to investigate the potential genetic causes in primary amenorrhea patients by high resolution array based comparative genomic hybridization (array-CGH) analysis.Methods Following the standard karyotyping analysis, genomic DNA from whole blood of 15 primary amenorrhea patients and 15 normal control women was hybridized with Affymetrix cytogenetic 2.7M arrays following the standard protocol. Copy number variations identified by array-CGH were confirmed by real time polymerase chain reaction.Results All the 30 samples were negative by conventional karyotyping analysis. Microdeletions on chromosome 17q21.31-q21.32 with approximately 1.3 Mb were identified in four patients by high resolution array-CGH analysis. This included the female reproductive secretory pathway related factor N-ethylmaleimide-sensitive factor (NSF) gene.Conclusions The results of the present study suggest that there may be critical regions regulating primary ovarian insufficiency in women with a 17q21.31-q21.32 microdeletion. This effect might be due to the loss of function of the NSF gene/genes within the deleted region or to effects on contiguous genes.

  3. Comparative genomic hybridization array study and its utility in detection of constitutional and acquired anomalies. (United States)

    Andrieux, Joris; Sheth, Frenny


    The last decade has witnessed an upsurge in the knowledge of cytogenetic disorders and putting the old technology in a new basket with molecular genetics. As conventional cytogenetic can detect the genetic alteration of 10-15 Mb, many of the micro-deletions and micro-duplications are missed. However, with the advent of technology of fluorescence in situ hybridization (FISH), the resolution of genetic aberrations can reach to 3-5 Mb, nonetheless the anomalies smaller than the above, need further precision which has been achieved using comparative genomic hybridization array (CGH-array). Introduction of array-CGH has brought higher sensitivity with automated DNA fragment analyzer and DNA chip for submicroscopic chromosomal anomalies that are missed till date in many of the acquired and constitutional genetic disorders. The resolution of the technology varies from several Kb to 1 Mb depending upon the type of array selected. With the recent improvement in the array-CGH technology, a link between cytogenetic and molecular biology has been established without replacing conventional cytogenetic technique. The wider accessibility of the technology shall certainly provide a clue to the many unidentified/unexplained genetic disorders which shall prove to be a boon to the clinicians.

  4. A comparison of alternative 60-mer probe designs in an in-situ synthesized oligonucleotide microarray

    Directory of Open Access Journals (Sweden)

    Fairbanks Benjamin D


    Full Text Available Abstract Background DNA microarrays have proven powerful for functional genomics studies. Several technologies exist for the generation of whole-genome arrays. It is well documented that 25mer probes directed against different regions of the same gene produce variable signal intensity values. However, the extent to which this is true for probes of greater length (60mers is not well characterized. Moreover, this information has not previously been reported for whole-genome arrays designed against bacteria, whose genomes may differ substantially in characteristics directly affecting microarray performance. Results We report here an analysis of alternative 60mer probe designs for an in-situ synthesized oligonucleotide array for the GC rich, β-proteobacterium Burkholderia cenocepacia. Probes were designed using the ArrayOligoSel3.5 software package and whole-genome microarrays synthesized by Agilent, Inc. using their in-situ, ink-jet technology platform. We first validated the quality of the microarrays as demonstrated by an average signal to noise ratio of >1000. Next, we determined that the variance of replicate probes (1178 total probes examined of identical sequence was 3.8% whereas the variance of alternative probes (558 total alternative probes examined designs was 9.5%. We determined that depending upon the definition, about 2.4% of replicate and 7.8% of alternative probes produced outlier conclusions. Finally, we determined none of the probe design subscores (GC content, internal repeat, binding energy and self annealment produced by ArrayOligoSel3.5 were predictive or probes that produced outlier signals. Conclusion Our analysis demonstrated that the use of multiple probes per target sequence is not essential for in-situ synthesized 60mer oligonucleotide arrays designed against bacteria. Although probes producing outlier signals were identified, the use of ratios results in less than 10% of such outlier conclusions. We also determined that

  5. Comprehensive array CGH of normal karyotype myelodysplastic syndromes reveals hidden recurrent and individual genomic copy number alterations with prognostic relevance

    National Research Council Canada - National Science Library

    Thiel, A; Beier, M; Ingenhag, D; Servan, K; Hein, M; Moeller, V; Betz, B; Hildebrandt, B; Evers, C; Germing, U; Royer-Pokora, B


    .... Recurrent hidden deletions overlapping with known cytogenetic aberrations or sites of known tumor-associated genes were identified in 4q24 (TET2, 2x), 5q31.2 (2x), 7q22.1 (3x) and 21q22.12 (RUNX1, 2x...

  6. Targeted Resequencing of 9p in Acute Lymphoblastic Leukemia Yields Concordant Results with Array CGH and Reveals Novel Genomic Alterations

    NARCIS (Netherlands)

    Sarhadi, V.K.; Lahti, L.M.; Scheinin, I.; Tyybäkinoja, A.; Savola, S.; Usvasalo, A.; Räty, R.; Elonen, E.; Saarinen-Pihkala, U.M.; Knuutila, S.


    Genetic alterations of the short arm of chromosome 9 are frequent in acute lymphoblastic leukemia. We performed targeted sequencing of 9p region in 35 adolescent and adult acute lymphoblastic leukemia patients and sought to investigate the sensitivity of detecting copy number alterations in comparis

  7. Guanine-tethered antisense oligonucleotides as synthetic riboregulators. (United States)

    Hagihara, Masaki


    Regulation of gene expression by short oligonucleotides (antisense oligonucleotides), which can modulate RNA structures and inhibit subsequent associations with the translation machinery, is a potential approach for gene therapy. This chapter describes an alternative antisense strategy using guanine-tethered antisense oligonucleotides (G-ASs) to introduce a DNA-RNA heteroquadruplex structure at a designated sequence on RNA targets. The feasibility of using G-ASs to modulate RNA conformation may allow control of RNA function by inducing biologically important quadruplex structures. This approach to manipulate quadruplex structures using G-ASs may expand the strategies for regulating RNA structures and the functions of short oligonucleotide riboregulators.

  8. Lipid Oligonucleotide Conjugates as Responsive Material (United States)


    U.S. Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS Amphiphiles, oligonucleotides, lipids...peer-reviewed journals: (c) Presentations 1. Philippe Barthélémy, « Hybrid Lipids for Biomedical Applications », Targeting and Triggering Basic Research ...Steadel C. ; Pierre, N. ; Barthélémy, P. : Oligonucléotides amphiphile : Journée Scientifique de l’IFR 66, Talence, le 2 décembre 2008, France 29. Taib

  9. Application of Oligonucleotide Microarrays for Bacterial Source Tracking of Environmental Enterococcus sp. Isolates

    Directory of Open Access Journals (Sweden)

    John S. Furey


    Full Text Available In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Enterococcus sp. isolates originating from deer, bovine, gull, and human sources were examined using microarrays. Isolates were subjected to Box PCR amplification and the resulting amplification products labeled with Cy5. Fluorescent-labeled templates were hybridized to in-house constructed nonamer oligonucleotide microarrays consisting of 198 probes. Microarray hybridization profiles were obtained using the ArrayPro image analysis software. Principal Components Analysis (PCA and Hierarchical Cluster Analysis (HCA were compared for their ability to visually cluster microarray hybridization profiles based on the environmental source from which the Enterococcus sp. isolates originated. The PCA was visually superior at separating origin-specific clusters, even for as few as 3 factors. A Soft Independent Modeling (SIM classification confirmed the PCA, resulting in zero misclassifications using 5 factors for each class. The implication of these results for the application of random oligonucleotide microarrays for BST is that, given the reproducibility issues, factor-based variable selection such as in PCA and SIM greatly outperforms dendrogram-based similarity measures such as in HCA and K-Nearest Neighbor KNN.

  10. Intragenic rearrangements in X-linked intellectual deficiency: results of a-CGH in a series of 54 patients and identification of TRPC5 and KLHL15 as potential XLID genes. (United States)

    Mignon-Ravix, Cécile; Cacciagli, Pierre; Choucair, Nancy; Popovici, Cornel; Missirian, Chantal; Milh, Mathieu; Mégarbané, André; Busa, Tiffany; Julia, Sophie; Girard, Nadine; Badens, Catherine; Sigaudy, Sabine; Philip, Nicole; Villard, Laurent


    High-resolution array comparative genomic hybridization (a-CGH) enables the detection of intragenic rearrangements, such as single exon deletion or duplication. This approach can lead to the identification of new disease genes. We report on the analysis of 54 male patients presenting with intellectual deficiency (ID) and a family history suggesting X-linked (XL) inheritance or maternal skewed X-chromosome inactivation (XCI), using a home-made X-chromosome-specific microarray covering the whole human X-chromosome at high resolution. The majority of patients had whole genome array-CGH prior to the selection and we did not include large rearrangements such as MECP2 and FMR1 duplications. We identified four rearrangements considered as causative or potentially pathogenic, corresponding to a detection rate of 8%. Two CNVs affected known XLID genes and were therefore considered as causative (IL1RAPL1 and OPHN1 intragenic deletions). Two new CNVs were considered as potentially pathogenic as they affected interesting candidates for ID. The first CNV is a deletion of the first exon of the TRPC5 gene, encoding a cation channel implicated in dendrite growth and patterning, in a child presenting with ID and an autism spectrum disorder (ASD). The second CNV is a partial deletion of KLHL15, in a patient with severe ID, epilepsy, and anomalies of cortical development. In both cases, in spite of strong arguments for clinical relevance, we were not able at this stage to confirm pathogenicity of the mutations, and the causality of the variants identified in XLID remains to be confirmed.

  11. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Chen Feng


    Full Text Available Abstract Background Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators. Results Taking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals and Plasmodium falciparum (a related parasite responsible for severe human malaria, we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at

  12. In situ synthesis of DNA micro-arrays using typography technique

    Institute of Scientific and Technical Information of China (English)


    A novel typography technique was developed to in situ synthesize oligonucleotide arrays on glass slide,which has the celerity,high spatial resolution,lower cost,reliable operation,and high synthetic efficiency.The principle and process of the typography technique for fabricating gene-chips have been described in detail.A suit of poly(terafluoroethylene)devices for synthesizing oligonucleotide arrays were designed and prepared,and the fiber tubes with a number of nano-or micron-channels were em- ployed.The oligonucleotide arrays of 16 and 160 features with four different probes were synthesized using the typography technique.The four specific oligonucleotide probes including the matched and the mismatched by the fluorescent target sequence gave obviously different hybridization fluorescent signals.It was indicated that the gene-chip fabricated by the typography method could be used to rapidly screen single-nucleotide polymorphisms(SNP)and to detect mutations.

  13. In situ synthesis of DNA micro-arrays using typography technique

    Institute of Scientific and Technical Information of China (English)

    TANG JianXin; HE NongYue; LI Song


    A novel typography technique was developed to in situ synthesize oligonucleotide arrays on glass slide, which has the celerity, high spatial resolution, lower cost, reliable operation, and high synthetic efficiency. The principle and process of the typography technique for fabricating gene-chips have been described in detail. A suit of poly(terafluoroethylene) devices for synthesizing oligonucleotide arrays were designed and prepared, and the fiber tubes with a number of nano- or micron-channels were employed. The oligonucleotide arrays of 16 and 160 features with four different probes were synthesized using the typography technique. The four specific oligonucleotide probes including the matched and the mismatched by the fluorescent target sequence gave obviously different hybridization fluorescent signals. It was indicated that the gene-chip fabricated by the typography method could be used to rapidly screen single-nucleotide polymorphisms (SNP) and to detect mutations.

  14. Clinical and cytogenetic features of a patient with partial trisomy 8q and partial monosomy 13q delineated by array comparative genomic hybridization. (United States)

    Sohn, Young Bae; Yun, Jun No; Park, Sang-Jin; Park, Moon Sung; Kim, Sung Hwan; Lee, Jang Hoon


    Partial trisomy 8q is rare and has distinctive clinical features, including severe mental retardation, growth impairment, dysmorphic facial appearances, cleft palate, congenital heart disease, and urogenital anomalies. Partial monosomy 13q is a rare genetic disorder displaying a variety of phenotypic characteristics including mental retardation, dysmorphic facial features, and congenital anomalies. Here, we describe for the first time clinical observations and cytogenetic analysis of a patient with a concomitant occurrence of partial trisomy of 8q (8q21.3→qter) and partial monosomy 13q(13q34→qter). The patient was a female neonate with facial dysmorphia, agenesis of the corpus callosum, cleft palate, and congenital heart disease. G-band standard karyotype was 46,XX,add(13)(q34). To determine the origin of additional genomic gain in chromosome 13, array comparative genomic hybridization (CGH) was performed. Array CGH showed a 56.8 Mb sized gain on chromosome 8q and a 0.28 Mb sized loss on chromosome 13q. Therefore, the final karyotype of the patient was defined as 46,XX, der(13)t(8;13)(q21.3;q34). In conclusion, we described the clinical and cytogenetic analysis of the patient with concomitant occurrence of partial trisomy 8q and partial monosomy 13q delineated by array CGH. This report suggests that the array CGH would be a valuable diagnostic tool for identifying the origin of small additional genetic materials.

  15. Using Array-Based Comparative Genomic Hybridization to Diagnose Pallister-Killian Syndrome. (United States)

    Lee, Mi Na; Lee, Jiwon; Yu, Hee Joon; Lee, Jeehun; Kim, Sun Hee


    Pallister-Killian syndrome (PKS) is a rare multisystem disorder characterized by isochromosome 12p and tissue-limited mosaic tetrasomy 12p. In this study, we diagnosed three pediatric patients who were suspicious of having PKS using array-based comparative genomic hybridization (array CGH) and FISH analyses performed on peripheral lymphocytes. Patients 1 and 2 presented with craniofacial dysmorphic features, hypotonia, and a developmental delay. Array CGH revealed two to three copies of 12p in patient 1 and three copies in patient 2. FISH analysis showed trisomy or tetrasomy 12p. Patient 3, who had clinical features comparable to those of patients 1 and 2, was diagnosed by using FISH analysis alone. Here, we report three patients with mosaic tetrasomy 12p. There have been only reported cases diagnosed by chromosome analysis and FISH analysis on skin fibroblast or amniotic fluid. To our knowledge, patient 1 was the first case diagnosed by using array CGH performed on peripheral lymphocytes in Korea.

  16. Antisense Oligonucleotide Therapy in Diabetic Retinopathy (United States)

    Hnik, Peter; Boyer, David S.; Grillone, Lisa R.; Clement, John G.; Henry, Scott P.; Green, Ellen A.


    Diabetic retinopathy is one of the leading causes of blindness in the United States and other parts of the world. Historically, laser photocoagulation and vitrectomy surgery have been used for the treatment of diabetic retinopathy, including diabetic macular edema. Both procedures have proven to be useful under certain conditions but have their limitations. New pathways and processes that promote diabetic retinopathy have been identified, and several new therapeutic approaches are under investigation. These new therapies may be beneficial in the treatment of diabetic retinopathy and include antivascular endothelial growth factor agents, corticosteroids, and therapies that may potentially target a number of additional diabetic retinopathy-related factors and processes, including antisense oligonucleotides. Second-generation antisense oligonucleotides, such as iCo-007, may offer a significant advantage in the treatment of diabetic retinopathy by downregulating the signal pathways of multiple growth factors that seem to play a critical role in the process of ocular angiogenesis and vascular leakage. Benefits of such molecules are expected to include the specificity of the kinase target and an extended half-life, resulting in less frequent intravitreal drug administration, resistance to molecule degradation, and a good safety profile. PMID:20144342

  17. Template switching between PNA and RNA oligonucleotides (United States)

    Bohler, C.; Nielsen, P. E.; Orgel, L. E.; Miller, S. L. (Principal Investigator)


    The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.

  18. Clinical trial involving sufferers and non-sufferers of cervicogenic headache (CGH): potential mechanisms of action of photobiomodulation (Conference Presentation) (United States)

    Liebert, Ann D.; Bicknell, Brian


    Photobiomodulation (PBM) is an effective tool for the management of spinal pain including inflammation of facet joints. Apart from cervical and lumbar joint pain the upper cervical spine facet joint inflammation can result in the CGH (traumatic or atraumatic in origin). This condition affects children, adults and elders and is responsible for 19% of chronic headache and up to 33% of patients in pain clinics. The condition responds well to physiotherapy, facet joint injection, radiofrequency neurotomy and surgery at a rate of 75%. The other 25% being unresponsive to treatment with no identified features of unresponsiveness. In other conditions of chronic unresponsive cervical pain have responded to photobiomodulation at a level of 80% in the short and medium term. A clinical trial was therefore conducted on a cohort of atraumatic patients from the ages of 5-93 (predominantly Neurologist referred / familial sufferers 2/3 generations vertically and laterally) who had responded to a course of PBM and physiotherapy. The CGH sufferers and their non CGH suffering relatives over these generations were then compared for features that distinguish the two groups. Fifty parameters were tested (anthropmetric, movement and neural tension tests included) and there was a noted difference in tandem stance between the groups (.04 significance with repeated measures). As this impairment is common to benign ataxia and migrainous vertigo and in these conditions there is an ion channelopathy (especially potassium channelopathy). A postulated mechanism of action of PBM would involve modulation of ion channels and this is discussed in this presentation.

  19. Oligonucleotides Containing Aminated 2′-Amino-LNA Nucleotides

    DEFF Research Database (Denmark)

    Lou, Chenguang; Samuelsen, Simone V.; Christensen, Niels Johan


    Mono- and diaminated 2′-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested...... that structural stabilization occurs via intrastrand electrostatic attraction between the protonated amino groups of the aminated 2′-amino-LNA monomers and the host oligonucleotide backbone....

  20. Voltage-gated calcium channel and antisense oligonucleotides thereto (United States)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)


    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  1. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. (United States)

    Geary, Richard S; Norris, Daniel; Yu, Rosie; Bennett, C Frank


    Pharmacokinetic properties of oligonucleotides are largely driven by chemistry of the backbone and thus are sequence independent within a chemical class. Tissue bioavailability (% of administered dose) is assisted by plasma protein binding that limits glomerular filtration and ultimate urinary excretion of oligonucleotides. The substitution of one non-bridging oxygen with the more hydrophobic sulfur atom (phosphorothioate) increases both plasma stability and plasma protein binding and thus, ultimately, tissue bioavailability. Additional modifications of the sugar at the 2' position, increase RNA binding affinity and significantly increase potency, tissue half-life and prolong RNA inhibitory activity. Oligonucleotides modified in this manner consistently exhibit the highest tissue bioavailability (>90%). Systemic biodistribution is broad, and organs typically with highest concentrations are liver and kidney followed by bone marrow, adipocytes, and lymph nodes. Cell uptake is predominantly mediated by endocytosis. Both size and charge for most oligonucleotides prevents distribution across the blood brain barrier. However, modified single-strand oligonucleotides administered by intrathecal injection into the CSF distribute broadly in the CNS. The majority of intracellular oligonucleotide distribution following systemic or local administration occurs rapidly in just a few hours following administration and is facilitated by rapid endocytotic uptake mechanisms. Further understanding of the intracellular trafficking of oligonucleotides may provide further enhancements in design and ultimate potency of antisense oligonucleotides in the future. Copyright © 2015. Published by Elsevier B.V.

  2. Enhanced fluorescence of silver nanoclusters stabilized with branched oligonucleotides. (United States)

    Latorre, Alfonso; Lorca, Romina; Zamora, Félix; Somoza, Álvaro


    DNA stabilized silver nanoclusters (AgNCs) are promising optical materials, whose fluorescence properties can be tuned by the selection of the DNA sequence employed. In this work we have used modified oligonucleotides in the preparation of AgNCs. The fluorescent intensity obtained was 60 times higher than that achieved with standard oligonucleotides.

  3. Noncoding oligonucleotides: the belle of the ball in gene therapy. (United States)

    Shum, Ka-To; Rossi, John J


    Gene therapy carries the promise of cures for many diseases based on manipulating the expression of a person's genes toward the therapeutic goal. The relevance of noncoding oligonucleotides to human disease is attracting widespread attention. Noncoding oligonucleotides are not only involved in gene regulation, but can also be modified into therapeutic tools. There are many strategies that leverage noncoding oligonucleotides for gene therapy, including small interfering RNAs, antisense oligonucleotides, aptamers, ribozymes, decoys, and bacteriophage phi 29 RNAs. In this chapter, we will provide a broad, comprehensive overview of gene therapies that use noncoding oligonucleotides for disease treatment. The mechanism and development of each therapeutic will be described, with a particular focus on its clinical development. Finally, we will discuss the challenges associated with developing nucleic acid therapeutics and the prospects for future success.

  4. Predicting oligonucleotide-directed mutagenesis failures in protein engineering. (United States)

    Wassman, Christopher D; Tam, Phillip Y; Lathrop, Richard H; Weiss, Gregory A


    Protein engineering uses oligonucleotide-directed mutagenesis to modify DNA sequences through a two-step process of hybridization and enzymatic synthesis. Inefficient reactions confound attempts to introduce mutations, especially for the construction of vast combinatorial protein libraries. This paper applied computational approaches to the problem of inefficient mutagenesis. Several results implicated oligonucleotide annealing to non-target sites, termed 'cross-hybridization', as a significant contributor to mutagenesis reaction failures. Test oligonucleotides demonstrated control over reaction outcomes. A novel cross-hybridization score, quickly computable for any plasmid and oligonucleotide mixture, directly correlated with yields of deleterious mutagenesis side products. Cross-hybridization was confirmed conclusively by partial incorporation of an oligonucleotide at a predicted cross-hybridization site, and by modification of putative template secondary structure to control cross-hybridization. Even in low concentrations, cross-hybridizing species in mixtures poisoned reactions. These results provide a basis for improved mutagenesis efficiencies and increased diversities of cognate protein libraries.

  5. Application of Array-based Comparative Genome Hybridization in Children with Developmental Delay or Mental Retardation

    Directory of Open Access Journals (Sweden)

    Jao-Shwann Liang


    Full Text Available Children with developmental delay or mental retardation (DD/MR are commonly en countered in child neurology clinics, and establishing an etiologic diagnosis is a challenge for child neurologists. Among the etiologies, chromosomal imbalance is one of the most important causes. However, many of these chromosomal imbalances are submicroscopic and cannot be detected by conventional cytogenetic methods. Microarray-based comparative genomic hybridization (array CGH is considered to be superior in the investigation of chromosomal deletions or duplications in children with DD/MR, and has been demonstrated to improve the diagnostic detection rate for these small chromosomal abnormalities. Here, we review the recent studies of array CGH in the evaluation of patients with idiopathic DD/MR.

  6. Mismatch oligonucleotides in human and yeast: guidelines for probe design on tiling microarrays

    Directory of Open Access Journals (Sweden)

    Jee Justin


    Full Text Available Abstract Background Mismatched oligonucleotides are widely used on microarrays to differentiate specific from nonspecific hybridization. While many experiments rely on such oligos, the hybridization behavior of various degrees of mismatch (MM structure has not been extensively studied. Here, we present the results of two large-scale microarray experiments on S. cerevisiae and H. sapiens genomic DNA, to explore MM oligonucleotide behavior with real sample mixtures under tiling-array conditions. Results We examined all possible nucleotide substitutions at the central position of 36-nucleotide probes, and found that nonspecific binding by MM oligos depends upon the individual nucleotide substitutions they incorporate: C→A, C→G and T→A (yielding purine-purine mispairs are most disruptive, whereas A→X were least disruptive. We also quantify a marked GC skew effect: substitutions raising probe GC content exhibit higher intensity (and vice versa. This skew is small in highly-expressed regions (± 0.5% of total intensity range and large (± 2% or more elsewhere. Multiple mismatches per oligo are largely additive in effect: each MM added in a distributed fashion causes an additional 21% intensity drop relative to PM, three-fold more disruptive than adding adjacent mispairs (7% drop per MM. Conclusion We investigate several parameters for oligonucleotide design, including the effects of each central nucleotide substitution on array signal intensity and of multiple MM per oligo. To avoid GC skew, individual substitutions should not alter probe GC content. RNA sample mixture complexity may increase the amount of nonspecific hybridization, magnify GC skew and boost the intensity of MM oligos at all levels.

  7. Validation and implementation of array comparative genomic hybridisation as a first line test in place of postnatal karyotyping for genome imbalance

    Directory of Open Access Journals (Sweden)

    Docherty Zoe


    Full Text Available Abstract Background Several studies have demonstrated that array comparative genomic hybridisation (CGH for genome-wide imbalance provides a substantial increase in diagnostic yield for patients traditionally referred for karyotyping by G-banded chromosome analysis. The purpose of this study was to demonstrate the feasibility of and strategies for, the use of array CGH in place of karyotyping for genome imbalance, and to report on the results of the implementation of this approach. Results Following a validation period, an oligoarray platform was chosen. In order to minimise costs and increase efficiency, a patient/patient hybridisation strategy was used, and analysis criteria were set to optimise detection of pathogenic imbalance. A customised database application with direct links to a number of online resources was developed to allow efficient management and tracking of patient samples and facilitate interpretation of results. Following introduction into our routine diagnostic service for patients with suspected genome imbalance, array CGH as a follow-on test for patients with normal karyotypes (n = 1245 and as a first-line test (n = 1169 gave imbalance detection rates of 26% and 22% respectively (excluding common, benign variants. At least 89% of the abnormalities detected by first line testing would not have been detected by standard karyotype analysis. The average reporting time for first-line tests was 25 days from receipt of sample. Conclusions Array CGH can be used in a diagnostic service setting in place of G-banded chromosome analysis, providing a more comprehensive and objective test for patients with suspected genome imbalance. The increase in consumable costs can be minimised by employing appropriate hybridisation strategies; the use of robotics and a customised database application to process multiple samples reduces staffing costs and streamlines analysis, interpretation and reporting of results. Array CGH provides a

  8. Cellular uptake and trafficking of antisense oligonucleotides. (United States)

    Crooke, Stanley T; Wang, Shiyu; Vickers, Timothy A; Shen, Wen; Liang, Xue-Hai


    Antisense oligonucleotides (ASOs) modified with phosphorothioate (PS) linkages and different 2' modifications can be used either as drugs (e.g., to treat homozygous familial hypercholesterolemia and spinal muscular atrophy) or as research tools to alter gene expression. PS-ASOs can enter cells without additional modification or formulation and can be designed to mediate sequence-specific cleavage of different types of RNA (including mRNA and non-coding RNA) targeted by endogenous RNase H1. Although PS-ASOs function in both the cytoplasm and nucleus, localization to different subcellular regions can affect their therapeutic potency. Cellular uptake and intracellular distribution of PS ASOs are mediated by protein interactions. The main proteins involved in these processes have been identified, and intracellular sites in which PS ASOs are active, or inactive, cataloged.

  9. Conjugation of fluorescent proteins with DNA oligonucleotides. (United States)

    Lapiene, Vidmantas; Kukolka, Florian; Kiko, Kathrin; Arndt, Andreas; Niemeyer, Christof M


    This work describes the synthesis of covalent ssDNA conjugates of six fluorescent proteins, ECFP, EGFP, E(2)GFP, mDsRed, Dronpa, and mCherry, which were cloned with an accessible C-terminal cystein residue to enable site-selective coupling using a heterobispecific cross-linker. The resulting conjugates revealed similar fluorescence emission intensity to the unconjugated proteins, and the functionality of the tethered oligonucleotide was proven by specific Watson-Crick base pairing to cDNA-modified gold nanoparticles. Fluorescence spectroscopy analysis indicated that the fluorescence of the FP is quenched by the gold particle, and the extent of quenching varied with the intrinsic spectroscopic properties of FP as well as with the configuration of surface attachment. Since this study demonstrates that biological fluorophores can be selectively incorporated into and optically coupled with nanoparticle-based devices, applications in DNA-based nanofabrication can be foreseen.

  10. Direct oligonucleotide-photosensitizer conjugates for photochemical delivery of antisense oligonucleotides. (United States)

    Yuan, Ahu; Laing, Brian; Hu, Yiqiao; Ming, Xin


    Activation of photosensitizers in endosomes enables release of therapeutic macromolecules into the cytosol of the target cells for pharmacological actions. In this study, we demonstrate that direct conjugation of photosensitizers to oligonucleotides (ONs) allows spatial and temporal co-localization of the two modalities in the target cells, and thus leads to superior functional delivery of ONs. Further, light-activated delivery of an anticancer ON caused cancer cell killing via modulation of an oncogene and photodynamic therapy.

  11. Preparation and application of triple helix forming oligonucleotides and single strand oligonucleotide donors for gene correction. (United States)

    Alam, Rowshon; Thazhathveetil, Arun Kalliat; Li, Hong; Seidman, Michael M


    Strategies for site-specific modulation of genomic sequences in mammalian cells require two components. One must be capable of recognizing and activating a specific target sequence in vivo, driving that site into an exploitable repair pathway. Information is transferred to the site via participation in the pathway by the second component, a donor nucleic acid, resulting in a permanent change in the target sequence. We have developed biologically active triple helix forming oligonucleotides (TFOs) as site-specific gene targeting reagents. These TFOs, linked to DNA reactive compounds (such as a cross-linking agent), activate pathways that can engage informational donors. We have used the combination of a psoralen-TFO and single strand oligonucleotide donors to generate novel cell lines with directed sequence changes at the target site. Here we describe the synthesis and purification of bioactive psoralen-linked TFOs, their co-introduction into mammalian cells with donor nucleic acids, and the identification of cells with sequence conversion of the target site. We have emphasized details in the synthesis and purification of the oligonucleotides that are essential for preparation of reagents with optimal activity.

  12. Design and analysis of mismatch probes for long oligonucleotide microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong


    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  13. Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease. (United States)

    Sardone, Valentina; Zhou, Haiyan; Muntoni, Francesco; Ferlini, Alessandra; Falzarano, Maria Sofia


    Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.

  14. Nucleic acid sequence detection using multiplexed oligonucleotide PCR (United States)

    Nolan, John P.; White, P. Scott


    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  15. Design and analysis of mismatch probes for long oligonucleotide microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong


    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  16. Antisense oligonucleotide induction of progerin in human myogenic cells.

    Directory of Open Access Journals (Sweden)

    Yue-Bei Luo

    Full Text Available We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA transcript in human myogenic cells. The progerin transcript (LMNA Δ150 lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS. HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model

  17. Genome-wide screening for genetic alterations in esophageal cancer by aCGH identifies 11q13 amplification oncogenes associated with nodal metastasis.

    Directory of Open Access Journals (Sweden)

    Jianming Ying

    Full Text Available BACKGROUND: Esophageal squamous cell carcinoma (ESCC is highly prevalent in China and other Asian countries, as a major cause of cancer-related mortality. ESCC displays complex chromosomal abnormalities, including multiple structural and numerical aberrations. Chromosomal abnormalities, such as recurrent amplifications and homozygous deletions, directly contribute to tumorigenesis through altering the expression of key oncogenes and tumor suppressor genes. METHODOLOGY/PRINCIPLE FINDINGS: To understand the role of genetic alterations in ESCC pathogenesis and identify critical amplification/deletion targets, we performed genome-wide 1-Mb array comparative genomic hybridization (aCGH analysis for 10 commonly used ESCC cell lines. Recurrent chromosomal gains were frequently detected on 3q26-27, 5p15-14, 8p12, 8p22-24, 11q13, 13q21-31, 18p11 and 20q11-13, with frequent losses also found on 8p23-22, 11q22, 14q32 and 18q11-23. Gain of 11q13.3-13.4 was the most frequent alteration in ESCC. Within this region, CCND1 oncogene was identified with high level of amplification and overexpression in ESCC, while FGF19 and SHANK2 was also remarkably over-expressed. Moreover, a high concordance (91.5% of gene amplification and protein overexpression of CCND1 was observed in primary ESCC tumors. CCND1 amplification/overexpression was also significantly correlated with the lymph node metastasis of ESCC. CONCLUSION: These findings suggest that genomic gain of 11q13 is the major mechanism contributing to the amplification. Novel oncogenes identified within the 11q13 amplicon including FGF19 and SHANK2 may play important roles in ESCC tumorigenesis.

  18. Anti-fouling characteristics of surface-confined oligonucleotide strands bioconjugated on streptavidin platforms in the presence of nanomaterials. (United States)

    Mir, Mònica; Cameron, Petra J; Zhong, Xinhua; Azzaroni, Omar; Alvarez, Marta; Knoll, Wolfgang


    This work describes our studies on the molecular design of interfacial architectures suitable for DNA sensing which could resist non-specific binding of nanomaterials commonly used as labels for amplifying biorecognition events. We observed that the non-specific binding of bio-nanomaterials to surface-confined oligonucleotide strands is highly dependent on the characteristics of the interfacial architecture. Thiolated double stranded oligonucleotide arrays assembled on Au surfaces evidence significant fouling in the presence of nanoparticles (NPs) at the nanomolar level. The non-specific interaction between the oligonucleotide strands and the nanomaterials can be sensitively minimized by introducing streptavidin (SAv) as an underlayer conjugated to the DNA arrays. The role of the SAv layer was attributed to the significant hydrophilic repulsion between the SAv-modified surface and the nanomaterials in close proximity to the interface, thus conferring outstanding anti-fouling characteristics to the interfacial architecture. These results provide a simple and straightforward strategy to overcome the limitations introduced by the non-specific binding of labels to achieve reliable detection of DNA-based biorecognition events.

  19. Optical Characterization of Oligonucleotide DNA Influenced by Magnetic Fields

    Directory of Open Access Journals (Sweden)

    Seyedeh Maryam Banihashemian


    Full Text Available UV-VIS spectroscopic analysis of oligonucleotide DNA exposed to different magnetic fields was performed in order to investigate the relationship between DNA extinction coefficients and optical parameters according to magnetic-field strength. The results with the oligonucleotides adenine-thymine 100 mer (AT-100 DNA and cytosine-guanine 100 mer (CG-100 DNA indicate that the magnetic field influences DNA molar extinction coefficients and refractive indexes. The imaginary parts of the refractive index and molar extinction coefficients of the AT-100 and CG-100 DNA decreased after exposure to a magnetic field of 750 mT due to cleavage of the DNA oligonucleotides into smaller segments.

  20. DNA microarray synthesis by using PDMS molecular stamp (II) -- Oligonucleotide on-chip synthesis using PDMS stamp

    Institute of Scientific and Technical Information of China (English)


    Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5′-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleotide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the conventional synthesis methods.

  1. FGX : a frequentist gene expression index for Affymetrix arrays

    NARCIS (Netherlands)

    Purutçuoğlu, Vilda; Wit, Ernst


    We consider a new frequentist gene expression index for Affymetrix oligonucleotide DNA arrays, using a similar probe intensity model as suggested previously, called the Bayesian gene expression index (BGX). According to this model, the perfect match and mismatch values are assumed to be correlated a

  2. Multilocus sequence typing of Staphylococcus aureus with DNA array technology

    NARCIS (Netherlands)

    W.B. van Leeuwen (Willem); C. Jay (Corinne); S.V. Snijders (Susan); N. Durin (Nathalia); B. Lacroix (Bruno); H.A. Verbrugh (Henri); M.C. Enright (Mark); A. Troesch (Alain); A.F. van Belkum (Alex)


    textabstractA newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of Staphylococcus aureus strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventiona

  3. Optimized design and assessment of whole genome tiling arrays.

    NARCIS (Netherlands)

    Graf, S.; Nielsen, F.G.G.; Kurtz, S.; Huynen, M.A.; Birney, E.; Stunnenberg, H.G.; Flicek, P.


    MOTIVATION: Recent advances in microarray technologies have made it feasible to interrogate whole genomes with tiling arrays and this technique is rapidly becoming one of the most important high-throughput functional genomics assays. For large mammalian genomes, analyzing oligonucleotide tiling arra

  4. Cluster Analysis of Comparative Genomic Hybridization (CGH Data Using Self-Organizing Maps: Application to Prostate Carcinomas

    Directory of Open Access Journals (Sweden)

    Torsten Mattfeldt


    Full Text Available Comparative genomic hybridization (CGH is a modern genetic method which enables a genome‐wide survey of chromosomal imbalances. For each chromosome region, one obtains the information whether there is a loss or gain of genetic material, or whether there is no change at that region. Usually it is not possible to evaluate all 46 chromosomes of a metaphase, therefore several (up to 20 or more metaphases are analyzed per individual, and expressed as average. Mostly one does not study one individual alone but groups of 20–30 individuals. Therefore, large amounts of data quickly accumulate which must be put into a logical order. In this paper we present the application of a self‐organizing map (Genecluster as a tool for cluster analysis of data from pT2N0 prostate cancer cases studied by CGH. Self‐organizing maps are artificial neural networks with the capability to form clusters on the basis of an unsupervised learning rule, i.e., in our examples it gets the CGH data as only information (no clinical data. We studied a group of 40 recent cases without follow‐up, an older group of 20 cases with follow‐up, and the data set obtained by pooling both groups. In all groups good clusterings were found in the sense that clinically similar cases were placed into the same clusters on the basis of the genetic information only. The data indicate that losses on chromosome arms 6q, 8p and 13q are all frequent in pT2N0 prostatic cancer, but the loss on 8p has probably the largest prognostic importance.

  5. Increased nuchal translucency with normal karyotype: a follow-up study of 100 cases supplemented with CGH and MLPA analyses

    DEFF Research Database (Denmark)

    Schou, K V; Kirchhoff, M; Nygaard, U;


    OBJECTIVE: To evaluate whether high-resolution comparative genomic hybridization (HR-CGH) and subtelomeric and syndrome-specific multiplex ligation-dependent probe amplification (MLPA) would detect minor chromosomal aberrations in fetuses with increased nuchal translucency thickness (NT) and norm...... disease, which supports the current approach of repeated ultrasound examinations in these high-risk pregnancies....... and subtelomeric regions. Pregnancy outcome was followed up. RESULTS: Among 80 liveborn children who were followed up, three (4%) had syndromes involving mental retardation, including a case of Sotos syndrome caused by a de novo mutation. 15% of fetuses were lost during pregnancy due to abnormalities...

  6. Multilocus sequence typing of Staphylococcus aureus with DNA array technology



    textabstractA newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of Staphylococcus aureus strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventional strain typing. Analysis of strains from the second collection revealed that chip-defined MLST was concordant with conventional MLST. Array-mediated MLST data were reproducible, exchangeable, and epidemiologically ...

  7. Hole hopping rates in single strand oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Borrelli, Raffaele [Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università di Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO (Italy); Capobianco, Amedeo [Dipartimento di Chimica e Biologia, Università di Salerno, Via Giovanni Paolo II, I-84084 Fisciano, SA (Italy); Peluso, Andrea, E-mail: [Dipartimento di Chimica e Biologia, Università di Salerno, Via Giovanni Paolo II, I-84084 Fisciano, SA (Italy)


    Highlights: • DNA hole transfer rates have been computed. • Delocalized adenine domains significantly affect hole transfer rates in DNA. • Franck–Condon weighted density of state from DFT normal modes. • DNA application in molecular electronics. - Abstract: The rates of hole transfer between guanine and adenine in single strand DNA have been evaluated by using Fermi’s golden rule and Kubo’s generating function approach for the Franck–Condon weighted density of states. The whole sets of the normal modes and vibrational frequencies of the two nucleobases, obtained at DFT/B3LYP level of calculation, have been considered in computations. The results show that in single strand the pyramidalization/planarization mode of the amino groups of both nucleobases plays the major role. At room temperature, the Franck–Condon density of states extends over a wide range of hole site energy difference, 0–1 eV, giving some hints about the design of oligonucleotides of potential technological interest.

  8. Oligonucleotide and Long Polymeric DNA Encoding

    Energy Technology Data Exchange (ETDEWEB)

    Miller, E; Mariella Jr., R P; Christian, A T; Gardner, S N; Williams, J M


    This report summarizes the work done at Lawrence Livermore National Laboratory for the Oligonucleotide and Long Polymeric DNA Encoding project, part of the Microelectronic Bioprocesses Program at DARPA. The goal of the project was to develop a process by which long (circa 10,000 base-pair) synthetic DNA molecules could be synthesized in a timely and economic manner. During construction of the long molecule, errors in DNA sequence occur during hybridization and/or the subsequent enzymatic process. The work done on this project has resulted in a novel synthesis scheme that we call the parallel pyramid synthesis protocol, the development of a suit of computational tools to minimize and quantify errors in the synthesized DNA sequence, and experimental proof of this technique. The modeling consists of three interrelated modules: the bioinformatics code which determines the specifics of parallel pyramid synthesis for a given chain of long DNA, the thermodynamics code which tracks the products of DNA hybridization and polymerase extension during the later steps in the process, and the kinetics model which examines the temporal and spatial processes during one thermocycle. Most importantly, we conducted the first successful syntheses of a gene using small starting oligomers (tetramers). The synthesized sequence, 813 base pairs long, contained a 725 base pair gene, modified green fluorescent protein (mGFP), which has been shown to be a functional gene by cloning into cells and observing its green fluorescent product.

  9. Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel.

    Directory of Open Access Journals (Sweden)

    Weizhong Tang

    Full Text Available To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA, (dC, (dG and (dT to silver staining could be ranged as (dA > (dG > (dC > (dT from high to low. It was unexpected that oligo (dT was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt. The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.

  10. Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.

    Energy Technology Data Exchange (ETDEWEB)

    Proudnikov, D.; Kirillov, E.; Chumakov, K.; Donion, J.; Rezapkin, G.; Mirzabekov, A.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Center for Biologics Evaluation and Research


    This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements. Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.

  11. Targeting of single stranded oligonucleotides through metal-induced cyclization of short complementary strands : Targeting of single stranded oligonucleotides


    Freville, Fabrice; Richard, Tristan; Bathany, Katell; Moreau, Serge


    International audience; A new strategy to cyclize a short synthetic oligonucleotide on a DNA or a RNA target strand is described. This one relies on a metal-mediated cyclization of short synthetic oligonucleotides conjugated with two chelating 2,2':6',2”-terpyridine moieties at their 3' and 5' ends. Cyclization following metal addition (Zn2+, Fe2+) was demonstrated using UV monitored thermal denaturation experiments, mass spectrometry analysis and gel shift assays. NMR experiments were used t...

  12. Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities

    Directory of Open Access Journals (Sweden)

    Reich Marlis


    Full Text Available Abstract Background In forest ecosystems, communities of ectomycorrhizal fungi (ECM are influenced by several biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera. Results We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products. Conclusion For the first time, we developed a custom phylochip that is specific for several ectomycorrhizal fungi. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot

  13. Reversing Antisense Oligonucleotide Activity with a Sense Oligonucleotide Antidote: Proof of Concept Targeting Prothrombin. (United States)

    Crosby, Jeff R; Zhao, Chenguang; Zhang, Hong; MacLeod, A Robert; Guo, Shuling; Monia, Brett P


    The tissue half-life of second-generation antisense oligonucleotide drugs (ASOs) is generally longer than traditional small molecule therapeutics. Thus, a strategy to reverse the activity of antisense drugs is warranted in certain settings. In this study, we describe a strategy employing the administration of a complementary sense oligonucleotide antidote (SOA). As a model system we have chosen to target the coagulation factor and antithrombotic drug target, prothrombin, to assess the feasibility of this approach. ASO targeting mouse prothrombin specifically suppressed >90% hepatic prothrombin mRNA levels and circulating prothrombin protein in mice. These effects were dose- and time-dependent, and as expected produced predictable increases in anticoagulation activity [prothrombin time/activated partial thromboplastin time (PT/aPTT)]. Treatment with prothrombin SOAs resulted in a dose-dependent reversal of ASO activity, as measured by a return in prothrombin mRNA levels and thrombin activity, and normalization of aPTT and PT. The antithrombotic activity of prothrombin ASOs was demonstrated in a FeCl3-induced thrombosis mouse model, and as predicted for this target, the doses required for antithrombotic activity were also associated with increased bleeding. Treatment with SOA was able to prevent prothrombin ASO-induced bleeding in a dose-dependent manner. These studies demonstrate for the first time the utility of SOAs to selectively and specifically reverse the intracellular effects of an antisense therapy.

  14. Aneuploidy screening by array comparative genomic hybridization improves success rates of in vitro fertilization: A multicenter Indian study

    Directory of Open Access Journals (Sweden)

    Aditi Kotdawala


    Full Text Available Objective: To evaluate the usefulness of preimplantation genetic screening (PGS using array comparative genomic hybridization (aCGH in the Indian population. Materials and Methods: This is a retrospective, multicenter study including 235 PGS cycles following intracytoplasmic sperm injection performed at six different infertility centers from September 2013 to June 2015. Patients were divided as per maternal age in several groups (40 years and as per indication for undergoing PGS. Indications for performing PGS were recurrent miscarriage, repetitive implantation failure, severe male factor, previous trisomic pregnancy, and advanced maternal age (≥35. Day 3 embryo biopsy was performed and analyzed by aCGH followed by day 5 embryo transfer in the same cycle or the following cycle. Outcomes such as pregnancy rates (PRs/transfer, implantation rates, miscarriage rates, percentage of abnormal embryos, and number of embryos with more than one aneuploidy and chaotic patterns were recorded for all the treated subjects based on different age and indication groups. Results: aCGH helped in identifying aneuploid embryos, thus leading to consistent implantation (range: 33.3%-42.9% and PRs per transfer (range: 31.8%-54.9% that were obtained for all the indications in all the age groups, after performing PGS. Conclusion: Aneuploidy is one of the major factors which affect embryo implantation. aCGH can be successfully employed for screening of aneuploid embryos. When euploid embryos are transferred, an increase in PRs can be achieved irrespective of the age or the indication.

  15. Antisense oligonucleotides for the treatment of dyslipidaemia. (United States)

    Visser, Maartje E; Witztum, Joseph L; Stroes, Erik S G; Kastelein, John J P


    Antisense oligonucleotides (ASOs) are short synthetic analogues of natural nucleic acids designed to specifically bind to a target messenger RNA (mRNA) by Watson-Crick hybridization, inducing selective degradation of the mRNA or prohibiting translation of the selected mRNA into protein. Antisense technology has the ability to inhibit unique targets with high specificity and can be used to inhibit synthesis of a wide range of proteins that could influence lipoprotein levels and other targets. A number of different classes of antisense agents are under development. To date, mipomersen, a 2'-O-methoxyethyl phosphorothioate 20-mer ASO, is the most advanced ASO in clinical development. It is a second-generation ASO developed to inhibit the synthesis of apolipoprotein B (apoB)-100 in the liver. In Phase 3 clinical trials, mipomersen has been shown to significantly reduce plasma low-density lipoprotein cholesterol (LDL-c) as well as other atherogenic apoB containing lipoproteins such as lipoprotein (a) [Lp(a)] and small-dense LDL particles. Although concerns have been raised because of an increase in intrahepatic triglyceride content, preliminary data from long-term studies suggest that with continued treatment, liver fat levels tend to stabilize or decline. Further studies are needed to evaluate potential clinical relevance of these changes. Proprotein convertase subtilisin/kexin-9 (PCSK9) is another promising novel target for lowering LDL-c by ASOs. Both second-generation ASOs and ASOs using locked nucleic acid technology have been developed to inhibit PCSK9 and are under clinical development. Other targets currently being addressed include apoC-III and apo(a) or Lp(a). By directly inhibiting the synthesis of specific proteins, ASO technology offers a promising new approach to influence the metabolism of lipids and to control lipoprotein levels. Its application to a wide variety of potential targets can be expected if these agents prove to be clinically safe and

  16. Biominetic High Density Lipoproteins for the Delivery of Therapeutic Oligonucleotides (United States)

    Tripathy, Sushant

    Advances in nanotechnology have brought about novel inorganic and hybrid nanoparticles with unique physico-chemical properties that make them suitable for a broad range of applications---from nano-circuitry to drug delivery. A significant part of those advancements have led to ground-breaking discoveries that have changed the approaches to formulation of therapeutics against diseases, such as cancer. Now-a-days the focus does not lie solely on finding a candidate small-molecule therapeutic with minimal adverse effects, but researchers are looking up to nanoparticles to improve biodistribution and biocompatibility profile of clinically proven therapeutics. The plethora of conjugation chemistries offered by currently extant inorganic nanoparticles have, in recent years, led to great leaps in the field of biomimicry---a modality that promises high biocompatibility. Further, in the pursuit of highly specific therapeutic molecules, researchers have turned to silencing oligonucleotides and some have already brought together the strengths of nanoparticles and silencing oligonucleotides in search of an efficacious therapy for cancer with minimal adverse effects. This dissertation work focuses on such a biomimetic platform---a gold nanoparticle based high density lipoprotein biomimetic (HDL NP), for the delivery of therapeutic oligonucleotides. The first chapter of this body of work introduces the molecular target of the silencing oligonucleotides---VEGFR2, and its role in the progression of solid tumor cancers. The background information also covers important aspects of natural high density lipoproteins (HDL), especially their innate capacity to bind and deliver exogenous and endogenous silencing oligonucleotides to tissues that express their high affinity receptor SRB1. We subsequently describe the synthesis of the biomimetic HDL NP and its oligonucleotide conjugates, and establish their biocompatibility. Further on, experimental data demonstrate the efficacy of silencing

  17. Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR). (United States)

    Wuyts, Véronique; Mattheus, Wesley; Roosens, Nancy H C; Marchal, Kathleen; Bertrand, Sophie; De Keersmaecker, Sigrid C J


    A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

  18. Copy Number Variation Analysis by Array Analysis of Single Cells Following Whole Genome Amplification. (United States)

    Dimitriadou, Eftychia; Zamani Esteki, Masoud; Vermeesch, Joris Robert


    Whole genome amplification is required to ensure the availability of sufficient material for copy number variation analysis of a genome deriving from an individual cell. Here, we describe the protocols we use for copy number variation analysis of non-fixed single cells by array-based approaches following single-cell isolation and whole genome amplification. We are focusing on two alternative protocols, an isothermal and a PCR-based whole genome amplification method, followed by either comparative genome hybridization (aCGH) or SNP array analysis, respectively.

  19. Characteristic archaebacterial 16S rRNA oligonucleotides (United States)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.


    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  20. Nano-topographic evaluation of highly disordered fractal-like structures of immobilized oligonucleotides using AFM

    Energy Technology Data Exchange (ETDEWEB)

    Sawant, P.D. [BioNanoEngineering Laboratory, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, Vic. 4122 (Australia)]. E-mail:; Nicolau, D.V. [BioNanoEngineering Laboratory, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, Vic. 4122 (Australia)


    In a recent trend of micro- and nano-array technologies, polymers are gaining preference over traditional substrates such as glass, silicates, etc. as a model biosurface to immobilize biomolecules. In present paper, we compared model polymeric surfaces such as cyclo olefin copolymer (COC) and polycarbonate (PC), with traditional surfaces such as silicone-wafer and mica. We used a 2D fractal dimension method, i.e. the perimeter-area relationship (PAR) to study the immobilization of 26 base pair oligonucleotide primer on surfaces which are imaged by AFM. Results revealed that the efficiency of the vertical immobilization is in the following order: COC > PC > mica > Si-wafer which can be contributed to chemical and nano-topographical heterogeneity. This study is useful for in-depth understanding of fundamental issues such as effects of manufacturing parameters and evaluation of surface nanotopographies for the development of high-density biochips.

  1. Improving oligonucleotide fingerprinting of rRNA genes by implementation of polony microarray technology (United States)

    Ruegger, Paul M.; Bent, Elizabeth; Li, Wei; Jeske, Daniel R.; Cui, Xinping; Braun, Jonathan; Jiang, Tao; Borneman, James


    Improvements to oligonucleotide fingerprinting of rRNA genes (OFRG) were obtained by implementing polony microarray technology. OFRG is an array-based method for analyzing microbial community composition. Polonies are discrete clusters of DNA, produced by solid-phase PCR in hydrogels, and derived from individual, spatially isolated DNA molecules. The advantages of a polony-based OFRG method include higher throughput and reductions in the PCR-induced errors and compositional skew inherent in all other PCR-based community composition methods, including high throughput sequencing of rRNA genes. Given the similarities between polony microarrays and certain aspects of sequencing methods such as the Illumina platform, we suggest that if concepts presented in this study were implemented in high throughput sequencing protocols, a reduction of PCR-induced errors and compositional skew may be realized. PMID:22640891

  2. Investigations of oligonucleotide usage variance within and between prokaryotes

    DEFF Research Database (Denmark)

    Bohlin, J.; Skjerve, E.; Ussery, David


    Oligonucleotide usage in archaeal and bacterial genomes can be linked to a number of properties, including codon usage (trinucleotides), DNA base-stacking energy (dinucleotides), and DNA structural conformation (di-to tetranucleotides). We wanted to assess the statistical information potential...... was that prokaryotic chromosomes can be described by hexanucleotide frequencies, suggesting that prokaryotic DNA is predominantly short range correlated, i. e., information in prokaryotic genomes is encoded in short oligonucleotides. Oligonucleotide usage varied more within AT-rich and host-associated genomes than...... in GC-rich and free-living genomes, and this variation was mainly located in non-coding regions. Bias (selectional pressure) in tetranucleotide usage correlated with GC content, and coding regions were more biased than non-coding regions. Non-coding regions were also found to be approximately 5.5% more...

  3. Delivery of RNAi-Based Oligonucleotides by Electropermeabilization

    Directory of Open Access Journals (Sweden)

    Muriel Golzio


    Full Text Available For more than a decade, understanding of RNA interference (RNAi has been a growing field of interest. The potent gene silencing ability that small oligonucleotides have offers new perspectives for cancer therapeutics. One of the present limits is that many biological barriers exist for their efficient delivery into target cells or tissues. Electropermeabilization (EP is one of the physical methods successfully used to transfer small oligonucleotides into cells or tissues. EP consists in the direct application of calibrated electric pulses to cells or tissues that transiently permeabilize the plasma membranes, allowing efficient in vitro and in vivo. cytoplasmic delivery of exogenous molecules. The present review reports on the type of therapeutic RNAi-based oligonucleotides that can be electrotransferred, the mechanism(s of their electrotransfer and the technical settings for pre-clinical purposes.

  4. Retro-1 Analogues Differentially Affect Oligonucleotide Delivery and Toxin Trafficking. (United States)

    Yang, Bing; Ming, Xin; Abdelkafi, Hajer; Pons, Valerie; Michau, Aurelien; Gillet, Daniel; Cintrat, Jean-Christophe; Barbier, Julien; Juliano, Rudy


    Retro-1 is a small molecule that displays two important biological activities: First, it blocks the actions of certain toxins by altering their intracellular trafficking. Second, it enhances the activity of oligonucleotides by releasing them from entrapment in endosomes. This raises the question of whether the two actions involve the same cellular target. Herein we report the effects of several Retro-1 analogues on both toxins and oligonucleotides. We found analogues that affect toxins but not oligonucleotides and vice-versa, while Retro-1 is the only compound that affects both. This indicates that the molecular target(s) involved in the two processes are distinct. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases

    Directory of Open Access Journals (Sweden)

    Wupeng Liao


    Full Text Available Inhaled oligonucleotide is an emerging therapeutic modality for various common respiratory diseases, including obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD and restrictive airway diseases like idiopathic pulmonary fibrosis (IPF. The advantage of direct accessibility for oligonucleotide molecules to the lung target sites, bypassing systemic administration, makes this therapeutic approach promising with minimized potential systemic side effects. Asthma, COPD, and IPF are common chronic respiratory diseases, characterized by persistent airway inflammation and dysregulated tissue repair and remodeling, although each individual disease has its unique etiology. Corticosteroids have been widely prescribed for the treatment of asthma, COPD, and IPF. However, the effectiveness of corticosteroids as an anti-inflammatory drug is limited by steroid resistance in severe asthma, the majority of COPD cases, and pulmonary fibrosis. There is an urgent medical need to develop target-specific drugs for the treatment of these respiratory conditions. Oligonucleotide therapies, including antisense oligonucleotide (ASO, small interfering RNA (siRNA, and microRNA (miRNA are now being evaluated both pre-clinically and clinically as potential therapeutics. The mechanisms of action of ASO and siRNA are highly target mRNA specific, ultimately leading to target protein knockdown. miRNA has both biomarker and therapeutic values, and its knockdown by a miRNA antagonist (antagomir has a broader but potentially more non-specific biological outcome. This review will compile the current findings of oligonucleotide therapeutic targets, verified in various respiratory disease models and in clinical trials, and evaluate different chemical modification approaches to improve the stability and potency of oligonucleotides for the treatment of respiratory diseases.

  6. Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases. (United States)

    Liao, Wupeng; Dong, Jinrui; Peh, Hong Yong; Tan, Lay Hong; Lim, Kah Suan; Li, Li; Wong, Wai-Shiu Fred


    Inhaled oligonucleotide is an emerging therapeutic modality for various common respiratory diseases, including obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD) and restrictive airway diseases like idiopathic pulmonary fibrosis (IPF). The advantage of direct accessibility for oligonucleotide molecules to the lung target sites, bypassing systemic administration, makes this therapeutic approach promising with minimized potential systemic side effects. Asthma, COPD, and IPF are common chronic respiratory diseases, characterized by persistent airway inflammation and dysregulated tissue repair and remodeling, although each individual disease has its unique etiology. Corticosteroids have been widely prescribed for the treatment of asthma, COPD, and IPF. However, the effectiveness of corticosteroids as an anti-inflammatory drug is limited by steroid resistance in severe asthma, the majority of COPD cases, and pulmonary fibrosis. There is an urgent medical need to develop target-specific drugs for the treatment of these respiratory conditions. Oligonucleotide therapies, including antisense oligonucleotide (ASO), small interfering RNA (siRNA), and microRNA (miRNA) are now being evaluated both pre-clinically and clinically as potential therapeutics. The mechanisms of action of ASO and siRNA are highly target mRNA specific, ultimately leading to target protein knockdown. miRNA has both biomarker and therapeutic values, and its knockdown by a miRNA antagonist (antagomir) has a broader but potentially more non-specific biological outcome. This review will compile the current findings of oligonucleotide therapeutic targets, verified in various respiratory disease models and in clinical trials, and evaluate different chemical modification approaches to improve the stability and potency of oligonucleotides for the treatment of respiratory diseases.

  7. A novel catechol-based universal support for oligonucleotide synthesis. (United States)

    Anderson, Keith M; Jaquinod, Laurent; Jensen, Michael A; Ngo, Nam; Davis, Ronald W


    A novel universal support for deoxyribo- and ribonucleic acid synthesis has been developed. The support, constructed from 1,4-dimethoxycatechol, represents an improvement over existing universal supports because of its ability to cleave and deprotect under mild conditions in standard reagents. Because no nonvolatile additives are required for cleavage and deprotection, the synthesized oligonucleotides do not require purification prior to use in biochemical assays. Using reverse phase HPLC and electrospray mass spectroscopy, it was determined that oligonucleotides synthesized on the universal support (UL1) 3'-dephosphorylate quickly (9 h in 28-30% ammonium hydroxide (NH4OH) at 55 degrees C, 2 h in 28-30% NH4OH at 80 degrees C, or <1 h in ammonium hydroxide/methylamine (1:1) (AMA) at 80 degrees C). Oligonucleotides used as primers for the polymerase chain reaction (PCR) assay were found to perform identically to control primers, demonstrating full biological compatibility. In addition, a method was developed for sintering the universal support directly into a filter plug which can be pressure fit into the synthesis column of a commercial synthesizer. The universal support plugs allow the synthesis of high-quality oligonucleotides at least 120 nucleotides in length, with purity comparable to non-universal commercial supports and approximately 50% lower reagent consumption. The universal support plugs are routinely used to synthesize deoxyribo-, ribo-, 3'-modified, 5'-modified, and thioated oligonucleotides. The flexibility of the universal support and the efficiency of 3'-dephosphorylation are expected to increase the use of universal supports in oligonucleotide synthesis.

  8. Lipid-modified G4-decoy oligonucleotide anchored to nanoparticles

    DEFF Research Database (Denmark)

    Cogoi, S; Jakobsen, U; Pedersen, E B


    KRAS is mutated in >90% of pancreatic ductal adenocarcinomas. As its inactivation leads to tumour regression, mutant KRAS is considered an attractive target for anticancer drugs. In this study we report a new delivery strategy for a G4-decoy oligonucleotide that sequesters MAZ, a transcription...... factor essential for KRAS transcription. It is based on the use of palmitoyl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with lipid-modified G4-decoy oligonucleotides and a lipid-modified cell penetrating TAT peptide. The potency of the strategy in pancreatic cancer cells is demonstrated...

  9. Inhibition of microRNA with antisense oligonucleotides. (United States)

    Esau, Christine C


    Antisense inhibition of microRNA (miRNA) function has been an important tool for uncovering miRNA biology. Chemical modification of anti-miRNA oligonucleotides (AMOs) is necessary to improve affinity for target miRNA, stabilize the AMO to nuclease degradation, and to promote tissue uptake for in vivo delivery. Here I summarize the work done to evaluate the effectiveness of various chemically modified AMOs for use in cultured cells and rodent models, and outline important issues to consider when inhibiting miRNAs with antisense oligonucleotides.

  10. Synthesis of Peptide-Oligonucleotide Conjugates Using a Heterobifunctional Crosslinker (United States)

    Williams, Berea A.R.; Chaput, John C.


    Peptide-oligonucleotide conjugates (POCs) are molecular chimeras composed of a nucleic acid moiety covalently attached to a polypeptide moiety. POCs have been used in numerous applications from therapeutics to nanotechnology, and most recently as combinatorial agents in the assembly of bivalent protein affinity reagents. This unit describes the synthesis and purification of POC molecules using the heterobifunctional crosslinking reagent succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), which enables amine-modified oligonucleotides to become covalently linked to cysteine-modified polypeptides. This solution-based protocol consists of a two-step synthesis followed by a single purification step. PMID:20827717

  11. Chemical phosphorylation of deoxyribonucleosides and thermolytic DNA oligonucleotides. (United States)

    Ausín, Cristina; Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L


    The phosphorylating reagent bis[S-(4,4'-dimethoxytrityl)-2-mercaptoethyl]-N,N-diisopropylphosphoramidite is prepared in three steps from commercial methyl thioglycolate and diisopropylphosphoramidous dichloride. The phosphorylating reagent has been used successfully in the solid-phase synthesis of deoxyribonucleoside 5'-/3'-phosphate or -thiophosphate monoesters and oligonucleotide 5'-phosphate/-thiophosphate monoesters. Bis[S-(4,4'-dimethoxytrityl)-2-mercaptoethyl]-N,N-diisopropylphosphoramidite has also been employed in the construction of a thermolytic dinucleotide prodrug model to evaluate the ability of the reagent to produce thermosentive oligonucleotide prodrugs under mild temperature conditions ( approximately 25 degrees C) for potential therapeutic applications.

  12. Versatile functionalization of nanoelectrodes by oligonucleotides via pyrrole electrochemistry. (United States)

    Descamps, Emeline; Nguyen, Khoa; Bouchain-Gautier, Christelle; Filoramo, Arianna; Goux-Capes, Laurence; Goffman, Marcello; Bourgoin, Jean-Philippe; Mailley, Pascal; Livache, Thierry


    Surface modification at the nanometer scale is a challenge for the future of molecular electronics. In particular, the precise anchoring and electrical addressing of biological scaffolds such as complex DNA nanonetworks is of importance for generating bio-directed assemblies of nano-objects for nanocircuit purposes. Herein, we consider the individual modification of nanoelectrodes with different oligonucleotide sequences by an electrochemically driven co-polymerization process of pyrrole and modified oligonucleotide sequences bearing pyrrole monomers. We demonstrate that this one-step technique presents the advantages of simplicity, localization of surface modification, mechanical, biological and chemical stability of the coatings, and high lateral resolution.

  13. Comparing temporally-focused GPC and CGH for two-photon excitation and optogenetics in turbid media

    DEFF Research Database (Denmark)

    Villangca, Mark Jayson; Bañas, Andrew Rafael; Aabo, Thomas


    Inherent inhomogeneity in turbid media not only hinders imaging but also projection of arbitrary light patterns for excitation or optical manipulation. In this work we compare two of the most popular phase modulation-based techniques in beam shaping. The Generalized Phase Contrast (GPC) method uses...... a 4f setup that directly converts phase information to intensity. The GPC method has been used with temporal focusing for excitation in two-photon optogenetics [1-3]. The computer generated hologram (CGH) is also used to generate arbitrary light patterns and has been used for optical manipulation...... and fabrication because of its high diffraction efficiency and axial confinement. We model the effect of the turbid media as a phase randomization process. We compare the quality and asses the degradation of the projected light pattern for both techniques as it propagates in the turbid media....

  14. “Distributed hybrid” MH–CGH2 system for hydrogen storage and its supply to LT PEMFC power modules

    Energy Technology Data Exchange (ETDEWEB)

    Lototskyy, M., E-mail: [HySA Systems Competence Centre, South African Institute for Advanced Materials Chemistry, Faculty of Natural Sciences, University of the Western Cape, Private Bag X17, Bellville 7535 (South Africa); Tolj, I.; Davids, M.W.; Bujlo, P. [HySA Systems Competence Centre, South African Institute for Advanced Materials Chemistry, Faculty of Natural Sciences, University of the Western Cape, Private Bag X17, Bellville 7535 (South Africa); Smith, F. [Impala Platinum Ltd, Springs (South Africa); Pollet, B.G. [HySA Systems Competence Centre, South African Institute for Advanced Materials Chemistry, Faculty of Natural Sciences, University of the Western Cape, Private Bag X17, Bellville 7535 (South Africa)


    Highlights: • Prototype hydrogen storage and supply system for LTPEMFC applications was developed. • Combination of MH and CGH2 tanks with common gas manifold was used. • Thermal coupling of fuel cell stack and MH tank was applied. • The system uses AB2-type MH; H2 equilibrium pressure ∼10 bar at room temperature. • Shorter H2 charge time and stable H2 supply at a fluctuating load were observed. - Abstract: This paper describes the layout and presents the results of the testing of a novel prototype “distributed hybrid” hydrogen storage and supply system that has the potential to be used for Low Temperature Proton Exchange Membrane Fuel Cell (LT-PEMFC) applications. The system consists of individual Metal Hydride (MH) and Compressed Gas (CGH2) tanks with common gas manifold, and a thermal management system where heat exchanger of the liquid heated-cooled MH tank is integrated with the cooling system of the LT-PEMFC BoP. The MH tank is filled with a medium-stability AB{sub 2}-type MH material (H{sub 2} equilibrium pressure of about 10 bar at room temperature). This innovative solution allows for (i) an increase in hydrogen storage capacity of the whole gas storage system and the reduction of H{sub 2} charge pressure; (ii) shorter charging times in the refuelling mode and smoother peaks of H{sub 2} consumption during its supply to the fuel cell stack; (iii) the use of standard parts with simple layout and lower costs; and (iv) adding flexibility in the layout and placement of the components of the hydrogen storage and supply system.

  15. Anti sense and sensibility : renal and skin effects of (antisense) oligonucleotides

    NARCIS (Netherlands)

    Meer, van L.


    This thesis describes the clinical investigation of a novel treatment strategy for type 2 diabetes mellitus (t2dm) using an antisense oligonucleotide(aon)to inhibit the sglt2 receptor. Furthermore it describes skin effects of oligonucleotides

  16. Delivery of antisense oligonucleotides using cholesterol-modified sense dendrimers and cationic lipids

    NARCIS (Netherlands)

    Chaltin, Patrick; Margineanu, Anca; Marchand, Damien; Aerschot, Arthur Van; Rozenski, Jef; Schryver, Frans De; Herrmann, Andreas; Müllen, Klaus; Juliano, Rudolph; Fisher, Michael H.; Kang, Hyunmin; Feyter, Steven De; Herdewijn, Piet


    Cholesterol modified mono-, di-, and tetrameric oligonucleotides were synthesized and hybridized with antisense oligonucleotides to study their incorporation in cationic liposomes together with the influence of this dendrimeric delivery system on biological activity. Electrostatic interactions seem

  17. The advantage of using SNP array in clinical testing for hematological malignancies--a comparative study of three genetic testing methods. (United States)

    Xu, Xinjie; Johnson, Eric B; Leverton, Lisa; Arthur, Ashley; Watson, Quinn; Chang, Faye L; Raca, Gordana; Laffin, Jennifer J


    Cytogenetic methods, including G-banded chromosome analysis and fluorescence in situ hybridization (FISH) analysis, serve as a critical part of routine clinical testing for hematological malignancies and provide important diagnostic and prognostic information; however, the limitations of cytogenetic methods, including the requirement for actively dividing cells and lower resolution of G-banded chromosome analysis as well as the inability of both G-banded chromosome analysis and FISH to detect copy number neutral loss of heterozygosity (CN-LOH), can result in a failure to detect genomic abnormalities with diagnostic and prognostic significance. Here, we compared the abnormality detection rate of clinically requested testing (i.e., G-banded chromosome analysis and FISH) with high-resolution oligo (i.e., array comparative genomic hybridization (aCGH)) and single-nucleotide polymorphism (SNP)/oligo hybrid (i.e., SNP-CGH) arrays in a series of patients, in an effort to assess the ability of newer technologies to overcome these limitations. This series found the detection rate for SNP-CGH to be 62.5% for myelodysplastic syndrome (MDS) cases and 72.7% for chronic lymphocytic leukemia (CLL) cases, which are significantly higher than the detection rates of aCGH (31.3% for MDS and 54.5% for CLL) and G-banding and/or FISH (43.8% for MDS and 54.5% for CLL). This demonstrates the advantages of combining SNP-CGH with conventional cytogenetics to provide comprehensive clinical information by detecting clonality, large balanced rearrangements, copy number aberrations, and CN-LOH.

  18. Array-based techniques for fingerprinting medicinal herbs

    Directory of Open Access Journals (Sweden)

    Xue Charlie


    Full Text Available Abstract Poor quality control of medicinal herbs has led to instances of toxicity, poisoning and even deaths. The fundamental step in quality control of herbal medicine is accurate identification of herbs. Array-based techniques have recently been adapted to authenticate or identify herbal plants. This article reviews the current array-based techniques, eg oligonucleotides microarrays, gene-based probe microarrays, Suppression Subtractive Hybridization (SSH-based arrays, Diversity Array Technology (DArT and Subtracted Diversity Array (SDA. We further compare these techniques according to important parameters such as markers, polymorphism rates, restriction enzymes and sample type. The applicability of the array-based methods for fingerprinting depends on the availability of genomics and genetics of the species to be fingerprinted. For the species with few genome sequence information but high polymorphism rates, SDA techniques are particularly recommended because they require less labour and lower material cost.

  19. Effects of fluid flow on the oligonucleotide folding in single-walled carbon nanotubes. (United States)

    Lim, M C G; Zhong, Z W


    This paper presents molecular-dynamics (MD) simulations of DNA oligonucleotide and water molecules translocating through carbon nanotube (CNT) channels. An induced pressure difference is applied to the system by pushing a layer of water molecules toward the flow direction to drive the oligonucleotide and other molecules. This MD simulation investigates the changes that occur in the conformation of the oligonucleotide due to water molecules in nanochannels while controlling the temperature and volume of the system in a canonical ensemble. The results show that the oligonucleotide in the (8,8)-(12,12) CNT channel forms a folded state at a lower pressure, whereas the oligonucleotide in the (10,10)-(14,14) CNT channel forms a folded state at a higher pressure instead. The van der Waals forces between the water molecules and the oligonucleotide suggest that the attraction between these two types of molecules results in the linear arrangements of the bases of the oligonucleotide. For a larger nanotube channel, the folding of the oligonucleotide is mainly dependent on the solvent (water molecules), whereas pressure, the size of the nanotube junction, and water molecules are the considering factors of the folding of the oligonucleotide at a smaller nanotube channel. For a folded oligonucleotide, the water distribution around the oligonucleotide is concentrated at a smaller range than that for the distribution around an unfolded oligonucleotide.

  20. Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex

    DEFF Research Database (Denmark)

    Lou, Chenguang; Christensen, Niels Johan; Martos Maldonado, Manuel Cristo


    by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex...

  1. Oligonucleotide-directed mutagenesis for precision gene editing. (United States)

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W


    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed.

  2. Differential oligonucleotide activity in cell culture versus mouse models. (United States)

    Wickstrom, E; Tyson, F L


    The usual course of drug discovery begins with the demonstration of compound activity in cells and, usually, a lower level of activity in animals. Successive rounds of drug design may result in a compound with sufficient activity in animals to justify clinical trials. The basic endpoints of therapeutic oligonucleotide experiments include target antigen reduction, target messenger reduction and inhibition of transformed cell proliferation or viral replication. However, one should expect oligonucleotides to exhibit pleiotropic behaviour, as do all other drugs. In an animal oligonucleotides will necessarily bind to and dissociate from all macromolecules encountered in the blood, in tissues, on cell surfaces and within cellular compartments. Contrary to expectations, oligonucleotides designed to be complementary to certain transcripts have sometimes been found moderately effective in cell-free extracts, more effective in cell culture and most effective in animal models. If greater potency against standard endpoints is reported in mouse models than was observed in cell culture, critical examination must consider alternate modes of action in animals that may not apply in cell culture. This counterintuitive paradox will be examined, based on studies of Ha-ras expression in bladder cancer, Ki-ras expression in pancreatic cancer, erbB2 expression in ovarian cancer and c-myc expression in B cell lymphoma.

  3. Chromosome-specific painting in Cucumis species using bulked oligonucleotides (United States)

    Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a sing...

  4. Splice-switching antisense oligonucleotides as therapeutic drugs

    National Research Council Canada - National Science Library

    Havens, Mallory A; Hastings, Michelle L


    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA...

  5. Antithrombotic effect of antisense factor XI oligonucleotide treatment in primates. (United States)

    Crosby, Jeffrey R; Marzec, Ulla; Revenko, Alexey S; Zhao, Chenguang; Gao, Dacao; Matafonov, Anton; Gailani, David; MacLeod, A Robert; Tucker, Erik I; Gruber, Andras; Hanson, Stephen R; Monia, Brett P


    During coagulation, factor IX (FIX) is activated by 2 distinct mechanisms mediated by the active proteases of either FVIIa or FXIa. Both coagulation factors may contribute to thrombosis; FXI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. We have reported that reducing FXI levels with FXI antisense oligonucleotides produces antithrombotic activity in mice, and that administration of FXI antisense oligonucleotides to primates decreases circulating FXI levels and activity in a dose-dependent and time-dependent manner. Here, we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present article, antisense oligonucleotides-mediated reduction of FXI plasma levels by ≥ 50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. These results indicate that reducing FXI levels using antisense oligonucleotides is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.

  6. Systematic design of mouse Vh gene family-specific oligonucleotides

    NARCIS (Netherlands)

    Seijen, AM; Seijen, HG; Bos, NA


    Kabat's database has often been used to design mouse Vh gene-specific 5 ' primers. The emphasis was mostly on constructing a universal (degenerate) 5 ' primer or 5 ' primer set, which would be able to match every mouse Vh gene. We were interested in finding oligonucleotides that could be used as pri

  7. Regioselective immobilization of short oligonucleotides to acrylic copolymer gels. (United States)

    Timofeev, E; Kochetkova, S V; Mirzabekov, A D; Florentiev, V L


    Four types of polyacrylamide or polydimethyl-acrylamide gels for regioselective (by immobilization at the 3' end) of short oligonucleotides have been designed for use in manufacturing oligonucleotide microchips. Two of these supports contain amino or aldehyde groups in the gel, allowing coupling with oligonucleotides bearing aldehyde or amino groups, respectively, in the presence of a reducing agent. The aldehyde gel support showed a higher immobilization efficiency relative to the amino gel. Of all reducing agents tested, the best results were obtained with a pyridine-borane complex. The other supports are based on an acrylamide gel activated with glutaraldehyde or a hydroxyalkyl-functionalized gel treated with mesyl chloride. The use of dimethylacrylamide instead of acrylamide allows subsequent gel modifications in organic solvents. All the immobilization methods are easy and simple to perform, give high and reproducible yields, allow long durations of storage of the activated support, and provide high stability of attachment and low non-specific binding. Although these gel supports have been developed for preparing oligonucleotide microchips, they may be used for other purposes as well. PMID:8774893

  8. LNA 5'-phosphoramidites for 5'→3'-oligonucleotide synthesis

    DEFF Research Database (Denmark)

    Madsen, Andreas Stahl; Kumar, Santhosh T.; Wengel, Jesper


    Hereby we report an efficient synthesis of LNA thymine and LNA 5-methylcytosine 5′-phosphoramidites, allowing incorporation of LNA thymine and LNA 5-methylcytosine into oligonucleotides synthesized in the 5′→3′ direction. Key steps include regioselective enzymatic benzoylation of the 5′-hydroxy...

  9. Systematic design of mouse Vh gene family-specific oligonucleotides

    NARCIS (Netherlands)

    Seijen, AM; Seijen, HG; Bos, NA


    Kabat's database has often been used to design mouse Vh gene-specific 5 ' primers. The emphasis was mostly on constructing a universal (degenerate) 5 ' primer or 5 ' primer set, which would be able to match every mouse Vh gene. We were interested in finding oligonucleotides that could be used as pri

  10. A strategy for detection of known and unknown SNP using a minimum number of oligonucleotides applicable in the clinical settings

    Directory of Open Access Journals (Sweden)

    Klein Harvey


    Full Text Available Abstract Detection of unknown single nucleotide polymorphism (SNP relies on large scale sequencing expeditions of genomic fragments or complex high-throughput chip technology. We describe a simplified strategy for fluorimetric detection of known and unknown SNP by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes. The array consists of two sets of 18-mer oligonucleotide probes. One set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected "consensus" sequence (consensus-oligos, the other includes oligos specific for known SNP within the same genomic region (variant-oligos. Fluorescence-labeled DNA amplified from a homozygous source identical to the consensus represents the reference target and is co-hybridized with a differentially-labeled test sample. Lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant-oligos designates a known allele. Lack of hybridization to consensus- and variant-oligos indicates a new allele. Detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal. This method can identify unknown SNP in heterozygous conditions with a sensitivity of 82% and specificity of 90%. This strategy should dramatically increase the efficiency of SNP detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials.

  11. Analphoid supernumerary marker chromosome characterized by aCGH and FISH as inv dup(3(q25.33qter de novo in a child with dysmorphic features and streaky pigmentation: case report

    Directory of Open Access Journals (Sweden)

    Pramathan R


    Full Text Available Abstract Background Small supernumerary marker chromosomes (sSMC occur in 0.075% of unselected prenatal and in 0.044% of consecutively studied postnatal cases. Individuals with sSMC present with varying phenotype, ranging from normal to extremely mild or severe depending on the chromosomal region involved, the euchromatic content present and degree of mosaicism. Except for chromosomes 15 and 22, the number of reported cases of sSMC is extremely small to provide us with a good genotype-phenotype correlation. Analphoid sSMC are even rarer. To our knowledge only eight cases of analphoid inversion-duplication 3q sSMC are reported so far. Results We describe here a one month old female child with several dysmorphic features and with a de novo analphoid supernumerary marker chromosome only in cultured skin fibroblast cells and not in lymphocytes. The marker was characterized as analphoid inversion-duplication 3q25.33-qter by oligo array comparative genomic hybridization (aCGH and fluorescence in situ hybridization (FISH studies. The final skin fibroblast karyotype was interpreted as 47,XX,+der(3.ish inv dup(3(qter-q25.33::q25.33-qter(subtel 3q+,subtel 3q+ de novo. Conclusion In addition to the eight reported cases of analphoid inversion-duplication 3q supernumerary marker in the literature, this is yet another case of 3q sSMC with a new breakpoint at 3q25.33 and with varying phenotype as described in the case report. Identification of more and more similar cases of analphoid inversion-duplication 3q marker will help in establishing a better genotype-phenotype correlation. The study further demonstrates that aCGH in conjunction with routine cytogenetics and FISH is very useful in precisely identifying and characterizing a marker chromosome, and more importantly help in providing with an accurate genetic diagnosis and better counseling to the family.

  12. Glycoclusters on oligonucleotide and PNA scaffolds: synthesis and applications. (United States)

    Spinelli, Nicolas; Defrancq, Eric; Morvan, François


    Conjugation of oligonucleotides (ONs) to a variety of reporter groups has been the subject of intensive research during the last decade. Conjugation is indeed of great interest because it can be used not only to improve the existing ONs properties but also to impart new ones. In this context tremendous efforts have been made to conjugate carbohydrate moieties to ONs. Indeed carbohydrates play an important role in biological processes such as signal transduction and cell adhesion through the recognition with sugar-binding proteins (i.e. lectins) located on the surface of cells. For this reason, carbohydrate-oligonucleotide conjugates (COCs) have been first developed for improving the poor cellular uptake or tissue specific delivery of ONs through receptor-mediated endocytosis. Besides the targeted ONs delivery, carbohydrate-oligonucleotide conjugates (COCs) are also evaluated in the context of carbohydrate biochips in which surface coating with carbohydrates is achieved by using the DNA-directed immobilization strategy (DDI). Peptide nucleic acids (PNAs) have also been extensively investigated as a surrogate of DNA for diverse applications. Therefore attachment of carbohydrate moieties to this class of molecules has been studied. The aforementioned applications of COCs require mimicking of the natural processes, in which the weak individual protein-carbohydrate binding is overcome by using multivalent interactions. This tutorial review focuses on the recent advances in carbohydrate-oligonucleotide conjugates and describes the major synthetic approaches available. In addition, an overview of applications that have been developed using various scaffolds allowing multivalent interactions is provided. Finally recent results on the use of peptide nucleic acids as oligonucleotides surrogate are described.

  13. Array comparative genomic hybridization-based characterization of genetic alterations in pulmonary neuroendocrine tumors. (United States)

    Voortman, Johannes; Lee, Jih-Hsiang; Killian, Jonathan Keith; Suuriniemi, Miia; Wang, Yonghong; Lucchi, Marco; Smith, William I; Meltzer, Paul; Wang, Yisong; Giaccone, Giuseppe


    The goal of this study was to characterize and classify pulmonary neuroendocrine tumors based on array comparative genomic hybridization (aCGH). Using aCGH, we performed karyotype analysis of 33 small cell lung cancer (SCLC) tumors, 13 SCLC cell lines, 19 bronchial carcinoids, and 9 gastrointestinal carcinoids. In contrast to the relatively conserved karyotypes of carcinoid tumors, the karyotypes of SCLC tumors and cell lines were highly aberrant. High copy number (CN) gains were detected in SCLC tumors and cell lines in cytogenetic bands encoding JAK2, FGFR1, and MYC family members. In some of those samples, the CN of these genes exceeded 100, suggesting that they could represent driver alterations and potential drug targets in subgroups of SCLC patients. In SCLC tumors, as well as bronchial carcinoids and carcinoids of gastrointestinal origin, recurrent CN alterations were observed in 203 genes, including the RB1 gene and 59 microRNAs of which 51 locate in the DLK1-DIO3 domain. These findings suggest the existence of partially shared CN alterations in these tumor types. In contrast, CN alterations of the TP53 gene and the MYC family members were predominantly observed in SCLC. Furthermore, we demonstrated that the aCGH profile of SCLC cell lines highly resembles that of clinical SCLC specimens. Finally, by analyzing potential drug targets, we provide a genomics-based rationale for targeting the AKT-mTOR and apoptosis pathways in SCLC.

  14. Array comparative genomic hybridization analysis of Trichoderma reesei strains with enhanced cellulase production properties

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    Penttilä Merja


    Full Text Available Abstract Background Trichoderma reesei is the main industrial producer of cellulases and hemicellulases that are used to depolymerize biomass in a variety of biotechnical applications. Many of the production strains currently in use have been generated by classical mutagenesis. In this study we characterized genomic alterations in high-producing mutants of T. reesei by high-resolution array comparative genomic hybridization (aCGH. Our aim was to obtain genome-wide information which could be utilized for better understanding of the mechanisms underlying efficient cellulase production, and would enable targeted genetic engineering for improved production of proteins in general. Results We carried out an aCGH analysis of four high-producing strains (QM9123, QM9414, NG14 and Rut-C30 using the natural isolate QM6a as a reference. In QM9123 and QM9414 we detected a total of 44 previously undocumented mutation sites including deletions, chromosomal translocation breakpoints and single nucleotide mutations. In NG14 and Rut-C30 we detected 126 mutations of which 17 were new mutations not documented previously. Among these new mutations are the first chromosomal translocation breakpoints identified in NG14 and Rut-C30. We studied the effects of two deletions identified in Rut-C30 (a deletion of 85 kb in the scaffold 15 and a deletion in a gene encoding a transcription factor on cellulase production by constructing knock-out strains in the QM6a background. Neither the 85 kb deletion nor the deletion of the transcription factor affected cellulase production. Conclusions aCGH analysis identified dozens of mutations in each strain analyzed. The resolution was at the level of single nucleotide mutation. High-density aCGH is a powerful tool for genome-wide analysis of organisms with small genomes e.g. fungi, especially in studies where a large set of interesting strains is analyzed.

  15. Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides (United States)

    Kolganova, N. A.; Shchyolkina, A. K.; Chudinov, A. V.; Zasedatelev, A. S.; Florentiev, V. L.; Timofeev, E. N.


    Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine. PMID:22641847

  16. Hepatotoxic Potential of Therapeutic Oligonucleotides Can Be Predicted from Their Sequence and Modification Pattern (United States)

    Hagedorn, Peter H.; Yakimov, Victor; Ottosen, Søren; Kammler, Susanne; Nielsen, Niels F.; Høg, Anja M.; Hedtjärn, Maj; Meldgaard, Michael; Møller, Marianne R.; Ørum, Henrik; Koch, Troels


    Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines. PMID:23952551

  17. High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides (United States)

    Yang, B.; Ming, X.; Cao, C.; Laing, B.; Yuan, A.; Porter, M. A.; Hull-Ryde, E. A.; Maddry, J.; Suto, M.; Janzen, W. P.; Juliano, R. L.


    The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics. PMID:25662226

  18. Biophysical and RNA Interference Inhibitory Properties of Oligonucleotides Carrying Tetrathiafulvalene Groups at Terminal Positions

    Directory of Open Access Journals (Sweden)

    Sónia Pérez-Rentero


    Full Text Available Oligonucleotide conjugates carrying a single functionalized tetrathiafulvalene (TTF unit linked through a threoninol molecule to the 3′ or 5′ ends were synthesized together with their complementary oligonucleotides carrying a TTF, pyrene, or pentafluorophenyl group. TTF-oligonucleotide conjugates formed duplexes with higher thermal stability than the corresponding unmodified oligonucleotides and pyrene- and pentafluorophenyl-modified oligonucleotides. TTF-modified oligonucleotides are able to bind to citrate-stabilized gold nanoparticles (AuNPs and produce stable gold AuNPs functionalized with oligonucleotides. Finally, TTF-oligoribonucleotides have been synthesized to produce siRNA duplexes carrying TTF units. The presence of the TTF molecule is compatible with the RNA interference mechanism for gene inhibition.

  19. A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp. (United States)

    Cao, Boyang; Liu, Xiangqian; Yu, Xiang; Chen, Min; Feng, Lu; Wang, Lei


    Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.

  20. Functionalization of magnetic gold/iron-oxide composite nanoparticles with oligonucleotides and magnetic separation of specific target

    Energy Technology Data Exchange (ETDEWEB)

    Kinoshita, Takuya [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)]. E-mail:; Seino, Satoshi [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Mizukoshi, Yoshiteru [Faculty of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan); Nakagawa, Takashi [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Yamamoto, Takao A. [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)


    Magnetic composite nanoparticles of gold and iron-oxide synthesized with gamma-rays or ultrasonics were functionalized with thiol-modified oligonucleotides. The amount of oligonucleotides bound to the functionalized nanoparticle probes via hybridization was quantified with fluorescently-labeled target oligonucleotides. Our composite nanoparticles magnetically separated the specific target oligonucleotides without the non-specific adsorption.

  1. aCGH Analysis to Estimate Genetic Variations among Domesticated Chickens

    Directory of Open Access Journals (Sweden)

    Tomoyoshi Komiyama


    Full Text Available Chickens have been familiar to humans since ancient times and have been used not only for culinary purposes but also for cultural purposes including ritual ceremonies and traditional entertainment. The various chicken breeds developed for these purposes often display distinct morphological and/or behavioural traits. For example, the Japanese Shamo is larger and more aggressive than other domesticated chickens, reflecting its role as a fighting cock breed, whereas Japanese Naganakidori breeds, which have long-crowing behaviour, were bred instead for their entertaining and aesthetic qualities. However, the genetic backgrounds of these distinct morphological and behavioural traits remain unclear. Therefore, the question arises as to which genomic regions in these chickens were acted upon by selective pressures through breeding. We compared the entire genomes of six chicken breeds domesticated for various cultural purposes by utilizing array comparative genomic hybridization. From these analyses, we identified 782 regions that underwent insertions, deletions, or mutations, representing man-made selection pressure in these chickens. Furthermore, we found that a number of genes diversified in domesticated chickens bred for cultural or entertainment purposes were different from those diversified in chickens bred for food, such as broilers and layers.

  2. Application of array-based comparative genomic hybridization in primary amenorrhea women%基于微阵列芯片的比较基因组杂交技术在原发性闭经患者中的应用

    Institute of Scientific and Technical Information of China (English)

    冯琼; 符芳; 廖灿; 杨昕; 张亮; 田峰; 蔡斌; 刘帅


    目的 采用array-CGH技术对原发性闭经患者进行检测,探讨原发性闭经的分子生物学机制.方法 选取原发性闭经患者10例,另外选择10名具有规则月经周期的同龄女性自愿者作为健康对照者.分别采用常规细胞核型分析技术及array-CGH技术对10例原发性闭经患者和健康对照者进行分析.细胞核型分析技术采用常规G显带染色体核型分析技术,array-CGH技术采用美国Affymetrix公司Cytogenetic 2.7M微阵列芯片技术.结果 10例原发性闭经病例经常规G显带染色体核型分析未发现异常,所有病例和健康对照标本均为正常女性染色体核型:46,XX.Array-CGH 分析结果显示,有5例原发性闭经患者的X染色体短臂末端发生了约110000 bp的细小缺失,定位到染色体上的位置为:46,X,del(X)(p22.33).所有健康对照者经array-CGH分析均未见异常的DNA拷贝数改变.结论 Array-CGH技术在DNA水平上提高了染色体病的诊断水平,对于常规方法不能明确诊断的原发性闭经患者,有必要应用array-CGH技术进行更高分辨率的遗传学分析.%Objective To explore the molecular mechanisms of primary amenorrhea by using arrayCGH technology. Methods Ten patients with primary amenorrhea and 10 female volunteers with regular menstrual cycles as healthy controls were selected. All patients and control samples were analyzed by conventional chromosome analysis (G-banding technology) and array-CGH technology, respectively. ArrayCGH was performed using Affymetrix Cytogenetic 2. 7M arrays following the manufacturer's standard protocol. Results Both the patient group and control group analyzed by conventional G-banding karyotype technology showed a negative result with a normal female karyotype: 46, XX. The result of array-CGH analysis demonstrated a microdeletion of approximately 110 000 bp located at the end of the short arm of X chromosome [46, X, del (X) (p22. 33 )] were identified in 5 patients, which was

  3. Fluorescence quenching of TMR by guanosine in oligonucleotides

    Institute of Scientific and Technical Information of China (English)


    Nucleotide-specific fluorescence quenching in fluorescently labeled DNA has many applications in biotechnology. We have studied the inter-and intra-molecular quenching of tetramethylrhodamine (TMR) by nucleotides to better understand their quenching mechanism and influencing factors. In agreement with previous work, dGMP can effectively quench TMR, while the quenching of TMR by other nucleotides is negligible. The Stern-Volmer plot between TMR and dGMP delivers a bimolecular quenching constant of Ks=52.3 M-1. The fluorescence of TMR in labeled oligonucleotides decreases efficiently through photoinduced electron transfer by guanosine. The quenching rate constant between TMR and guanosine was measured using fluorescence correlation spectroscopy (FCS). In addition, our data show that the steric hindrance by bases around guanosine has significant effect on the G-quenching. The availability of these data should be useful in designing fluorescent oligonucleotides and understanding the G-quenching process.

  4. Electrochemical uranyl cation biosensor with DNA oligonucleotides as receptor layer. (United States)

    Jarczewska, Marta; Ziółkowski, Robert; Górski, Łukasz; Malinowska, Elżbieta


    The present study aims at the further development of the uranyl oligonucleotide-based voltammetric biosensor, which takes advantage of strong interaction between UO2(2+) and phosphate DNA backbone. Herein we report the optimization of working parameters of previously elaborated electrochemical DNA biosensor. It is shown that the sensor sensitivity is highly dependent on the oligonucleotide probe length and the incubation time of sensor in a sample solution. Consequently, the highest sensitivity was obtained for 10-nucleotide sequence and 60 min incubation time. The lower detection limit towards uranyl cation for developed biosensor was 30 nM. The influence of mixed monolayers and the possibility of developing a non-calibration device were also investigated. The selectivity of the proposed biosensor was significantly improved via elimination of adenine nucleobases from the DNA probe. Moreover, the regeneration procedure was elaborated and tested to prolong the use of the same biosensor for 4 subsequent determinations of UO2(2+).

  5. Palladium-Catalyzed Modification of Unprotected Nucleosides, Nucleotides, and Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Kevin H. Shaughnessy


    Full Text Available Synthetic modification of nucleoside structures provides access to molecules of interest as pharmaceuticals, biochemical probes, and models to study diseases. Covalent modification of the purine and pyrimidine bases is an important strategy for the synthesis of these adducts. Palladium-catalyzed cross-coupling is a powerful method to attach groups to the base heterocycles through the formation of new carbon-carbon and carbon-heteroatom bonds. In this review, approaches to palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides are reviewed. Polar reaction media, such as water or polar aprotic solvents, allow reactions to be performed directly on the hydrophilic nucleosides and nucleotides without the need to use protecting groups. Homogeneous aqueous-phase coupling reactions catalyzed by palladium complexes of water-soluble ligands provide a general approach to the synthesis of modified nucleosides, nucleotides, and oligonucleotides.

  6. One-oligonucleotide method for constructing vectors for RNA interference

    Institute of Scientific and Technical Information of China (English)



    Aim: To develop an easy, fast, automated, and inexpensive method for constructing short-hairpin-RNA cassettes for RNAi studies. Methods: Using single oligonucleotides, a variety of DNA cassettes for RNAi vectors were constructed in only few minutes in an automated manner. The cassettes, targeting the eGFP,were cloned into plasmids driven by RNA polymerase Ⅲ promoter H 1. Then, the plasmids were transfected into HeLa cells that were later infected with a recombinant adenovirus encoding the eGFP gene. The level of eGFP fluorescence was evaluated by confocal imaging and flow cytometry. Results: The plasmids constructed with the DNA cassettes made by the one-oligonucleotide method inhibited eGFP with different potencies, ranging from 55% to 75%. Conclusion: By using the method reported here, it is possible to simultaneously construct hundreds of different DNA cassettes for RNAi experiments in an inexpensive, automated way. This method will facilitate functional genomics studies on mammalian cells.

  7. Inhibition of HTLV-III by exogenous oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Goodchild, J.; Zamecnik, P.C.


    A method is described of detecting the presence of HTLV-III virus in a sample by demonstrating inhibition of replication of the virus in cells which are normally killed by the HTLV-III virus after the cells have been (a) combined with the sample and an oligonucleotide complementary to at least one highly conserved region of the HTLV-III genome necessary for HTLV-III replication and capable of hybridizing with at least the highly conserved region, the highly conserved region of the HTLV-III genome being a nucleotide sequence present in the genomes of HTLV-III isolates and the oligonucleotide complementary to at least one highly conserved region of the HTLV-III genome necessary for HTLV-III replication being complementary to a region of the HTLV-III genome.

  8. Solid-phase synthesis of siRNA oligonucleotides. (United States)

    Beaucage, Serge L


    Since the discovery of RNA interference (RNAi) as a means to silence the expression of specific genes, small interfering RNA (siRNA) oligonucleotides have been recognized as powerful tools for targeting therapeutically important mRNAs and eliciting their destruction. This discovery has created a high demand for synthetic oligoribonucleotides as potential therapeutics and has spurred a renaissance in the development of rapid, efficient methods for solid-phase RNA synthesis. The design and implementation of 2'-hydroxyl protecting groups that provide ribonucleoside phosphoramidites with coupling kinetics and coupling efficiencies comparable to those of deoxyribonucleoside phosphoramidites are key to the production of RNA oligonucleotides in sufficient quantity and purity for pharmaceutical applications. In this context, various siRNAs were chemically modified to identify the biophysical and biochemical parameters necessary for effective and stable RNAi-mediated gene-silencing activities.

  9. PAB-1, a Caenorhabditis elegans poly(A-binding protein, regulates mRNA metabolism in germline by interacting with CGH-1 and CAR-1.

    Directory of Open Access Journals (Sweden)

    Sunhee Ko

    Full Text Available Poly(A-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.

  10. PAB-1, a Caenorhabditis elegans poly(A)-binding protein, regulates mRNA metabolism in germline by interacting with CGH-1 and CAR-1. (United States)

    Ko, Sunhee; Kawasaki, Ichiro; Shim, Yhong-Hee


    Poly(A)-binding proteins are highly conserved among eukaryotes and regulate stability of mRNA and translation. Among C. elegans homologues, pab-1 mutants showed defects in germline mitotic proliferation. Unlike pab-1 mutants, pab-1 RNAi at every larval stage caused arrest of germline development at the following stage, indicating that pab-1 is required for the entire postembryonic germline development. This idea is supported by the observations that the mRNA level of pab-1 increased throughout postembryonic development and its protein expression was germline-enriched. PAB-1 localized to P granules and the cytoplasm in the germline. PAB-1 colocalized with CGH-1 and CAR-1 and affected their localization, suggesting that PAB-1 is a component of processing (P)-bodies that interacts with them. The mRNA and protein levels of representative germline genes, rec-8, GLP-1, rme-2, and msp-152, were decreased after pab-1 RNAi. Although the mRNA level of msp-152 was increased in cgh-1 mutant, it was also significantly reduced by pab-1 RNAi. Our results suggest that PAB-1 positively regulates the mRNA levels of germline genes, which is likely facilitated by the interaction of PAB-1 with other P-body components, CGH-1 and CAR-1.

  11. High-Resolution Analysis of Gene Copy Number Alterations in Human Prostate Cancer Using CGH on cDNA Microarrays: Impact of Copy Number on Gene Expression

    Directory of Open Access Journals (Sweden)

    Maija Wolf


    Full Text Available Identification of target genes for genetic rearrangements in prostate cancer and the impact of copy number changes on gene expression are currently not well understood. Here, we applied high-resolution comparative genomic hybridization (CGH on cDNA microarrays for analysis of prostate cancer cell lines. CGH microarrays identified most of the alterations detected by classical chromosomal CGH, as well as a number of previously unreported alterations. Specific recurrent regions of gain (28 and loss (18 were found, their boundaries defined with sub-megabasepair accuracy. The most common changes included copy number decreases at 13% and gains at iq and 5p. Refined mapping identified several sites, such as at 13q (33-44, 49-51, 74-76 Mbp from the p-telomere, which matched with minimal regions of loss seen in extensive loss of heterozygosity mapping studies of large numbers of tumors. Previously unreported recurrent changes were found at 2p, 2q, 3p, 17q (losses, at 3q, 5p, 6p (gains. Integration of genomic and transcriptomic data revealed the role of individual candidate target genes for genomic alterations as well as a highly significant (P < .0001 overall association between copy number levels and the percentage of differentially expressed genes. Across the genome, the overall impact of copy number on gene expression levels was, to a large extent, attributable to low-level gains and losses of copy number, corresponding to common deletions and gains of often large chromosomal regions.

  12. Thermoplastic polymers surfaces for Dip-Pen Nanolithography of oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Suriano, Raffaella [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy); Biella, Serena, E-mail: [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy); Cesura, Federico; Levi, Marinella; Turri, Stefano [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy)


    Different thermoplastic polymers were spin-coated to prepare smooth surfaces for the direct deposition of end-group modified oligonucleotides by Dip-Pen Nanolithography. A study of the diffusion process was done in order to investigate the dependence of calibration coefficient and quality of deposited features on environmental parameters (temperature, relative humidity) and ink's molecular weight and functionality. The optimization of the process parameters led to the realization of high quality and density nanoarrays on plastics.

  13. Cardiovascular and Metabolic Effects of ANGPTL3 Antisense Oligonucleotides. (United States)

    Graham, Mark J; Lee, Richard G; Brandt, Teresa A; Tai, Li-Jung; Fu, Wuxia; Peralta, Raechel; Yu, Rosie; Hurh, Eunju; Paz, Erika; McEvoy, Bradley W; Baker, Brenda F; Pham, Nguyen C; Digenio, Andres; Hughes, Steven G; Geary, Richard S; Witztum, Joseph L; Crooke, Rosanne M; Tsimikas, Sotirios


    Epidemiologic and genomewide association studies have linked loss-of-function variants in ANGPTL3, encoding angiopoietin-like 3, with low levels of plasma lipoproteins. We evaluated antisense oligonucleotides (ASOs) targeting Angptl3 messenger RNA (mRNA) for effects on plasma lipid levels, triglyceride clearance, liver triglyceride content, insulin sensitivity, and atherosclerosis in mice. Subsequently, 44 human participants (with triglyceride levels of either 90 to 150 mg per deciliter [1.0 to 1.7 mmol per liter] or >150 mg per deciliter, depending on the dose group) were randomly assigned to receive subcutaneous injections of placebo or an antisense oligonucleotide targeting ANGPTL3 mRNA in a single dose (20, 40, or 80 mg) or multiple doses (10, 20, 40, or 60 mg per week for 6 weeks). The main end points were safety, side-effect profile, pharmacokinetic and pharmacodynamic measures, and changes in levels of lipids and lipoproteins. The treated mice had dose-dependent reductions in levels of hepatic Angptl3 mRNA, Angptl3 protein, triglycerides, and low-density lipoprotein (LDL) cholesterol, as well as reductions in liver triglyceride content and atherosclerosis progression and increases in insulin sensitivity. After 6 weeks of treatment, persons in the multiple-dose groups had reductions in levels of ANGPTL3 protein (reductions of 46.6 to 84.5% from baseline, Pantisense oligonucleotide and three who received placebo reported dizziness or headache. There were no serious adverse events. Oligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in mice. Use of the same strategy to target human ANGPTL3 reduced levels of atherogenic lipoproteins in humans. (Funded by Ionis Pharmaceuticals; number, NCT02709850 .).

  14. Voltammetric behaviour of oligonucleotide lipoplexes adsorbed onto glassy carbon electrodes


    Piedade, J. A. P.; M. Mano; Lima, M. C. Pedroso de; Oretskaya, T S; Oliveira-Brett, A. M.


    The voltammetric behaviour of oligonucleotide lipoplexes (ODN-lipoplexes) prepared from short oligodeoxynucleotides (ODN), with different base compositions, and liposomes of the cationic lipid DOTAP, was studied by differential pulse voltammetry with a glassy carbon mini-electrode. It was found that the ODN base composition influences the ODN-lipoplex voltammetric response. Differential pulse voltammograms for ODN-lipoplexes of the ODN adenosine nucleotides present two different features when...

  15. Thermodynamic treatment of oligonucleotide duplex–simplex equilibria (United States)

    Owczarzy, Richard; Dunietz, Isard; Behlke, Mark A.; Klotz, Irving M.; Walder, Joseph A.


    Thermodynamic formulations have been devised to obtain ΔG° values directly from spectroscopic data at a fixed common temperature in nucleic acid duplex–simplex melting curves. In addition, the dependence of melting on salt concentration has been expressed in terms of a stepwise stoichiometric representation, which leads to a specific equation for the partition of the added sodium ions between the different oligonucleotide forms. PMID:14657395

  16. Anti-tumor activity of splice-switching oligonucleotides


    Bauman, John A; Li, Shyh-Dar; Yang, Angela; Huang, Leaf; Kole, Ryszard


    Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. T...

  17. Triplex-forming oligonucleotide target sequences in the human genome


    Goñi, J Ramon; de la Cruz, Xavier; Orozco, Modesto


    The existence of sequences in the human genome which can be a target for triplex formation, and accordingly are candidates for anti-gene therapies, has been studied by using bioinformatics tools. It was found that the population of triplex-forming oligonucleotide target sequences (TTS) is much more abundant than that expected from simple random models. The population of TTS is large in all the genome, without major differences between chromosomes. A wide analysis along annotated regions of th...

  18. Differentiation of regions with atypical oligonucleotide composition in bacterial genomes

    Directory of Open Access Journals (Sweden)

    Reva Oleg N


    Full Text Available Abstract Background Complete sequencing of bacterial genomes has become a common technique of present day microbiology. Thereafter, data mining in the complete sequence is an essential step. New in silico methods are needed that rapidly identify the major features of genome organization and facilitate the prediction of the functional class of ORFs. We tested the usefulness of local oligonucleotide usage (OU patterns to recognize and differentiate types of atypical oligonucleotide composition in DNA sequences of bacterial genomes. Results A total of 163 bacterial genomes of eubacteria and archaea published in the NCBI database were analyzed. Local OU patterns exhibit substantial intrachromosomal variation in bacteria. Loci with alternative OU patterns were parts of horizontally acquired gene islands or ancient regions such as genes for ribosomal proteins and RNAs. OU statistical parameters, such as local pattern deviation (D, pattern skew (PS and OU variance (OUV enabled the detection and visualization of gene islands of different functional classes. Conclusion A set of approaches has been designed for the statistical analysis of nucleotide sequences of bacterial genomes. These methods are useful for the visualization and differentiation of regions with atypical oligonucleotide composition prior to or accompanying gene annotation.

  19. G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents. (United States)

    Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela


    Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding processes to external stimuli, high structural compactness and chemo-enzymatic and thermodynamic stability. A number of G-quadruplex-forming oligonucleotides having relevant in vitro anti-HIV activity have been discovered in the last two decades through either SELEX or rational design approaches. Improved aptamers have been obtained by chemical modifications of natural oligonucleotides, as terminal conjugations with large hydrophobic groups, replacement of phosphodiester linkages with phosphorothioate bonds or other surrogates, insertion of base-modified monomers, etc. In turn, detailed structural studies have elucidated the peculiar architectures adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. An overview of the state-of-the-art knowledge of the relevance of putative G-quadruplex forming sequences within the viral genome and of the most studied G-quadruplex-forming aptamers, selectively targeting HIV proteins, is here presented.

  20. Recursive construction of perfect DNA molecules from imperfect oligonucleotides. (United States)

    Linshiz, Gregory; Yehezkel, Tuval Ben; Kaplan, Shai; Gronau, Ilan; Ravid, Sivan; Adar, Rivka; Shapiro, Ehud


    Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error-prone oligonucleotides are recursively combined in vitro, forming error-prone DNA molecules; error-free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms.

  1. Therapeutic Antisense Oligonucleotides against Cancer: Hurdling to the Clinic (United States)

    Moreno, Pedro; Pêgo, Ana


    Under clinical development since the early 90’s and with two successfully approved drugs (Fomivirsen and Mipomersen), oligonucleotide-based therapeutics have not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given towards a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field.


    Directory of Open Access Journals (Sweden)

    Pedro Miguel Duarte Moreno


    Full Text Available Under clinical development since the early 90’s and with two successfully approved drugs (Fomivirsen and Mipomersen, oligonucleotide-based therapeutics have not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given towards a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field.

  3. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing (United States)

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P.; Low, Audrey; Yoshida, Masayuki; Bennett, C. Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori


    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  4. Targeting several CAG expansion diseases by a single antisense oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Melvin M Evers

    Full Text Available To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2'-O-methyl phosphorothioate (CUGn triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG(7, also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well.

  5. Characterization of self-assembled DNA concatemers from synthetic oligonucleotides

    Directory of Open Access Journals (Sweden)

    Lu Sun


    Full Text Available Studies of DNA–ligand interaction on a single molecule level provide opportunities to understand individual behavior of molecules. Construction of DNA molecules with repetitive copies of the same segments of sequences linked in series could be helpful for enhancing the interaction possibility for sequence-specific binding ligand to DNA. Here we report on the use of synthetic oligonucleotides to self-assembly into duplex DNA concatemeric molecules. Two strands of synthetic oligonucleotides used here were designed with 50-mer in length and the sequences are semi-complimentary so to hybridize spontaneously into concatemers of double stranded DNA. In order to optimize the length of the concatemers the oligonucleotides were incubated at different oligomer concentrations, ionic strengths and temperatures for different durations. Increasing the salt concentration to 200 mM NaCl was found to be the major optimizing factor because at this enhanced ionic strength the concatemers formed most quickly and the other parameters had no detectable effect. The size and shape of formed DNA concatemers were studied by gel electrophoresis in agarose, polyacrylamide gels and by AFM. Our results show that linear DNA constructs up to several hundred base pairs were formed and could be separated from a substantial fraction of non-linear constructs.

  6. Synthesis of triazole-linked oligonucleotides with high affinity to DNA complements and an analysis of their compatibility with biosystems. (United States)

    Varizhuk, Anna M; Kaluzhny, Dmitry N; Novikov, Roman A; Chizhov, Alexandr O; Smirnov, Igor P; Chuvilin, Andrey N; Tatarinova, Olga N; Fisunov, Gleb Y; Pozmogova, Galina E; Florentiev, Vladimir L


    New oligonucleotide analogues with triazole internucleotide linkages were synthesized, and their hybridization properties were studied. The analogues demonstrated DNA binding affinities similar to those of unmodified oligonucleotides. The modification was shown to protect the oligonucleotides from nuclease hydrolysis. The modified oligonucleotides were tested as PCR primers. Modifications remote from the 3'-terminus were tolerated by polymerases. Our results suggest that these new oligonucleotide analogues are among the most promising triazole DNA mimics characterized to date.

  7. [Sequencing by hybridization methods to generate large arrays of oligonucleotides]. Final technical report

    Energy Technology Data Exchange (ETDEWEB)



    The subject of this project is to address a pressing need for custom DNA microarrays (chips) which can be easily and at low cost formatted and revised for research. In this sense, the term custom means chips for which there is need for limited quantities (less than hundreds) of any particular chip design which contains a large number of different, users defined sequences. Of the three principal approaches to fabricate DNA microarrays, the two which have been commercialized (a and b below) are not particularly suited to research purposes because of the significant time and costs required, once a result is obtained, to utilize that result in the design of a new and better chip: (a) the photodeprotection scheme used by Affymetrix; and (b) the spotting of pre-synthesized oligos or c-DNA onto surfaces.

  8. A study of oligonucleotide occurrence distributions in DNA coding segments. (United States)

    Castrignanò, T; Colosimo, A; Morante, S; Parisi, V; Rossi, G C


    In this paper we present a general strategy designed to study the occurrence frequency distributions of oligonucleotides in DNA coding segments and to deal with the problem of detecting possible patterns of genomic compositional inhomogeneities and disuniformities. Identifying specific tendencies or peculiar deviations in the distributions of the effective occurrence frequencies of oligonucleotides, with respect to what can be a priori expected, is of the greatest importance in biology. Differences between expected and actual distributions may in fact suggest or confirm the existence of specific biological mechanisms related to them. Similarly, a marked deviation in the occurrence frequency of an oligonucleotide may suggest that it belongs to the class of so-called "DNA signal (target) sequences". The approach we have elaborated is innovative in various aspects. Firstly, the analysis of the genomic data is carried out in the light of the observation that the distribution of the four nucleotides along the coding regions of the genoma is biased by the existence of a well-defined "reading frame". Secondly, the "experimental" numbers found by counting the occurrences of the various oligonucleotide sequences are appropriately corrected for the many kinds of mistakes and redundancies present in the available genetic Data Bases. A methodologically significant further improvement of our approach over the existing searching strategies is represented by the fact that, in order to decide whether or not the (corrected) "experimental" value of the occurrence frequency of a given oligonucleotide is within statistical expectations, a measure of the strength of the selective pressure, having acted on it in the course of the evolution, is assigned to the sequence, in a way that takes into account both the value of the "experimental" occurrence frequency of the sequence and the magnitude of the probability that this number might be the result of statistical fluctuations. If the

  9. Construction and Evaluation of Desulfovibrio vulgaris Whole-Genome Oligonucleotide Microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Z. He; Q. He; L. Wu; M.E. Clark; J.D. Wall; Jizhong Zhou; Matthew W. Fields


    Desulfovibrio vulgaris Hildenborough has been the focus of biochemical and physiological studies in the laboratory, and the metabolic versatility of this organism has been largely recognized, particularly the reduction of sulfate, fumarate, iron, uranium and chromium. In addition, a Desulfovibrio sp. has been shown to utilize uranium as the sole electron acceptor. D. vulgaris is a d-Proteobacterium with a genome size of 3.6 Mb and 3584 ORFs. The whole-genome microarrays of D. vulgaris have been constructed using 70mer oligonucleotides. All ORFs in the genome were represented with 3471 (97.1%) unique probes and 103 (2.9%) non-specific probes that may have cross-hybridization with other ORFs. In preparation for use of the experimental microarrays, artificial probes and targets were designed to assess specificity and sensitivity and identify optimal hybridization conditions for oligonucleotide microarrays. The results indicated that for 50mer and 70mer oligonucleotide arrays, hybridization at 45 C to 50 C, washing at 37 C and a wash time of 2.5 to 5 minutes obtained specific and strong hybridization signals. In order to evaluate the performance of the experimental microarrays, growth conditions were selected that were expected to give significant hybridization differences for different sets of genes. The initial evaluations were performed using D. vulgaris cells grown at logarithmic and stationary phases. Transcriptional analysis of D. vulgaris cells sampled during logarithmic phase growth indicated that 25% of annotated ORFs were up-regulated and 3% of annotated ORFs were downregulated compared to stationary phase cells. The up-regulated genes included ORFs predicted to be involved with acyl chain biosynthesis, amino acid ABC transporter, translational initiation factors, and ribosomal proteins. In the stationary phase growth cells, the two most up-regulated ORFs (70-fold) were annotated as a carboxynorspermidine decarboxylase and a 2C-methyl-D-erythritol-2

  10. Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore (United States)

    Cao, Chan; Ying, Yi-Lun; Hu, Zheng-Li; Liao, Dong-Fang; Tian, He; Long, Yi-Tao


    Protein nanopores offer an inexpensive, label-free method of analysing single oligonucleotides. The sensitivity of the approach is largely determined by the characteristics of the pore-forming protein employed, and typically relies on nanopores that have been chemically modified or incorporate molecular motors. Effective, high-resolution discrimination of oligonucleotides using wild-type biological nanopores remains difficult to achieve. Here, we show that a wild-type aerolysin nanopore can resolve individual short oligonucleotides that are 2 to 10 bases long. The sensing capabilities are attributed to the geometry of aerolysin and the electrostatic interactions between the nanopore and the oligonucleotides. We also show that the wild-type aerolysin nanopores can distinguish individual oligonucleotides from mixtures and can monitor the stepwise cleavage of oligonucleotides by exonuclease I.

  11. Filter arrays

    Energy Technology Data Exchange (ETDEWEB)

    Page, Ralph H.; Doty, Patrick F.


    The various technologies presented herein relate to a tiled filter array that can be used in connection with performance of spatial sampling of optical signals. The filter array comprises filter tiles, wherein a first plurality of filter tiles are formed from a first material, the first material being configured such that only photons having wavelengths in a first wavelength band pass therethrough. A second plurality of filter tiles is formed from a second material, the second material being configured such that only photons having wavelengths in a second wavelength band pass therethrough. The first plurality of filter tiles and the second plurality of filter tiles can be interspersed to form the filter array comprising an alternating arrangement of first filter tiles and second filter tiles.

  12. Modulating anti-MicroRNA-21 activity and specificity using oligonucleotide derivatives and length optimization

    DEFF Research Database (Denmark)

    Munoz-Alarcon, Andres; Guterstam, Peter; Romero, Cristian


    but reduced specificity when incorporating locked nucleic acid monomers, whereas the opposite was observed when introducing unlocked nucleic acid monomers. Our data suggest that phosphorothioate anti-microRNA oligonucleotides yield a greater activity than their phosphodiester counterparts and that a moderate...... truncation of the anti-microRNA oligonucleotide improves specificity without significantly losing activity. These results provide useful insights for design of anti-microRNA oligonucleotides to achieve both high activity as well as efficient mismatch discrimination....

  13. Synthesis of triazole-nucleoside phosphoramidites and their use in solid-phase oligonucleotide synthesis. (United States)

    Peel, Brandon J; Efthymiou, Tim C; Desaulniers, Jean-Paul


    Triazole-backbone oligonucleotides are macromolecules that have one or more triazole units that are acting as a backbone mimic. Triazoles within the backbone have been used within oligonucleotides for a variety of applications. This unit describes the preparation and synthesis of two triazole-nucleoside phosphoramidites [uracil-triazole-uracil (UtU) and cytosine-triazole-uracil (CtU)] based on a PNA-like scaffold, and their incorporation within oligonucleotides.

  14. A New Achiral Linker Reagent for the Incorporation of Multiple Amino Groups Into Oligonucleotides

    DEFF Research Database (Denmark)


    The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide wit......, to a method for preparing a labelled oligonucleotide, and to the use of the labelled oligonucleotide as hybridisation probe, in polymerase chain reactions (PCR), in nucleic acid sequencing, in cloning recombinant DNA and $i(in vitro) mutagenesis....

  15. Hemopoiesis-stimulating activity of immobilized oligonucleotides and hyaluronidase during cytostatic-induced myelosuppression. (United States)

    Dygai, A M; Skurikhin, E G; Pershina, O V; Zhdanov, V V; Khmelevskaya, A M; Andreeva, T V; Poponina, A M; Zjuzkov, G N; Udut, E V; Khrichkova, T Ju; Simanina, E V; Miroshnichenko, L A; Stavrova, L A; Tchaikovsky, A S; Markova, T S; Gurto, R V; Brjushinina, O S; Slepichev, V A


    The hemopoiesis-stimulating effect of combined treatment with immobilized oligonucleotides and hyaluronidase preparations was studied during cytostatic-induced myelosuppression caused by cyclophosphamide administration. Immobilized hyaluronidase was shown to increase the efficiency of correction of changes in the erythroid and granulocytic hemopoietic stems with immobilized oligonucleotides. This potentiation of the effect of immobilized oligonucleotides by immobilized hyaluronidase was related to an increase in functional activity of committed hemopoietic precursors.

  16. FISH and array-CGH analysis of a complex chromosome 3 aberration suggests that loss of CNTN4 and CRBN contributes to mental retardation in 3pter deletions. (United States)

    Dijkhuizen, Trijnie; van Essen, Ton; van der Vlies, Pieter; Verheij, Joke B G M; Sikkema-Raddatz, Birgit; van der Veen, Anneke Y; Gerssen-Schoorl, Klasien B J; Buys, Charles H C M; Kok, Klaas


    Imbalances of 3p telomeric sequences cause 3p- and trisomy 3p syndrome, respectively, showing distinct, but also shared clinical features. No causative genes have been identified in trisomy 3p patients, but for the 3p- syndrome, there is growing evidence that monosomy for one or more of four genes at 3pter, CHL1, CNTN4, CRBN, and MEGAP/srGAP3, may play a causative role. We describe here an analysis of a complex chromosome 3p aberration in a severely mentally retarded patient that revealed two adjacent segments with different copy number gains and a distal deletion. The deletion in this patient included the loci for CHL1, CNTN4, and CRBN, and narrowed the critical segment associated with the 3p- syndrome to 1.5 Mb, including the loci for CNTN4 and CRBN. We speculate that the deletion contributes more to this patient's phenotype than the gains that were observed. We suggest that 3p- syndrome associated features are primarily caused by loss of CNTN4 and CRBN, with loss of CHL1 probably having an additional detrimental effect on the cognitive functioning of the present patient.

  17. FISH and array-CGH analysis of a complex chromosome 3 aberration suggests that loss of CNTN4 and CRBN contributes to mental retardation in 3pter deletions

    NARCIS (Netherlands)

    Dijkhuizen, Trijnie; van Essen, Ton; van der Vlies, Pieter; Verheij, Joke B. G. M.; Sikkema-Raddatz, Birgit; van der Veen, Anneke Y.; Gerssen-Schoorl, Klasien B. J.; Buys, Charles H. C. M.; Kok, Klaas


    Imbalances of 3p telomeric sequences cause 3p- and trisomy 3p syndrome, respectively, showing distinct, but also shared clinical features. No causative genes have been identified in trisomy 3p patients, but for the 3p- syndrome, there is growing evidence that monosomy for one or more of four genes a

  18. Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    Directory of Open Access Journals (Sweden)

    Holt Robert A


    Full Text Available Abstract Background In principle, a pre-constructed library of all possible short oligonucleotides could be used to construct many distinct gene sequences. In order to assess the feasibility of such an approach, we characterized T4 DNA Ligase activity on short oligonucleotide substrates and defined conditions suitable for assembly of a plurality of oligonucleotides. Findings Ligation by T4 DNA Ligase was found to be dependent on the formation of a double stranded DNA duplex of at least five base pairs surrounding the site of ligation. However, ligations could be performed effectively with overhangs smaller than five base pairs and oligonucleotides as small as octamers, in the presence of a second, complementary oligonucleotide. We demonstrate the feasibility of simultaneous oligonucleotide phosphorylation and ligation and, as a proof of principle for DNA synthesis through the assembly of short oligonucleotides, we performed a hierarchical ligation procedure whereby octamers were combined to construct a target 128-bp segment of the beta-actin gene. Conclusions Oligonucleotides as short as 8 nucleotides can be efficiently assembled using T4 DNA Ligase. Thus, the construction of synthetic genes, without the need for custom oligonucleotide synthesis, appears feasible.

  19. Determination of optimal sites of antisense oligonucleotide cleavage within TNFα mRNA (United States)

    Lloyd, B. H.; Giles, R. V.; Spiller, D. G.; Grzybowski, J.; Tidd, D. M.; Sibson, D. R.


    Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity. PMID:11522838

  20. Label-free detection of hybridization of oligonucleotides by oblique-incidence reflectivity difference method

    Institute of Scientific and Technical Information of China (English)


    The microarrays of 20-base oligonucleotide with different concentrations are detected before and after hybridization by the oblique-incidence reflectivity difference (OI-RD) method. The experimental results prove that OI-RD is a label-free method which can not only distinguish the concentration difference of oligonucleotides before and after the hybridization but also detect the hybridization of short oligonucleotides. At present the OI-RD method can detect 0.39 μmol/L 20-base oligonucleotide or less. These results suggest that the OI-RD method is a promising and potential technique for label-free detection of biological microarrays.

  1. Chemically modified oligonucleotides with efficient RNase H response

    DEFF Research Database (Denmark)

    Vester, Birte; Boel, Anne Marie; Lobedanz, Sune;


    Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly...... in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage....

  2. Splice-switching antisense oligonucleotides as therapeutic drugs


    Havens, Mallory A.; Hastings, Michelle L.


    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipu...

  3. Tandem Oligonucleotide Probe Annealing and Elongation To Discriminate Viral Sequence

    DEFF Research Database (Denmark)

    Taskova, Maria; Uhd, Jesper; Miotke, Laura


    followed by click assembly and analysis of the read sequence by various techniques. As we demonstrate in this paper, using our new approach, a viral RNA sequence can be detected in less than 2 h without the need for cDNA synthesis or any other enzymatic reactions and with a sensitivity of ... opportunities in transcriptome analysis, virology, and other fields. Herein, we report for the first time a "click" chemistry approach to oligonucleotide probe elongation as a novel approach to specifically detect a viral sequence. We hybridized a library of short, terminally labeled probes to Ebola virus RNA...

  4. Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray.

    Directory of Open Access Journals (Sweden)

    Michael J Lodes

    Full Text Available Bacterial and viral upper respiratory infections (URI produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

  5. Strong position-dependent effects of sequence mismatches on signal ratios measured using long oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Hulme Helen


    Full Text Available Abstract Background Microarrays are an important and widely used tool. Applications include capturing genomic DNA for high-throughput sequencing in addition to the traditional monitoring of gene expression and identifying DNA copy number variations. Sequence mismatches between probe and target strands are known to affect the stability of the probe-target duplex, and hence the strength of the observed signals from microarrays. Results We describe a large-scale investigation of microarray hybridisations to murine probes with known sequence mismatches, demonstrating that the effect of mismatches is strongly position-dependent and for small numbers of sequence mismatches is correlated with the maximum length of perfectly matched probe-target duplex. Length of perfect match explained 43% of the variance in log2 signal ratios between probes with one and two mismatches. The correlation with maximum length of perfect match does not conform to expectations based on considering the effect of mismatches purely in terms of reducing the binding energy. However, it can be explained qualitatively by considering the entropic contribution to duplex stability from configurations of differing perfect match length. Conclusion The results of this study have implications in terms of array design and analysis. They highlight the significant effect that short sequence mismatches can have upon microarray hybridisation intensities even for long oligonucleotide probes. All microarray data presented in this study are available from the GEO database 1, under accession number [GEO: GSE9669

  6. Human transcriptome array for high-throughput clinical studies. (United States)

    Xu, Weihong; Seok, Junhee; Mindrinos, Michael N; Schweitzer, Anthony C; Jiang, Hui; Wilhelmy, Julie; Clark, Tyson A; Kapur, Karen; Xing, Yi; Faham, Malek; Storey, John D; Moldawer, Lyle L; Maier, Ronald V; Tompkins, Ronald G; Wong, Wing Hung; Davis, Ronald W; Xiao, Wenzhong


    A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays.

  7. Design, validation and annotation of transcriptome-wide oligonucleotide probes for the oligochaete annelid Eisenia fetida.

    Directory of Open Access Journals (Sweden)

    Ping Gong

    Full Text Available High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs. Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59% probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1 does not require a major genomics core facility; (2 allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3 is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4 if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is

  8. aCGH detects partial tetrasomy of 12p in blood from Pallister-Killian syndrome cases without invasive skin biopsy. (United States)

    Theisen, Aaron; Rosenfeld, Jill A; Farrell, Sandra A; Harris, Catharine J; Wetzel, Heather H; Torchia, Beth A; Bejjani, Bassem A; Ballif, Blake C; Shaffer, Lisa G


    Pallister-Killian syndrome (PKS) is a genetic disorder characterized by mental retardation, seizures, streaks of hypo- or hyperpigmentation and dysmorphic features. PKS is associated with tissue-limited mosaic partial tetrasomy of 12p, usually caused by an isochromosome 12p. The mosaicism is usually detected in cultured skin fibroblasts or amniotic cells and rarely in phytohemagluttinin-stimulated lymphocytes, which suggests stimulation of T-lymphocytes may distort the percentage of abnormal cells. We recently reported on the identification by microarray-based comparative genomic hybridization (aCGH) of a previously unsuspected case of partial tetrasomy of 12p caused by an isochromosome 12p. Here we report on seven additional individuals with partial tetrasomy of 12p characterized by our laboratory. All individuals were referred for mental retardation/developmental delay and/or dysmorphic features. In each case, aCGH using genomic DNA extracted from whole peripheral blood detected copy-number gain for all clones for the short arm of chromosome 12. In all but one case, FISH on metaphases from cultured lymphocytes did not detect the copy-number gain; in the remaining case, metaphase FISH on cultured lymphocytes showed an isochromosome in 10% of cells. However, interphase FISH using probes to 12p on peripheral blood smears showed additional hybridization signals in 18-70% of cells. Microarray and FISH analysis on cultured skin biopsies from four individuals confirmed the presence of an isochromosome 12p. Our results demonstrate the usefulness of aCGH with genomic DNA from whole peripheral blood to detect chromosome abnormalities that are not present in stimulated blood cultures and would otherwise require invasive skin biopsies for identification.

  9. True 3q chromosomal amplification in squamous cell lung carcinoma by FISH and aCGH molecular analysis: impact on targeted drugs.

    Directory of Open Access Journals (Sweden)

    Matteo Brunelli

    Full Text Available Squamous lung carcinoma lacks specific "ad hoc" therapies. Amplification of chromosome 3q is the most common genomic aberration and this region harbours genes having role as novel targets for therapeutics. There is no standard definition on how to score and report 3q amplification. False versus true 3q chromosomal amplification in squamous cell lung carcinoma may have tremendous impact on trials involving drugs which target DNA zones mapping on 3q. Forty squamous lung carcinomas were analyzed by FISH to assess chromosome 3q amplification. aCGH was performed as gold-standard to avoid false positive amplifications. Three clustered patterns of fluorescent signals were observed. Eight cases out of 40 (20% showed ≥8 3q signals. Twenty out of 40 (50% showed from 3 to 7 signals. The remaining showed two fluorescent signals (30%. When corrected by whole chromosome 3 signals, only cases with ≥8 signals maintained a LSI 3q/CEP3 ratio >2. Only the cases showing 3q amplification by aCGH (+3q25.3-3q27.3 showed ≥8 fluorescent signals at FISH evidencing a 3q/3 ratio >2. The remaining cases showed flat genomic portrait at aCGH on chromosome 3. We concluded that: 1 absolute copy number of 3q chromosomal region may harbour false positive interpretation of 3q amplification in squamous cell carcinoma; 2 a case results truly "amplified for chromosome 3q" when showing ≥8 fluorescent 3q signals; 3 trials involving drugs targeting loci on chromosome 3q in squamous lung carcinoma therapy have to consider false versus true 3q chromosomal amplification.

  10. An overview of sugar-modified oligonucleotides for antisense therapeutics. (United States)

    Prakash, Thazha P


    Among the multitude of chemical modifications that have been described over the past two decades, oligonucleotide analogs that are modified at the 2'-position of the furanose sugar have been especially useful for improving the drug-like properties of antisense oligonucleotides (ASOs). These modifications bias the sugar pucker towards the 3'-endo-conformation and improve ASO affinity for its biological target (i.e., mRNA). In addition, antisense drugs incorporating 2'-modified nucleotides exhibit enhanced metabolic stability, and improved pharmacokinetic and toxicological properties. Further conformational restriction of the 2'-substituent to the 4'-position of the furanose ring yielded the 2',4'-bridged nucleic acid (BNA) analogs. ASOs containing BNA modifications showed unprecedented increase in binding affinity for target RNA, while also improved nuclease resistance, in vitro and in vivo potency. Several ASO drug candidates containing 2'-modified nucleotides have entered clinical trials and continue to make progress in the clinic for a variety of therapeutic indications. 2011 Verlag Helvetica Chimica Acta AG, Zürich.

  11. Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Hongguang Sun


    Full Text Available Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed “chemical antibodies.” In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

  12. The use of oligonucleotide probes for meningococcal serotype characterization

    Directory of Open Access Journals (Sweden)

    SACCHI Claudio Tavares


    Full Text Available In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs. Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs12. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A and 615U (VR3-B used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

  13. Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates. (United States)

    Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M


    The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

  14. Array comparative genomic hybridization analysis of small supernumerary marker chromosomes in human infertility. (United States)

    Guediche, N; Tosca, L; Kara Terki, A; Bas, C; Lecerf, L; Young, J; Briand-Suleau, A; Tou, B; Bouligand, J; Brisset, S; Misrahi, M; Guiochon-Mantel, A; Goossens, M; Tachdjian, G


    Small supernumerary marker chromosomes (sSMC) are structurally abnormal chromosomes that cannot be unambiguously identified by conventional banding cytogenetics. This study describes four patients with sSMC in relation with infertility. Patient 1 had primary infertility. His brother, fertile, carried the same sSMC (patient 2). Patient 3 presented polycystic ovary syndrome and patient 4 primary ovarian insufficiency. Cytogenetic studies, array comparative genomic hybridization (CGH) and sperm analyses were compared with cases previously reported. sSMC corresponded to the 15q11.2 region (patients 1 and 2), the centromeric chromosome 15 region (patient 3) and the 21p11.2 region (patient 4). Array CGH showed 3.6-Mb gain for patients 1 and 2 and 0.266-Mb gain for patient 4. Sperm fluorescent in-situ hybridization analyses found ratios of 0.37 and 0.30 of sperm nuclei with sSMC(15) for patients 1 and 2, respectively (P < 0.001). An increase of sperm nuclei with disomy X, Y and 18 was noted for patient 1 compared with control and patient 2 (P < 0.001). Among the genes mapped in the unbalanced chromosomal regions, POTE B and BAGE are related to the testis and ovary, respectively. The implication of sSMC in infertility could be due to duplication, but also to mechanical effects perturbing meiosis.

  15. Custom Array Comparative Genomic Hybridization: the Importance of DNA Quality, an Expert Eye, and Variant Validation

    Directory of Open Access Journals (Sweden)

    Francesca Lantieri


    Full Text Available The presence of false positive and false negative results in the Array Comparative Genomic Hybridization (aCGH design is poorly addressed in literature reports. We took advantage of a custom aCGH recently carried out to analyze its design performance, the use of several Agilent aberrations detection algorithms, and the presence of false results. Our study provides a confirmation that the high density design does not generate more noise than standard designs and, might reach a good resolution. We noticed a not negligible presence of false negative and false positive results in the imbalances call performed by the Agilent software. The Aberration Detection Method 2 (ADM-2 algorithm with a threshold of 6 performed quite well, and the array design proved to be reliable, provided that some additional filters are applied, such as considering only intervals with average absolute log2ratio above 0.3. We also propose an additional filter that takes into account the proportion of probes with log2ratio exceeding suggestive values for gain or loss. In addition, the quality of samples was confirmed to be a crucial parameter. Finally, this work raises the importance of evaluating the samples profiles by eye and the necessity of validating the imbalances detected.

  16. The MOX/SUC precursor strategies: robust ways to construct functionalized oligonucleotides. (United States)

    Polushin, N


    The use of phosphoramidites bearing one or more methoxyoxalamido (MOX) or succinimido (SUC) reactive groups for construction of functionalized oligonucleotides is described. The efficiency of the new precursor strategy was demonstrated in the synthesis of oligonucleotide containing up to 16 imidazole residues.

  17. Multicellular Tumor Spheroids as a Model for Assessing Delivery of Oligonucleotides in Three Dimensions (United States)

    Carver, Kyle; Ming, Xin; Juliano, Rudolph L


    Oligonucleotides have shown promise in selectively manipulating gene expression in vitro, but that success has not translated to the clinic for cancer therapy. A potential reason for this is that cells behave differently in monolayer than in the three-dimensional tumor, resulting in limited penetration and distribution of oligonucleotides in the tumor. This may be especially true when oligonucleotides are associated with nanocarriers such as lipoplexes and polyplexes, commonly used delivery vehicles for oligonucleotides. The multicellular tumor spheroid (MCTS), a three-dimensional model that closely resembles small avascular tumors and micrometastases, has been utilized as an intermediate between monolayer culture and in vivo studies for the screening of small-molecule drugs. However, spheroids have been little used for the study of various oligonucleotide delivery formulations. Here, we have evaluated the uptake and efficacy of splice-switching antisense oligonucleotides using various delivery modalities in two- and three-dimensional culture models. We find that the size of the delivery agent dramatically influences penetration into the spheroid and thus the biological effect of the oligonucleotides. We hypothesize that the MCTS model will prove to be a useful tool in the future development of oligonucleotide delivery formulations. PMID:24618852

  18. Nucleobase azide-ethynylribose click chemistry contributes to stabilizing oligonucleotide duplexes and stem-loop structures. (United States)

    Kitamura, Yoshiaki; Asakura, Ryo; Terazawa, Koki; Shibata, Aya; Ikeda, Masato; Kitade, Yukio


    The formation of 1,4-disubstituted 1,2,3-triazoles through copper-catalyzed azide-alkyne cycloaddition (CuAAC) in oligonucleotides bearing 1-deoxy-1-ethynyl-β-d-ribofuranose (R(E)) can have a positive impact on the stability of oligonucleotide duplexes and stem-loop structures. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Studies on the Syntheses and Properties of 5'-Branched-sugar Isonucleosides and the Related Oligonucleotides

    Institute of Scientific and Technical Information of China (English)

    Tian Xiaobing; Zhang Lihe; Min Jimei


    @@ The chemistry of nucleosides and oligonucleotides is an actively investigated field in the search for new drugs. Thesyntheses and the properties of isonucleosides and oligonucleotides have been investigated to improve their stability,antitumor and antiviral activities, and to reduce their toxicity.

  20. Synthesis of 3'-, or 5'-, or internal methacrylamido-modified oligonucleotides (United States)

    Golova, Julia B.; Chernov, Boris K.


    New modifiers were synthesized for incorporation of a methacrylic function in 3'-, 5'- and internal positions of oligonucleotides during solid phase synthesis. A modifier was used for synthesis of 5'-methacrylated oligonucleotides for preparation of microarrays by a co-polymerization method.

  1. Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: hybridization assays on polymer particles obtained by direct solid phase assembly of the oligonucleotide probes. (United States)

    Hakala, H; Heinonen, P; Iitiä, A; Lönnberg, H


    Oligodeoxyribonucleotides were assembled by conventional phosphoramidite chemistry on uniformly sized (50 microns) porous glycidyl methacrylate/ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatized with DMTrO-acetyl groups. The linker was completely resistant toward ammonolytic deprotection of the base moieties. The quality of oligonucleotides was assessed by repeating the synthesis on the same particles derivatized with a cleavable ester linker. The ability of the oligonucleotide-coated particles to bind complementary sequences via hybridization was examined by following the attachment of oligonucleotides bearing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hybridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentration of the target oligonucleotide in solution, length of the hybridizing sequence, presence of noncomplementary sequences, and ionic strength. The fluorescence signal measured on a single particle after hybridization was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.

  2. Cellular Uptake and Intracellular Trafficking of Antisense and siRNA Oligonucleotides (United States)

    Juliano, RL; Ming, Xin; Nakagawa, Osamu


    Significant progress is being made concerning the development of oligonucleotides as therapeutic agents. Studies with antisense, siRNA, and other forms of oligonucleotides have shown promise in cellular and animal models and in some clinical studies. Nonetheless our understanding of how oligonucleotides function in cells and tissues is really quite limited. One major issue concerns the modes of uptake and intracellular trafficking of oligonucleotides, whether as ‘free’ molecules, or linked to various delivery moieties such as nanoparticles or targeting ligands. In this review we examine the recent literature on oligonucleotide internalization and subcellular trafficking in the context of current insights into the basic machinery for endocytosis and intracellular vesicular traffic. PMID:21992697

  3. Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides. (United States)

    Soontornworajit, Boonchoy; Zhou, Jing; Snipes, Matthew P; Battig, Mark R; Wang, Yong


    Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.

  4. High-density rhesus macaque oligonucleotide microarray design using early-stage rhesus genome sequence information and human genome annotations

    Directory of Open Access Journals (Sweden)

    Magness Charles L


    Full Text Available Abstract Background Until recently, few genomic reagents specific for non-human primate research have been available. To address this need, we have constructed a macaque-specific high-density oligonucleotide microarray by using highly fragmented low-pass sequence contigs from the rhesus genome project together with the detailed sequence and exon structure of the human genome. Using this method, we designed oligonucleotide probes to over 17,000 distinct rhesus/human gene orthologs and increased by four-fold the number of available genes relative to our first-generation expressed sequence tag (EST-derived array. Results We constructed a database containing 248,000 exon sequences from 23,000 human RefSeq genes and compared each human exon with its best matching sequence in the January 2005 version of the rhesus genome project list of 486,000 DNA contigs. Best matching rhesus exon sequences for each of the 23,000 human genes were then concatenated in the proper order and orientation to produce a rhesus "virtual transcriptome." Microarray probes were designed, one per gene, to the region closest to the 3' untranslated region (UTR of each rhesus virtual transcript. Each probe was compared to a composite rhesus/human transcript database to test for cross-hybridization potential yielding a final probe set representing 18,296 rhesus/human gene orthologs, including transcript variants, and over 17,000 distinct genes. We hybridized mRNA from rhesus brain and spleen to both the EST- and genome-derived microarrays. Besides four-fold greater gene coverage, the genome-derived array also showed greater mean signal intensities for genes present on both arrays. Genome-derived probes showed 99.4% identity when compared to 4,767 rhesus GenBank sequence tag site (STS sequences indicating that early stage low-pass versions of complex genomes are of sufficient quality to yield valuable functional genomic information when combined with finished genome information from

  5. Identification of chromosome aberrations in sporadic microsatellite stable and unstable colorectal cancers using array comparative genomic hybridization

    DEFF Research Database (Denmark)

    Jensen, Thomas Dyrsø; Li, Jian; Wang, Kai;


    Colorectal cancer (CRC) is one of the most common cancers in Denmark and in the western world in general, and the prognosis is generally poor. According to the traditional molecular classification of sporadic colorectal cancer, microsatellite stable (MSS)/chromosome unstable (CIN) colorectal...... cancers constitute approximately 85% of sporadic cases, whereas microsatellite unstable (MSI) cases constitute the remaining 15%. In this study, we used array comparative genomic hybridization (aCGH) to identify genomic hotspot regions that harbor recurrent copy number changes. The study material...

  6. Recommendations for safety pharmacology evaluations of oligonucleotide-based therapeutics. (United States)

    Berman, Cindy L; Cannon, Keri; Cui, Yi; Kornbrust, Douglas J; Lagrutta, Armando; Sun, Sunny Z; Tepper, Jeff; Waldron, Gareth; Younis, Husam S


    This document was prepared by the Safety Pharmacology Subcommittee of the Oligonucleotide Safety Working Group (OSWG), a group of industry and regulatory scientists involved in the development and regulation of therapeutic oligonucleotides. The mission of the Subcommittee was to develop scientific recommendations for the industry regarding the appropriate scope and strategies for safety pharmacology evaluations of oligonucleotides (ONs). These recommendations are the consensus opinion of the Subcommittee and do not necessarily reflect the current expectations of regulatory authorities. 1) Safety pharmacology testing, as described in the International Conference on Harmonisation (ICH) S7 guidance, is as applicable to ONs as it is to small molecule drugs and biotherapeutics. 2) Study design considerations for ONs are similar to those for other classes of drugs. In general, as with other therapeutics, studies should evaluate the drug product administered via the clinical route. Species selection should ideally consider relevance of the model with regard to the endpoints of interest, pharmacological responsiveness, and continuity with the nonclinical development program. 3) Evaluation of potential effects in the core battery (cardiovascular, central nervous, and respiratory systems) is recommended. In general: a. In vitro human ether-a-go-go-related gene (hERG) testing does not provide any specific value and is not warranted. b. Emphasis should be placed on in vivo evaluation of cardiovascular function, typically in nonhuman primates (NHPs). c. Due to the low level of concern, neurologic and respiratory function can be assessed concurrently with cardiovascular safety pharmacology evaluation in NHPs, within repeat-dose toxicity studies, or as stand-alone studies. In the latter case, rodents are most commonly used. 4) Other dedicated safety pharmacology studies, beyond the core battery, may have limited value for ONs. Although ONs can accumulate in the kidney and liver

  7. Global Arrays

    Energy Technology Data Exchange (ETDEWEB)

    Krishnamoorthy, Sriram; Daily, Jeffrey A.; Vishnu, Abhinav; Palmer, Bruce J.


    Global Arrays (GA) is a distributed-memory programming model that allows for shared-memory-style programming combined with one-sided communication, to create a set of tools that combine high performance with ease-of-use. GA exposes a relatively straightforward programming abstraction, while supporting fully-distributed data structures, locality of reference, and high-performance communication. GA was originally formulated in the early 1990’s to provide a communication layer for the Northwest Chemistry (NWChem) suite of chemistry modeling codes that was being developed concurrently.

  8. Estudio de las alteraciones genéticas en adenocarcinomas gástricos mediante aCGH y FISH en su contexto clínico-patológico


    Otero Motta, Ana Pastora


    [ES] Antecedentes Hasta ahora los estudios genómicos de alto rendimiento (aCGH) no se han correlacionado con las características clínico-patológicas en series amplias de cáncer gástrico ni se ha estudiado adecuadamente la heterogeneidad intratumoral. Los objetivos fueron: 1) determinar la incidencia de alteraciones cromosómicas mediante aCGH 2) correlacionarlas con el comportamiento clínico-patológico y evolución 3) determinar las vías de evolución clonal intratumorales. Material y método...

  9. Oligonucleotides with 1,4-dioxane-based nucleotide monomers

    DEFF Research Database (Denmark)

    Madsen, Andreas S; Wengel, Jesper


    An epimeric mixture of H-phosphonates 5R and 5S has been synthesized in three steps from known secouridine 1. Separation of the epimers has been accomplished by RP-HPLC, allowing full characterization and incorporation of monomers X and Y into 9-mer oligonucleotides using H-phosphonates building...... blocks 5R and 5S, respectively. A single incorporation of either monomer X or monomer Y in the central position of a DNA 9-mer results in decreased thermal affinity toward both DNA and RNA complements (ΔT(m) = -3.5 °C/-3.5 °C for monomer X and ΔT(m) = -11.0 °C/-6.5 °C for monomer Y). CD measurements do...

  10. Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

    Directory of Open Access Journals (Sweden)

    Andrea Pauli

    Full Text Available Antisense oligonucleotides (ASOs are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.

  11. Antisense Oligonucleotides: Translation from Mouse Models to Human Neurodegenerative Diseases. (United States)

    Schoch, Kathleen M; Miller, Timothy M


    Multiple neurodegenerative diseases are characterized by single-protein dysfunction and aggregation. Treatment strategies for these diseases have often targeted downstream pathways to ameliorate consequences of protein dysfunction; however, targeting the source of that dysfunction, the affected protein itself, seems most judicious to achieve a highly effective therapeutic outcome. Antisense oligonucleotides (ASOs) are small sequences of DNA able to target RNA transcripts, resulting in reduced or modified protein expression. ASOs are ideal candidates for the treatment of neurodegenerative diseases, given numerous advancements made to their chemical modifications and delivery methods. Successes achieved in both animal models and human clinical trials have proven ASOs both safe and effective. With proper considerations in mind regarding the human applicability of ASOs, we anticipate ongoing in vivo research and clinical trial development of ASOs for the treatment of neurodegenerative diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Antisense oligonucleotide targeting midkine suppresses in vivo angiogenesis

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Xiang Wang; Xing Yao; Yong-Liang Lu; Jin-Liang Ping; Jian-Fang He


    AIM: To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) andin situ human hepatocellular carcinoma (HCC).METHODS: An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylineosin (HE) staining.RESULTS: MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues.CONCLUSION: MK-AS is an effective antiangiogenesis agent in vivo.

  13. Tetramerization of an RNA oligonucleotide containing a GGGG sequence. (United States)

    Kim, J; Cheong, C; Moore, P B


    Poly rG can form four-stranded helices. The Hoogsteen-paired quartets of G residues on which such structures depend are so stable that they will form in 5'-GMP solutions, provided that Na+ or K+ are present (see for example, refs 2-4). Telomeric DNA sequences, which are G-rich, adopt four-stranded antiparallel G-quartet conformations in vitro, and parallel tetramerization of G-rich sequences may be involved in meiosis. Here we show that RNAs containing short runs of Gs can also tetramerize. A 19-base oligonucleotide derived from the 5S RNA of Escherichia coli (strand III), 5'GCCGAUGGUAGUGUGGGGU3', forms a K(+)-stabilized tetrameric aggregate that depends on the G residues at its 3' end. This complex is so stable that it would be surprising if similar structures do not occur in nature.

  14. Design and development of thermolytic DNA oligonucleotide prodrugs. (United States)

    Grajkowski, Andrzej; Pedras-Vasconcelos, Joao; Ausín, Cristina; Verthelyi, Daniela; Beaucage, Serge L


    Deoxyribonucleoside phosphoramidites functionalized with the thermolytic 2-(N-formyl-N-methyl)aminoethyl group for phosphorus protection (1a-d) have been prepared and employed in the solid-phase synthesis of CpG ODN fma1555. Given that this modified oligonucleotide can be converted to the immunomodulatory CpG ODN 1555 under neutral conditions at 37 degrees C, its biologic activity was demonstrated in vivo by studies showing that intraperitoneal administration of CpG ODN fma1555 in mice resulted in the activation of cytokine-secreting splenocytes. Furthermore, administration of CpG ODN fma1555 to mice that were challenged intradermally in the ear with live L. major metacyclic promastigotes, reduced the severity of Leishmania skin lesions over time to an extent similar to that obtained with CpG ODN 1555. In another infectious model experiment, CpG ODN fma1555 protected newborn mice from death (65% survival) when administered 3 days before infection with the aggressive Tacaribe (TCRV) virus. A comparable immunoprotection was obtained by treatment of TCRV-infected mice with CpG ODN 1555 administered on the same day of infection (45% survival). However, when TCRV-infected mice were treated with CpG ODN fma1555 on the day of infection, they died as a consequence of the relatively slow conversion of the oligonucleotide prodrug to the bioactive CpG ODN 1555. Co-administration of both CpG ODN 1555 and CpG ODN fma1555 to mice 3 days prior to TCRV infection or on the day of infection provided protection from death (45-65% survival) and thus widened the immunoprotection window against TCRV-infection.

  15. Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

    Directory of Open Access Journals (Sweden)

    Kai Hoehlig

    Full Text Available A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.

  16. Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides (United States)

    Wang, Xiaolong; Gou, Deming; Xu, Shuang-yong


    Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR), for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI) cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs. PMID:20062528

  17. Managing the sequence-specificity of antisense oligonucleotides in drug discovery. (United States)

    Hagedorn, Peter H; Hansen, Bo R; Koch, Troels; Lindow, Morten


    All drugs perturb the expression of many genes in the cells that are exposed to them. These gene expression changes can be divided into effects resulting from engaging the intended target and effects resulting from engaging unintended targets. For antisense oligonucleotides, developments in bioinformatics algorithms, and the quality of sequence databases, allow oligonucleotide sequences to be analyzed computationally, in terms of the predictability of their interactions with intended and unintended RNA targets. Applying these tools enables selection of sequence-specific oligonucleotides where no- or only few unintended RNA targets are expected. To evaluate oligonucleotide sequence-specificity experimentally, we recommend a transcriptomics protocol where two or more oligonucleotides targeting the same RNA molecule, but with entirely different sequences, are evaluated together. This helps to clarify which changes in cellular RNA levels result from downstream processes of engaging the intended target, and which are likely to be related to engaging unintended targets. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell- and animal models before clinical testing. Since potential adverse effects related to unintended targeting are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are especially relevant in oligonucleotide drug discovery. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Stereospecificity of Oligonucleotide Interactions Revisited: No Evidence for Heterochiral Hybridization and Ribozyme/DNAzyme Activity (United States)

    Hoehlig, Kai; Bethge, Lucas; Klussmann, Sven


    A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications. PMID:25679211

  19. Release of DNA oligonucleotides and their conjugates from controlled-pore glass under thermolytic conditions. (United States)

    Grajkowski, Andrzej; Cieślak, Jacek; Norris, Scott; Freedberg, Darón I; Kauffman, Jon S; Duff, Robert J; Beaucage, Serge L


    The sequential functionalization of long-chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and a dinucleoside phosphorotetrazolide leads to a uniquely engineered support for solid-phase synthesis. Unlike conventional succinylated-CPG supports, this support is designed to allow oligonucleotide deprotection and elimination of deprotection side-products to proceed without release of the oligonucleotide. When needed, the DNA oligonucleotide can be thermolytically released in 2 hr under essentially neutral conditions. The modified CPG support has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. On the basis of reversed-phase HPLC and electrophoretic analyses, the purity of the released oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated-CPG supports, in terms of both shorter-than-full-length oligonucleotide contaminants and overall yields. The detailed preparation of DNA oligonucleotides conjugated with exemplary reporter or functional groups, either at the 3'-terminus or at both 3'- and 5'-termini, is also described.

  20. Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

    Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

  1. A Brassica exon array for whole-transcript gene expression profiling.

    Directory of Open Access Journals (Sweden)

    Christopher G Love

    Full Text Available Affymetrix GeneChip® arrays are used widely to study transcriptional changes in response to developmental and environmental stimuli. GeneChip® arrays comprise multiple 25-mer oligonucleotide probes per gene and retain certain advantages over direct sequencing. For plants, there are several public GeneChip® arrays whose probes are localised primarily in 3' exons. Plant whole-transcript (WT GeneChip® arrays are not yet publicly available, although WT resolution is needed to study complex crop genomes such as Brassica, which are typified by segmental duplications containing paralogous genes and/or allopolyploidy. Available sequence data were sampled from the Brassica A and C genomes, and 142,997 gene models identified. The assembled gene models were then used to establish a comprehensive public WT exon array for transcriptomics studies. The Affymetrix GeneChip® Brassica Exon 1.0 ST Array is a 5 µM feature size array, containing 2.4 million 25-base oligonucleotide probes representing 135,201 gene models, with 15 probes per gene distributed among exons. Discrimination of the gene models was based on an E-value cut-off of 1E(-5, with ≤98% sequence identity. The 135 k Brassica Exon Array was validated by quantifying transcriptome differences between leaf and root tissue from a reference Brassica rapa line (R-o-18, and categorisation by Gene Ontologies (GO based on gene orthology with Arabidopsis thaliana. Technical validation involved comparison of the exon array with a 60-mer array platform using the same starting RNA samples. The 135 k Brassica Exon Array is a robust platform. All data relating to the array design and probe identities are available in the public domain and are curated within the BrassEnsembl genome viewer at

  2. The Use of Gel Electrophoresis to Study the Reactions of Activated Amino Acids with Oligonucleotides (United States)

    Zieboll, Gerhard; Orgel, Leslie E.


    We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonu-cleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5 inches phosphates and ethylenediamine. We find that arginine and amino acids can interact with oligonucleotidesl through stacking interactions react most efficiently. D- and L-amino acids give indistinguishable families of products.

  3. Chimeric RNA Oligonucleotides with Triazole and Phosphate Linkages: Synthesis and RNA Interference. (United States)

    Fujino, Tomoko; Kogashi, Kanako; Okada, Koudai; Mattarella, Martin; Suzuki, Takeru; Yasumoto, Kenichi; Sogawa, Kazuhiro; Isobe, Hiroyuki


    Chimeric RNA oligonucleotides with an artificial triazole linker were synthesized using solution-phase click chemistry and solid-phase automated synthesis. Scalable synthesis methods for jointing units for the chimeric structure have been developed, and after click-coupling of the jointing units with triazole linkers, a series of chimeric oligonucleotides was prepared by utilizing the well-established phosphoramidite method for the elongation. The series of chimeric 21-mer oligonucleotides that possessed the triazole linker at different strands and positions allowed for a screening study of the RNA interference to clarify the preference of the triazole modifications in small-interfering RNA molecules.

  4. Solid-phase synthesis of 2{sup '}-O-methoxyethyl oligonucleotides using dimeric phosphoramidate blocks

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Gi Weon; Kang, Yong Han [Dept. of Applied Chemistry, Hanyang University, Ansan (Korea, Republic of)


    This research focused on the method of using dimeric phosphoramidite blocks to synthesize oligonucleotides for development as oligonucleotide drugs. A 16-mer oligonucleotide with the randomly selected sequence of C*C*T*C*G*C *T*C*T*C*G*C*C* C*G*C was synthesized using CC, GC, and TC dimers, a combination of monomers and dimers, or only monomers as building blocks. Using dimer blocks in this synthetic method provided a significant decrease in critical impurities that had similar properties to the main product, which was confirmed by LC-MS and HPLC analysis.

  5. Major copy proportion analysis of tumor samples using SNP arrays

    Directory of Open Access Journals (Sweden)

    Li Cheng


    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs are the most common genetic variations in the human genome and are useful as genomic markers. Oligonucleotide SNP microarrays have been developed for high-throughput genotyping of up to 900,000 human SNPs and have been used widely in linkage and cancer genomics studies. We have previously used Hidden Markov Models (HMM to analyze SNP array data for inferring copy numbers and loss-of-heterozygosity (LOH from paired normal and tumor samples and unpaired tumor samples. Results We proposed and implemented major copy proportion (MCP analysis of oligonucleotide SNP array data. A HMM was constructed to infer unobserved MCP states from observed allele-specific signals through emission and transition distributions. We used 10 K, 100 K and 250 K SNP array datasets to compare MCP analysis with LOH and copy number analysis, and showed that MCP performs better than LOH analysis for allelic-imbalanced chromosome regions and normal contaminated samples. The major and minor copy alleles can also be inferred from allelic-imbalanced regions by MCP analysis. Conclusion MCP extends tumor LOH analysis to allelic imbalance analysis and supplies complementary information to total copy numbers. MCP analysis of mixing normal and tumor samples suggests the utility of MCP analysis of normal-contaminated tumor samples. The described analysis and visualization methods are readily available in the user-friendly dChip software.

  6. Carboranyl Nucleosides & Oligonucleotides for Neutron Capture Therapy Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Schinazi, Raymond F.


    This proposal enabled us to synthesize and develop boron-rich nucleosides and oligonucleotide analogues for boron neutron capture therapy (BNCT) and the treatment of various malignancies. First, we determined the relationship between structure, cellular accumulation and tissue distribution of 5-o-carboranyl-2'-deoxyuridine (D-CDU) and its derivatives D-ribo-CU and 5-o-carboranyluracil (CU), to potentially target brain and other solid tumors for neutron capture therapy. Synthesized carborane containing nucleoside derivatives of CDU, D- and L-enantiomers of CDU, D-ribo-CU and CU were used. We measured tissue disposition in xenografted mice bearing 9479 human prostate tumors xenografts and in rats bearing 9L gliosarcoma isografts in their flanks and intracranially. The accumulation of D-CDU, 1-({beta}-L-arabinosyl)-5-o-carboranyluracil, D-ribo-CU, and CU were also studied in LnCap human prostate tumor cells and their retention was measured in male nude mice bearing LnCap and 9479 human prostate tumor xenografts. D-CDU, D-ribo-CU and CU levels were measured after administration in mice bearing 9479 human prostate tumors in their flanks. D-CDU achieved high cellular concentrations in LnCap cells and up to 2.5% of the total cellular compound was recovered in the 5'-monophosphorylated form. D-CDU cellular concentrations were similar in LnCap and 9479 tumor xenografts. Studies in tumor bearing animals indicated that increasing the number of hydroxyl moieties in the sugar constituent of the carboranyl nucleosides lead to increased rate and extent of renal elimination, a decrease in serum half-lives and an increased tissue specificity. Tumor/brain ratios were greatest for CDU and D-ribo-CU, while tumor/prostate ratios were greatest with CU. CDU and D-ribo-CU have potential for BNCT of brain malignancies, while CU may be further developed for prostate cancer. A method was developed for the solid phase synthesis of oligonucleotides containing (ocarboran-1-yl

  7. Microfluidic gene arrays for rapid genomic profiling (United States)

    West, Jay A.; Hukari, Kyle W.; Hux, Gary A.; Shepodd, Timothy J.


    Genomic analysis tools have recently become an indispensable tool for the evaluation of gene expression in a variety of experiment protocols. Two of the main drawbacks to this technology are the labor and time intensive process for sample preparation and the relatively long times required for target/probe hybridization. In order to overcome these two technological barriers we have developed a microfluidic chip to perform on chip sample purification and labeling, integrated with a high density genearray. Sample purification was performed using a porous polymer monolithic material functionalized with an oligo dT nucleotide sequence for the isolation of high purity mRNA. These purified mRNA"s can then rapidly labeled using a covalent fluorescent molecule which forms a selective covalent bond at the N7 position of guanine residues. These labeled mRNA"s can then released from the polymer monolith to allow for direct hybridization with oligonucletide probes deposited in microfluidic channel. To allow for rapid target/probe hybridization high density microarray were printed in microchannels. The channels can accommodate array densities as high as 4000 probes. When oligonucleotide deposition is complete, these channels are sealed using a polymer film which forms a pressure tight seal to allow sample reagent flow to the arrayed probes. This process will allow for real time target to probe hybridization monitoring using a top mounted CCD fiber bundle combination. Using this process we have been able to perform a multi-step sample preparation to labeled target/probe hybridization in less than 30 minutes. These results demonstrate the capability to perform rapid genomic screening on a high density microfluidic microarray of oligonucleotides.

  8. Attachment of DNA to microfabricated arrays with self-assembled monolayer

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Guojun; Tanii, Takashi; Miyake, Takeo; Funatsu, Takashi; Ohdomari, Iwao


    A novel approach of fabricating sub-10-{mu}m patterns on silicon surfaces by electron beam (EB) lithography for attachment of oligonucleotides was described. The shape of the microfabricated arrays was observed to be regular by optical microscopy. An octadecyltrimethoxysilane (ODS) monolayer was deposited on the regions outside the patterned areas to minimize the nonspecific binding of biomolecules. Cy 5-labeled target DNA was hybridized to both complementary and noncomplementary oligonucleotides that were covalently anchored to micropatterns. As a result, the micropatterns where specific binding occurred show strong signals, whereas no signals are observed in the case of nonspecific binding. These data indicate that miniature micro- and nano-arrays will find applications in biochips and biosensors.

  9. Conjugation with receptor-targeted histidine-rich peptides enhances the pharmacological effectiveness of antisense oligonucleotides. (United States)

    Nakagawa, Osamu; Ming, Xin; Carver, Kyle; Juliano, Rudy


    Ineffective delivery to intracellular sites of action is one of the key limitations to the use of antisense and siRNA oligonucleotides as therapeutic agents. Here, we describe molecular scale antisense oligonucleotide conjugates that bind selectively to a cell surface receptor, are internalized, and then partially escape from nonproductive endosomal locations to reach their sites of action in the nucleus. Peptides that include bombesin sequences for receptor targeting and a run of histidine residues for endosomal disruption were covalently linked to a splice switching antisense oligonucleotide. The conjugates were tested for their ability to correct splicing and up-regulate expression of a luciferase reporter in prostate cancer cells that express the bombesin receptor. We found that trivalent conjugates that included both the targeting sequence and several histidine residues were substantially more effective than conjugates containing only the bombesin or histidine moieties. This demonstrates the potential of creating molecular scale oligonucleotide conjugates with both targeting and endosome escape capabilities.

  10. Chemically robust fluoroalkyl phthalocyanine-oligonucleotide bioconjugates and their GRP78 oncogene photocleavage activity. (United States)

    Patel, Pradeepkumar; Patel, Hemantbhai H; Borland, Emily; Gorun, Sergiu M; Sabatino, David


    The first representative of functionalized fluoroalkyl phthalocyanines, F48H7(COOH)PcZn, is reported. The complex generates (1)O2 affording long-lasting photooxidation of an external substrate without self-decomposition. The carboxylic group couples with an antisense oligonucleotide targeting GRP78 oncogenes, resulting in the F48H7PcZn-cancer targeting oligonucleotide (CTO). The bioconjugated fluorophthalocyanine effectively hybridizes complementary GRP78 DNA and mRNA sequences. Piperidine cleavage assays reveal desired photochemical oligonucleotide oxidative degradation for both F48H7PcZn-CTO:DNA and F48H7PcZn-CTO:mRNA hybrids. This new materials strategy could be extended to other functional fluorinated phthalocyanines-antisense oligonucleotide combinations for long-lasting oncogene-targeting photodynamic therapy.

  11. Oligonucleotide-based biosensors for in vitro diagnostics and environmental hazard detection. (United States)

    Jung, Il Young; Lee, Eun Hee; Suh, Ah Young; Lee, Seung Jin; Lee, Hyukjin


    Oligonucleotide-based biosensors have drawn much attention because of their broad applications in in vitro diagnostics and environmental hazard detection. They are particularly of interest to many researchers because of their high specificity as well as excellent sensitivity. Recently, oligonucleotide-based biosensors have been used to achieve not only genetic detection of targets but also the detection of small molecules, peptides, and proteins. This has further broadened the applications of these sensors in the medical and health care industry. In this review, we highlight various examples of oligonucleotide-based biosensors for the detection of diseases, drugs, and environmentally hazardous chemicals. Each example is provided with detailed schematics of the detection mechanism in addition to the supporting experimental results. Furthermore, future perspectives and new challenges in oligonucleotide-based biosensors are discussed.

  12. Exploiting Protected Maleimides to Modify Oligonucleotides, Peptides and Peptide Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Clément Paris


    Full Text Available This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols.

  13. Nucleoside, nucleotide and oligonucleotide based amphiphiles: a successful marriage of nucleic acids with lipids. (United States)

    Gissot, Arnaud; Camplo, Michel; Grinstaff, Mark W; Barthélémy, Philippe


    Amphiphilic molecules based on nucleosides, nucleotides and oligonucleotides are finding more and more biotechnological applications. This Perspective highlights their synthesis, supramolecular organization as well as their applications in the field of biotechnology.

  14. High Precision Metrology on the Ultra-Lightweight W 50.8 cm f/1.25 Parabolic SHARPI Primary Mirror using a CGH Null Lens (United States)

    Antonille, Scott


    For potential use on the SHARPI mission, Eastman Kodak has delivered a 50.8cm CA f/1.25 ultra-lightweight UV parabolic mirror with a surface figure error requirement of 6nm RMS. We address the challenges involved in verifying and mapping the surface error of this large lightweight mirror to +/-3nm using a diffractive CGH null lens. Of main concern is removal of large systematic errors resulting from surface deflections of the mirror due to gravity as well as smaller contributions from system misalignment and reference optic errors. We present our efforts to characterize these errors and remove their wavefront error contribution in post-processing as well as minimizing the uncertainty these calculations introduce. Data from Kodak and preliminary measurements from NASA Goddard will be included.

  15. Sheath liquid effects in capillary high-performance liquid chromatography-electrospray mass spectrometry of oligonucleotides. (United States)

    Huber, C G; Krajete, A


    Fused-silica capillary columns of 200 microm inner diameter were packed with micropellicular, octadecylated, 2.3 microm poly(styrene-divinylbenzene) particles and applied to the separation of oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography. Oligonucleotides were eluted at 50 degrees C with gradients of 3-13% acetonitrile in 50 mM triethylammonium bicarbonate. Addition of sheath liquid to the column effluent allowed the detection of oligonucleotides by electrospray ionization mass spectrometry using full-scan data acquisition with a detectability comparable to that obtained with UV detection. The signal-to-noise ratios with different sheath liquids increased in the order isopropanololigonucleotides longer than 20 nucleotide units whereas no significant effect was observed with shorter oligonucleotides. Organic acids and bases in the sheath liquid generally deteriorated the signal-to-noise ratios in the chromatograms and mass spectra mainly because of increased background noise. Only a few charge states were observed in the mass spectra of oligonucleotides because of charge state reduction due to the presence of carbonic acid in the eluent. With triethylammonium hydrogencarbonate as chromatographic eluent and acetonitrile as sheath liquid, very few cation adducts of oligonucleotides were observed in the mass spectra. However, the presence of small amounts of monopotassium adducts enabled the calculation of the charge state of multiply charged ions. With acetonitrile as sheath liquid, 710 amol of a 16-mer oligonucleotide were detected using selected ion monitoring data acquisition with a signal-to-noise ratio of 3:1. Finally, capillary ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was

  16. Complexes of carbon nanotubes with oligonucleotides in thin Langmuir-Blodgett films to detect electrochemically hybridization (United States)

    Egorov, A. S.; Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Orekhovskaya, T. I.; Veligura, A. A.; Govorov, M. I.; Shulitsky, B. G.


    Self-assembled complexes consisting of thin multi-walled carbon nanotubes (MWCNTs) and DNA-oligonucleotides which are able to a cooperative binding to complementary oligonucleotides have been investigated. It was establised a high-performance charge transport in nanostructured Langmuir-Blodgett complexes thin MWCNTs/DNA. A method to electrochemically detect DNA hybridization on the self-organized structures has been proposed.

  17. Respirable antisense oligonucleotides: a new drug class for respiratory disease

    Directory of Open Access Journals (Sweden)

    Tanaka Makoto


    Full Text Available Abstract Respirable antisense oligonucleotides (RASONs, which attenuate specific disease-associated mRNAs, represent a new class of respiratory therapeutics with considerable potential. RASONs overcome previous obstacles that have impeded the development of antisense therapeutics targeting diseases in other organ systems. RASONs are delivered directly to the target tissue via inhalation; their uptake seems to be enhanced by cationic properties inherent in pulmonary surfactant, and, because of the markedly different target properties of mRNA and proteins, they can have very long durations of effect compared with traditional drugs targeting the protein of the same gene. RASONs contain chemical modifications that decrease their degradation by cellular nucleases. However, total insensitivity to nucleases is probably not an optimal design criterion for RASONs, because moderate nuclease sensitivity can prevent their systemic delivery, decreasing the potential for systemic toxicity. EPI-2010 is a 21-mer phosphorothioate RASON that attenuates bronchoconstriction, inflammation and surfactant depletion in preclinical models of human asthma, has a duration of effect of seven days, and seems to undergo minimal systemic delivery.

  18. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation (United States)

    Kim, Jinho; Olsen, Timothy R.; Zhu, Jing; Hilton, John P.; Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N.; Lin, Qiao


    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours.

  19. Enzymic synthesis of oligonucleotides containing methylphosphonate internucleotide linkages. (United States)

    Higuchi, H; Endo, T; Kaji, A


    Thymidine 5'-O-(pyrophosphoryl methylphosphonate) (dTTP alpha CH3) has been chemically synthesized by condensation of thymidine 5'-O-(methylphosphonate) with pyrophosphate. This novel nucleotide, which contained an alpha-phosphorus atom as methylphosphonate, was used as a substrate of terminal deoxynucleotidyltransferase (TDTase) in the presence of oligonucleotide (5'-GCTGTATCGTCAAGGCACTC-3') as an initiator. The reaction products were separated into two components by reverse-phase high-performance liquid chromatography (RP-HPLC). These products were, after purification, digested with nuclease P1 and alkaline phosphatase followed by separation of digested products by RP-HPLC. The result showed the presence of one of the isomers of 2'-deoxycytidyl-3'-methylphosphonyl-5'-thymidine (dCpCH3T) and 2'-deoxycytidyl-3'-methylphosphonyl-5'-thymidyl-3'-methyl phosphonyl-5'-thymidin e (dCpCH3TpCH3T), respectively. Fast atom bombardment mass spectrometry of these products further supported identification of the dinucleotide and the trinucleotide. These results indicated that dTTP alpha CH3 was used as a substrate of TDTase, resulting in methylphosphonate linkages. Produced oligomers were resistant to hydrolysis by snake venom phosphodiesterase I.

  20. Advancements of antisense oligonucleotides in treatment of breast cancer

    Institute of Scientific and Technical Information of China (English)

    YANGShuan-Ping; SONGSan-Tai; 等


    Breast cancer is one kind of multi-gene related malignancy.Overexpression of some oncogenes such as HER-2(c-erbB-2,Neu),bcl-2/bcl-xL,protein kinase A(PKA),and transferrin receptor gene(TfR gene),etc significantly affect the prognosis of breast cancer.It was shown that specific suppression of the overexpressed genes above resulted in the improvement of the therapy of breast cancer.Antisense of useful tools for inhibiting the overexpression of specific oncogenes,was involved in the therapy of breast cancer in recent years. Data indicated that antisense oligonucleotides(ON)could inhibit specially the expression of the target genes on mRNA or protein levels in most of cases;some ON candidates showed encouraging therapeutic effects in vitro and in vivo on breast cancer cell lines or xenografts.Furthermore,the combination use of the antisense ON and normal chemotherapeutic agents indicated synergistic antitumor effects,which was probably the best utilization of antisense ON in the treatment of breast cancer.

  1. Antineoplastic effect of decoy oligonucleotide derived from MGMT enhancer.

    Directory of Open Access Journals (Sweden)

    Tamar Canello

    Full Text Available Silencing of O(6-methylguanine-DNA-methyltransferase (MGMT in tumors, mainly through promoter methylation, correlates with a better therapeutic response and with increased survival. Therefore, it is conceivable to consider MGMT as a potential therapeutic target for the treatment of cancers. Our previous results demonstrated the pivotal role of NF-kappaB in MGMT expression, mediated mainly through p65/NF-kappaB homodimers. Here we show that the non-canonical NF-KappaB motif (MGMT-kappaB1 within MGMT enhancer is probably the major inducer of MGMT expression following NF-kappaB activation. Thus, in an attempt to attenuate the transcription activity of MGMT in tumors we designed locked nucleic acids (LNA modified decoy oligonucleotides corresponding to the specific sequence of MGMT-kappaB1 (MGMT-kB1-LODN. Following confirmation of the ability of MGMT-kB1-LODN to interfere with the binding of p65/NF-kappaB to the NF-KappaB motif within MGMT enhancer, the efficacy of the decoy was studied in-vitro and in-vivo. The results of these experiments show that the decoy MGMT-kB1-LODN have a substantial antineoplastic effect when used either in combination with temozolomide or as monotherapy. Our results suggest that MGMT-kB1-LODN may provide a novel strategy for cancer therapy.

  2. Nanoexplosive gene therapy using triplex-forming oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Eun Jung; Min, Hye Jung; Choe, Jae Gol; Park, Gil Hong; Kim, Meyoung Kon [College of Medicine, Korea Univ., Seoul (Korea, Republic of)


    Triplex forming oligonucleotides (TFO) labeled with Auger emitter could be ideal vehicles for delivering radiation energy to specific DNA sequences, and followed by double-stranded DNA breaks and subsequent inactivation of targeted genes. We designed TFOs targeting the selected DNA fragments (i.e., estrogen receptors and N-myc promoter) and labeled with {sup 125}I and {sup 111}In. Various Cancer cells, e.g., MCF-7 (breast adenocarcinoma), MCF-10A (immortalized breast cells), Jurkat (T-cell leukemia), ARO (thyroid cancer), SNU-449 (Colon Caner), and HL-60 (polymyelocytic leukemia), were prepared and treated with radiolabeled TFO for 24 h. After the incubation, subcellular fractions (i.e., cell nucleus, cytoplasm and cultured medium) were collected and measured radioactivity by a gamma scintillation counter, respectively. The mean value of % injected dose for each fraction was ranged as follows: nucleus, 4.4-20%; cytoplasm, 8.2-29%; and medium, 64-87%. Therefore, we speculated that TFO labeled with Auger emitter could be a next-generation therapeutic tool in nanoexplosive gene therapy.

  3. Efficient in vivo delivery of antisense oligonucleotide to choroid plexus. (United States)

    Piao, Wenying; Nishina, Kazutaka; Yoshida-Tanaka, Kie; Kuwahara, Hiroya; Nishina, Tomoko; Sakata, Mina; Mizusawa, Hidehiro; Yokota, Takanori


    The choroid plexus (CP) is present on the ventricular walls of the brain, produces cerebrospinal fluid (CSF), contains many blood vessels, and is a major functional component of the blood-CSF barrier. The CP is an important site in the pathophysiology of various neurological diseases, including Alzheimer's disease and meningeal amyloidosis. We performed gene silencing in the CP in vivo by using an antisense oligonucleotide (ASO). A short ASO of length 12 nucleotides was intravenously injected into rats. The ASO was not delivered to neurons or glia in the central nervous system, but was successfully delivered into the CP, and resulted in a significant reduction of endogenous target gene expression in epithelial cells within the CP. Although the mechanism of uptake of the ASO by the CP was not elucidated, the ASO bound to albumin in vivo, and the distribution of ASO delivery was similar to that of albumin delivery. These findings suggest that we inhibited target gene expression in the epithelial cells of the CP via albumin-ASO conjugates. This strategy should be useful for investigations of the function of CP, and for the development of new gene-silencing therapies for diseases with pathophysiology related to the CP.

  4. Antineoplastic effect of decoy oligonucleotide derived from MGMT enhancer. (United States)

    Canello, Tamar; Ovadia, Haim; Refael, Miri; Zrihan, Daniel; Siegal, Tali; Lavon, Iris


    Silencing of O(6)-methylguanine-DNA-methyltransferase (MGMT) in tumors, mainly through promoter methylation, correlates with a better therapeutic response and with increased survival. Therefore, it is conceivable to consider MGMT as a potential therapeutic target for the treatment of cancers. Our previous results demonstrated the pivotal role of NF-kappaB in MGMT expression, mediated mainly through p65/NF-kappaB homodimers. Here we show that the non-canonical NF-KappaB motif (MGMT-kappaB1) within MGMT enhancer is probably the major inducer of MGMT expression following NF-kappaB activation. Thus, in an attempt to attenuate the transcription activity of MGMT in tumors we designed locked nucleic acids (LNA) modified decoy oligonucleotides corresponding to the specific sequence of MGMT-kappaB1 (MGMT-kB1-LODN). Following confirmation of the ability of MGMT-kB1-LODN to interfere with the binding of p65/NF-kappaB to the NF-KappaB motif within MGMT enhancer, the efficacy of the decoy was studied in-vitro and in-vivo. The results of these experiments show that the decoy MGMT-kB1-LODN have a substantial antineoplastic effect when used either in combination with temozolomide or as monotherapy. Our results suggest that MGMT-kB1-LODN may provide a novel strategy for cancer therapy.

  5. Chimeric Antisense Oligonucleotide Conjugated to α-Tocopherol

    Directory of Open Access Journals (Sweden)

    Tomoko Nishina


    Full Text Available We developed an efficient system for delivering short interfering RNA (siRNA to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid-DNA gapmer antisense oligonucleotide (ASO was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS molecules are bound to ASO with UNA sequences.

  6. Kinetic Hairpin Oligonucleotide Blockers for Selective Amplification of Rare Mutations (United States)

    Jia, Yanwei; Sanchez, J. Aquiles; Wangh, Lawrence J.


    Detection of rare mutant alleles in an excess of wild type alleles is increasingly important in cancer diagnosis. Several methods for selective amplification of a mutant allele via the polymerase chain reaction (PCR) have been reported, but each of these methods has its own limitations. A common problem is that Taq DNA polymerase errors early during amplification generate false positive mutations which also accumulate exponentially. In this paper, we described a novel method using hairpin oligonucleotide blockers that can selectively inhibit the amplification of wild type DNA during LATE-PCR amplification. LATE-PCR generates double-stranded DNA exponentially followed by linear amplification of single-stranded DNA. The efficiency of the blocker is optimized by adjusting the LATE-PCR temperature cycling profile. We also demonstrate that it is possible to minimize false positive signals caused by Taq DNA polymerase errors by using a mismatched excess primer plus a modified PCR profile to preferentially enrich for mutant target sequences prior to the start of the exponential phase of LATE-PCR amplification. In combination these procedures permit amplification of specific KRAS mutations in the presence of more than 10,000 fold excess of wild type DNA without false positive signals. PMID:25082368

  7. The development of bioactive triple helix-forming oligonucleotides. (United States)

    Seidman, Michael M; Puri, Nitin; Majumdar, Alokes; Cuenoud, Bernard; Miller, Paul S; Alam, Rowshon


    We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in mammalian cells. We have described psoralen-conjugated TFOs containing 2'-O-methyl (2'OMe) and 2'-O-aminoethoxy (AE) ribose substitutions. TFOs with a cluster of 3-4 AE residues, with all other sugars as 2'OMe, were bioactive in a gene knockout assay in mammalian cells. In contrast, TFOs with one or two clustered, or three dispersed, AE residues were inactive. Thermal stability analysis of the triplexes indicated that there were only incremental differences between the active and inactive TFOs. However the active and inactive TFOs could be distinguished by their association kinetics. The bioactive TFOs showed markedly greater on-rates than the inactive TFOs. It appears that the on-rate is a better predictor of TFO bioactivity than thermal stability. Our data are consistent with a model in which a cluster of 3-4 AE residues stabilizes the nucleation event that precedes formation of a complete triplex. It is likely that triplexes in cells are much less stable than triplexes in vitro probably as a result of elution by chromatin-associated translocases and helicases. Consequently the biologic assay will favor TFOs that can bind and rebind genomic targets quickly.

  8. Effect of oligonucleotide primers in determining viral variability within hosts

    Directory of Open Access Journals (Sweden)

    Moya Andrés


    Full Text Available Abstract Background Genetic variability in viral populations is usually estimated by means of polymerase chain reaction (PCR based methods in which the relative abundance of each amplicon is assumed to be proportional to the frequency of the corresponding template in the initial sample. Although bias in template-to-product ratios has been described before, its relevance in describing viral genetic variability at the intrapatient level has not been fully assessed yet. Results To investigate the role of oligonucleotide design in estimating viral variability within hosts, genetic diversity in hepatitis C virus (HCV populations from eight infected patients was characterised by two parallel PCR amplifications performed with two slightly different sets of primers, followed by cloning and sequencing (mean = 89 cloned sequences per patient. Population genetics analyses of viral populations recovered by pairs of amplifications revealed that in seven patients statistically significant differences were detected between populations sampled with different set of primers. Conclusions Genetic variability analyses demonstrates that PCR selection due to the choice of primers, differing in their degeneracy degree at some nucleotide positions, can eclipse totally or partially viral variants, hence yielding significant different estimates of viral variability within a single patient and therefore eventually producing quite different qualitative and quantitative descriptions of viral populations within each host.

  9. Advantages of ion-exchange chromatography for oligonucleotide analysis. (United States)

    Cook, Ken; Thayer, Jim


    The rapid development of therapeutic oligonucleotides (ONs) has created a need for in-depth characterization of ONs, beyond previous requirements. The natural migration to LC-MS requires the use of chromatography with MS-compatible eluents to introduce the large, highly charged biopolymers into the mass spectrometer. Most frequently this employs ion-pair reversed-phase liquid chromatography, which may leave gaps in the characterization, but these can be filled with the use of high-resolution ion-exchange chromatography. Several classes of isobaric isomers are among the impurities that will require further separation prior to MS analysis. This review shows how the use of ion exchange as an additional orthogonal analytical method can be used as standalone or interfaced with MS to achieve the highest possible analytical coverage in the characterization and quantification of impurities present in single- and double-stranded ON formulations. Some of these techniques have been in use for some time and the importance of others is just being recognized.

  10. Genomic Alterations in Sporadic Synchronous Primary Breast Cancer Using Array and Metaphase Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Arezou A. Ghazani


    Full Text Available Synchronous primary breast cancer describes the occurrence of multiple tumors affecting one or both breasts at initial diagnosis. This provides a unique opportunity to identify tissue-specific genomic markers that characterize each tumor while controlling for the individual genetic background of a patient. The aim of this study was to examine the genomic alterations and degree of similarity between synchronous cancers. Using metaphase comparative genomic hybridization and array comparative genomic hybridization (aCGH, the genomic alterations of 23 synchronous breast cancers from 10 patients were examined at both chromosomal and gene levels. Synchronous breast cancers, when compared to their matched counterparts, were found to have a common core set of genetic alterations, with additional unique changes present in each. They also frequently exhibited features distinct from the more usual solitary primary breast cancers. The most frequent genomic alterations included chromosomal gains of 1q, 3p, 4q, and 8q, and losses of 11q, 12q, 16q, and 17p. aCGH identified copy number amplification in regions that are present in all 23 tumor samples, including 1p31.3–1p32.3 harboring JAK1. Our findings suggest that synchronous primary breast cancers represent a unique type of breast cancer and, at least in some instances, one tumor may give rise to the other.

  11. New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Lorena Rodrigo


    Full Text Available The objective of this study was to evaluate the usefulness of comprehensive chromosome screening (CCS using array comparative genomic hybridization (aCGH. The study included 1420 CCS cycles for recurrent miscarriage (n=203; repetitive implantation failure (n=188; severe male factor (n=116; previous trisomic pregnancy (n=33; and advanced maternal age (n=880. CCS was performed in cycles with fresh oocytes and embryos (n=774; mixed cycles with fresh and vitrified oocytes (n=320; mixed cycles with fresh and vitrified day-2 embryos (n=235; and mixed cycles with fresh and vitrified day-3 embryos (n=91. Day-3 embryo biopsy was performed and analyzed by aCGH followed by day-5 embryo transfer. Consistent implantation (range: 40.5–54.2% and pregnancy rates per transfer (range: 46.0–62.9% were obtained for all the indications and independently of the origin of the oocytes or embryos. However, a lower delivery rate per cycle was achieved in women aged over 40 years (18.1% due to the higher percentage of aneuploid embryos (85.3% and lower number of cycles with at least one euploid embryo available per transfer (40.3%. We concluded that aneuploidy is one of the major factors which affect embryo implantation.

  12. Porous silicon-cell penetrating peptide hybrid nanocarrier for intracellular delivery of oligonucleotides. (United States)

    Rytkönen, Jussi; Arukuusk, Piret; Xu, Wujun; Kurrikoff, Kaido; Langel, Ulo; Lehto, Vesa-Pekka; Närvänen, Ale


    The largest obstacle to the use of oligonucleotides as therapeutic agents is the delivery of these large and negatively charged biomolecules through cell membranes into intracellular space. Mesoporous silicon (PSi) is widely recognized as a potential material for drug delivery purposes due to its several beneficial features like large surface area and pore volume, high loading capacity, biocompatibility, and biodegradability. In the present study, PSi nanoparticles stabilized by thermal oxidation or thermal carbonization and subsequently modified by grafting aminosilanes on the surface are utilized as an oligonucleotide carrier. Splice correcting oligonucleotides (SCOs), a model oligonucleotide drug, were loaded into the positively charged PSi nanoparticles with a loading degree as high as 14.3% (w/w). Rapid loading was achieved by electrostatic interactions, with the loading efficiencies reaching 100% within 5 min. The nanoparticles were shown to deliver and release SCOs, in its biologically active form, inside cells when formulated together with cell penetrating peptides (CPP). The biological effect was monitored with splice correction assay and confocal microscopy utilizing HeLa pLuc 705 cells. Furthermore, the use of PSi carrier platform in oligonucleotide delivery did not reduce the cell viability. Additionally, the SCO-CPP complexes formed in the pores of the carrier were stabilized against proteolytic digestion. The advantageous properties of protecting and releasing the cargo and the possibility to further functionalize the carrier surface make the hybrid nanoparticles a potential system for oligonucleotide delivery.

  13. Investigation of the structural organization of cationic nanoemulsion/antisense oligonucleotide complexes. (United States)

    Bruxel, Fernanda; Vilela, José Mario Carneiro; Andrade, Margareth Spangler; Malachias, Ângelo; Perez, Carlos A; Magalhães-Paniago, Rogério; Oliveira, Mônica Cristina; Teixeira, Helder F


    Atomic force microscopy image analysis and energy dispersive X-ray diffraction experiments were used to investigate the structural organization of cationic nanoemulsion/oligonucleotide complexes. Oligonucleotides targeting topoisomerase II gene were adsorbed on cationic nanoemulsions obtained by means of spontaneous emulsification procedure. Topographical analysis by atomic force microscopy allowed the observation of the nanoemulsion/oligonucleotide complexes through three-dimensional high-resolution images. Flattening of the oil droplets was observed, which was reduced in the complexes obtained at high amount of adsorbed oligonucleotides. In such conditions, complexes exhibit droplet size in the 600nm range. The oligonucleotides molecules were detected on the surface of the droplets, preventing their fusion during aggregation. A lamellar structure organization was identified by energy dispersive X-ray diffraction experiments. The presence of the nucleic acid molecules led to a disorganization of the lipid arrangement and an expansion in the lattice spacing, which was proportional to the amount of oligonucleotides added. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands. (United States)

    Dirin, Mehrdad; Urban, Ernst; Noe, Christian R; Winkler, Johannes


    Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment coupling procedures for producing oligonucleotide conjugates are often tedious, produce moderate yields and reaction byproducts are frequently difficult to remove. We have developed an improved method for solid-phase coupling of ligands to oligonucleotides via disulfides directly after solid-phase synthesis. A 2'-thiol introduced using a modified nucleotide building block was orthogonally deprotected on the controlled pore glass solid support with N-butylphosphine. Oligolysine peptides and a short monodisperse ethylene glycol chain were successfully coupled to the deprotected thiol. Cleavage from the resin and full removal of oligonucleotide protection groups were achieved using methanolic ammonia. After standard desalting, and without further purification, homogenous conjugates were obtained as demonstrated by HPLC, gel electrophoresis, and mass spectrometry. The attachment of both amphiphilic and cationic ligands proves the versatility of the conjugation procedure. An antisense oligonucleotide conjugate with hexalysine showed pronounced gene silencing in a cell culture tumor model in the absence of a transfection reagent and the corresponding ethylene glycol conjugate resulted in down regulation of the target gene to nearly 50% after naked application. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. The small molecule Retro-1 enhances the pharmacological actions of antisense and splice switching oligonucleotides. (United States)

    Ming, Xin; Carver, Kyle; Fisher, Michael; Noel, Romain; Cintrat, Jean-Christophe; Gillet, Daniel; Barbier, Julien; Cao, Canhong; Bauman, John; Juliano, Rudolph L


    The attainment of strong pharmacological effects with oligonucleotides is hampered by inefficient access of these molecules to their sites of action in the cytosol or nucleus. Attempts to address this problem with lipid or polymeric delivery systems have been only partially successful. Here, we describe a novel alternative approach involving the use of a non-toxic small molecule to enhance the pharmacological effects of oligonucleotides. The compound Retro-1 was discovered in a screen for small molecules that reduce the actions of bacterial toxins and has been shown to block the retrograde trafficking pathway. We demonstrate that Retro-1 can also substantially enhance the effectiveness of antisense and splice switching oligonucleotides in cell culture. This effect occurs at the level of intracellular trafficking or processing and is correlated with increased oligonucleotide accumulation in the nucleus but does not involve the perturbation of lysosomal compartments. We also show that Retro-1 can alter the effectiveness of splice switching oligonucleotides in the in vivo setting. These observations indicate that it is possible to enhance the pharmacological actions of oligonucleotides using non-toxic and non-lysosomotropic small molecule adjuncts.

  16. Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Yong-Zhong Wang; Guo-Xiang Wu; Li-Bo Luo; Min Chen; Li-Hua Ruan


    AIM: To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B.METHODS: Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated.RESULTS: Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41(33%), 41 (33%) and 45 (36%) samples, and genotype C in 76 (60%), 76 (60%) and 81 (64%) samples, by oligonucleotide chip, real-time PCR and sequencing,respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Realtime PCR was the rapidest and cheapest among the three assays.CONCLUSION: Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.

  17. Conceptual "Heat-Driven" approach to the synthesis of DNA oligonucleotides on microarrays. (United States)

    Grajkowski, A; Cieślak, J; Chmielewski, M K; Marchán, V; Phillips, L R; Wilk, A; Beaucage, S L


    The discovery of deoxyribonucleoside cyclic N-acylphosphoramidites, a novel class of phosphoramidite monomers for solid-phase oligonucleotide synthesis, has led to the development of a number of phosphate protecting groups that can be cleaved from DNA oligonucleotides under thermolytic neutral conditions. These include the 2-(N-formyl-N-methyl)aminoethyl, 4-oxopentyl, 3-(N-tert-butyl)carboxamido-1-propyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, and 4-methythiobutyl groups. When used for 5'-hydroxyl protection of nucleosides, the analogous 1-phenyl-2-[N-methyl-N-(2-pyridyl)]aminoethyloxycarbonyl group exhibited excellent thermolytic properties, which may permit an iterative "heat-driven" synthesis of DNA oligonucleotides on microarrays. In this regard, progress has been made toward the use of deoxyribonucleoside cyclic N-acylphosphoramidites in solid-phase oligonucleotide syntheses without nucleobase protection. Given that deoxyribonucleoside cyclic N-acylphosphoramidites produce oligonucleotides with heat-sensitive phosphate protecting groups, blocking the 5'-hydroxyl of these monomers with, for example, the thermolabile 1-phenyl-2-[N-methyl-N-(2-pyridyl)]aminoethyloxycarbonyl group may provide a convenient thermo-controlled method for the synthesis of oligonucleotides on microarrays.

  18. 2-O-[2-(Methylthio)ethyl]-Modified Oligonucleotide: An Analog of 2-O-[2-(Methoxy)ethyl]-Modified Oligonucleotide with Improved Protein Binding Properties and High Binding Affinity to Target RNA

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, T.P.; Manoharan, M.; Fraser, A.S.; Kawasaki, A.M.; Lesnik, E.; Sioufi, N.; Leeds, J.M.; Teplova, M.; Egli, M.


    A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.

  19. DNA sequence analysis by hybridization with oligonucleotide microchips : MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides.

    Energy Technology Data Exchange (ETDEWEB)

    Stomakhin, A. A.; Vasiliskov, V. A.; Timofeev, E.; Schulga, D.; Cotter, R. J.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Moscow Inst. of Physics and Technology; Middle Atlantic Mass Spectrometry Lab.; Johns Hopkins Univ. School of Medicine


    Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA-8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10-15{sup o}C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA-8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.

  20. Generation of a genomic tiling array of the human Major Histocompatibility Complex (MHC and its application for DNA methylation analysis

    Directory of Open Access Journals (Sweden)

    Ottaviani Diego


    Full Text Available Abstract Background The major histocompatibility complex (MHC is essential for human immunity and is highly associated with common diseases, including cancer. While the genetics of the MHC has been studied intensively for many decades, very little is known about the epigenetics of this most polymorphic and disease-associated region of the genome. Methods To facilitate comprehensive epigenetic analyses of this region, we have generated a genomic tiling array of 2 Kb resolution covering the entire 4 Mb MHC region. The array has been designed to be compatible with chromatin immunoprecipitation (ChIP, methylated DNA immunoprecipitation (MeDIP, array comparative genomic hybridization (aCGH and expression profiling, including of non-coding RNAs. The array comprises 7832 features, consisting of two replicates of both forward and reverse strands of MHC amplicons and appropriate controls. Results Using MeDIP, we demonstrate the application of the MHC array for DNA methylation profiling and the identification of tissue-specific differentially methylated regions (tDMRs. Based on the analysis of two tissues and two cell types, we identified 90 tDMRs within the MHC and describe their characterisation. Conclusion A tiling array covering the MHC region was developed and validated. Its successful application for DNA methylation profiling indicates that this array represents a useful tool for molecular analyses of the MHC in the context of medical genomics.

  1. Coupling in reflector arrays

    DEFF Research Database (Denmark)

    Appel-Hansen, Jørgen


    In order to reduce the space occupied by a reflector array, it is desirable to arrange the array antennas as close to each other as possible; however, in this case coupling between the array antennas will reduce the reflecting properties of the reflector array. The purpose of the present communic...

  2. Diffusion of Oligonucleotides from within Iron-Crosslinked Polyelectrolyte-Modified Alginate Beads: A Model System for Drug Release

    CERN Document Server

    Privman, Vladimir; Luz, Roberto A S; Guz, Nataliia; Glasser, M Lawrence; Katz, Evgeny


    We developed and experimentally verified an analytical model to describe diffusion of oligonucleotides from stable hydrogel beads. The synthesized alginate beads are Fe3+-cross-linked as well as polyelectrolyte-doped for uniformity and stability at physiological pH. Data on diffusion of oligonucleotides from inside the beads provide physical insights into the volume nature of the immobilization of a fraction of oligonucleotides due to polyelectrolyte cross-linking, i.e., the absence of the surface-layer barrier in this case. Furthermore, our results suggest a new simple approach to measuring the diffusion coefficient of the mobile oligonucleotide molecules inside hydrogel. The considered alginate beads provide a model for a well-defined component in drug release systems and for the oligonucleotide-release transduction steps in drug-delivering and biocomputing applications. This is illustrated by destabilizing the beads with citrate that induces full oligonucleotide release with non-diffusional kinetics.

  3. A simple and rapid method for the preparation of homologous DNA oligonucleotide hybridization probes from heterologous gene sequences and probes. (United States)

    Maxwell, E S; Sarge, K D


    We describe a simple and rapid method for the preparation of homologous DNA oligonucleotide probes for hybridization analysis and/or cDNA/genomic library screening. With this method, a synthetic DNA oligonucleotide derived from a known heterologous DNA/RNA/protein sequence is annealed to an RNA preparation containing the gene transcript of interest. Any unpaired 3'-terminal oligonucleotides of the heterologous DNA primer are then removed using the 3' exonuclease activity of the DNA Polymerase I Klenow fragment before primer extension/dideoxynucleotide sequencing of the annealed RNA species with AMV reverse transcriptase. From the determined RNA sequence, a completely homologous DNA oligonucleotide probe is then prepared. This approach has been used to prepare a homologous DNA oligonucleotide probe for the successful library screening of the yeast hybRNA gene starting with a heterologous mouse hybRNA DNA oligonucleotide probe.

  4. 'Specific' oligonucleotides often recognize more than one gene: the limits of in situ hybridization applied to GABA receptors. (United States)

    Mladinic, M; Didelon, F; Cherubini, E; Bradbury, A


    As exquisite probes for gene sequences, oligonucleotides are one of the most powerful tools of recombinant molecular biology. In studying the GABA receptor subunits in the neonatal hippocampus we have used oligonucleotide probes in in situ hybridization and cloning techniques. The oligonucleotides used and assumed to be specific for the target gene, actually recognized more than one gene, leading to surprising and contradictory results. In particular, we found that a GABA(A)-rho specific oligonucleotide recognized an abundant, previously unknown, transcription factor in both in situ and library screening, while oligos 'specific' for GABA(A) subunits were able to recognize 30 additional unrelated genes in library screening. This suggests that positive results obtained with oligonucleotides should be interpreted with caution unless confirmed by identical results with oligonucleotides from different parts of the same gene, or cDNA library screening excludes the presence of other hybridizing species.

  5. A Novel Family of Small Molecules that Enhance the Intracellular Delivery and Pharmacological Effectiveness of Antisense and Splice Switching Oligonucleotides. (United States)

    Wang, Ling; Ariyarathna, Yamuna; Ming, Xin; Yang, Bing; James, Lindsey I; Kreda, Silvia M; Porter, Melissa; Janzen, William; Juliano, Rudolph L


    The pharmacological effectiveness of oligonucleotides has been hampered by their tendency to remain entrapped in endosomes, thus limiting their access to cytosolic or nuclear targets. We have previously reported a group of small molecules that enhance the effects of oligonucleotides by causing their release from endosomes. Here, we describe a second novel family of oligonucleotide enhancing compounds (OECs) that is chemically distinct from the compounds reported previously. We demonstrate that these molecules substantially augment the actions of splice switching oligonucleotides (SSOs) and antisense oligonucleotides (ASOs) in cell culture. We also find enhancement of SSO effects in a murine model. These new compounds act by increasing endosome permeability and causing partial release of entrapped oligonucleotides. While they also affect the permeability of lysosomes, they are clearly different from typical lysosomotropic agents. Current members of this compound family display a relatively narrow window between effective dose and toxic dose. Thus, further improvements are necessary before these agents can become suitable for therapeutic use.

  6. Profiled support vector machines for antisense oligonucleotide efficacy prediction

    Directory of Open Access Journals (Sweden)

    Martín-Guerrero José D


    Full Text Available Abstract Background This paper presents the use of Support Vector Machines (SVMs for prediction and analysis of antisense oligonucleotide (AO efficacy. The collected database comprises 315 AO molecules including 68 features each, inducing a problem well-suited to SVMs. The task of feature selection is crucial given the presence of noisy or redundant features, and the well-known problem of the curse of dimensionality. We propose a two-stage strategy to develop an optimal model: (1 feature selection using correlation analysis, mutual information, and SVM-based recursive feature elimination (SVM-RFE, and (2 AO prediction using standard and profiled SVM formulations. A profiled SVM gives different weights to different parts of the training data to focus the training on the most important regions. Results In the first stage, the SVM-RFE technique was most efficient and robust in the presence of low number of samples and high input space dimension. This method yielded an optimal subset of 14 representative features, which were all related to energy and sequence motifs. The second stage evaluated the performance of the predictors (overall correlation coefficient between observed and predicted efficacy, r; mean error, ME; and root-mean-square-error, RMSE using 8-fold and minus-one-RNA cross-validation methods. The profiled SVM produced the best results (r = 0.44, ME = 0.022, and RMSE= 0.278 and predicted high (>75% inhibition of gene expression and low efficacy ( Conclusions The SVM approach is well suited to the AO prediction problem, and yields a prediction accuracy superior to previous methods. The profiled SVM was found to perform better than the standard SVM, suggesting that it could lead to improvements in other prediction problems as well.

  7. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides. (United States)

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D


    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing.

  8. Liposome-coated lipoplex-based carrier for antisense oligonucleotides. (United States)

    Wyrozumska, Paulina; Meissner, Justyna; Toporkiewicz, Monika; Szarawarska, Marta; Kuliczkowski, Kazimierz; Ugorski, Maciej; Walasek, Marta A; Sikorski, Aleksander F


    The chemical nature of genetic drugs (e.g. antisense oligonucleotides, siRNA, vectors) requires a suitable carrier system to protect them from enzymatic degradation without changing their properties and enable efficient delivery into target cells. Lipid vectors for nucleic acid delivery that have been widely investigated for years can be very effective. As the majority of attempts made in the field of cancer gene therapy have focused on solid tumors, while blood cancer cells have attracted less attention, the latter became the subject of our investigation. The lipid carrier proposed here is based on liposomes constructed by others but the lipid composition is original. A liposome-coated lipoplex (L-cL) consists of a core arising from complexation of positively charged lipid and negatively charged oligodeoxynucleotide (ODN) or plasmid DNA coated by a neutral or anionic lipid bilayer. Moreover, our lipid vector demonstrates size stability and is able to retain a high content of enclosed plasmid DNA or antisense oligodeoxynucleotides (asODNs). Observed transfection efficacies of the tested preparation using a plasmid coding for fluorescent protein were up to 60-85% of examined leukemia cells (Jurkat T and HL-60 lines) in the absence or the presence of serum. When BCL‑2 asODN was encapsulated in the L-cL, specific silencing of this gene product at both the mRNA and protein level and also a markedly decreased cell survival rate were observed in vitro. Moreover, biodistribution analysis in mice indicates prolonged circulation characteristic for PEG-modified liposomal carriers. Experiments on tumor-engrafted animals indicate substantial inhibition of tumor growth.

  9. Mongersen, an oral SMAD7 antisense oligonucleotide, and Crohn's disease. (United States)

    Monteleone, Giovanni; Neurath, Markus F; Ardizzone, Sandro; Di Sabatino, Antonio; Fantini, Massimo C; Castiglione, Fabiana; Scribano, Maria L; Armuzzi, Alessandro; Caprioli, Flavio; Sturniolo, Giacomo C; Rogai, Francesca; Vecchi, Maurizio; Atreya, Raja; Bossa, Fabrizio; Onali, Sara; Fichera, Maria; Corazza, Gino R; Biancone, Livia; Savarino, Vincenzo; Pica, Roberta; Orlando, Ambrogio; Pallone, Francesco


    Crohn's disease-related inflammation is characterized by reduced activity of the immunosuppressive cytokine transforming growth factor β1 (TGF-β1) due to high levels of SMAD7, an inhibitor of TGF-β1 signaling. Preclinical studies and a phase 1 study have shown that an oral SMAD7 antisense oligonucleotide, mongersen, targets ileal and colonic SMAD7. In a double-blind, placebo-controlled, phase 2 trial, we evaluated the efficacy of mongersen for the treatment of persons with active Crohn's disease. Patients were randomly assigned to receive 10, 40, or 160 mg of mongersen or placebo per day for 2 weeks. The primary outcomes were clinical remission at day 15, defined as a Crohn's Disease Activity Index (CDAI) score of less than 150, with maintenance of remission for at least 2 weeks, and the safety of mongersen treatment. A secondary outcome was clinical response (defined as a reduction of 100 points or more in the CDAI score) at day 28. The proportions of patients who reached the primary end point were 55% and 65% for the 40-mg and 160-mg mongersen groups, respectively, as compared with 10% for the placebo group (P<0.001). There was no significant difference in the percentage of participants reaching clinical remission between the 10-mg group (12%) and the placebo group. The rate of clinical response was significantly greater among patients receiving 10 mg (37%), 40 mg (58%), or 160 mg (72%) of mongersen than among those receiving placebo (17%) (P=0.04, P<0.001, and P<0.001, respectively). Most adverse events were related to complications and symptoms of Crohn's disease. We found that study participants with Crohn's disease who received mongersen had significantly higher rates of remission and clinical response than those who received placebo. (Funded by Giuliani; EudraCT number, 2011-002640-27.).

  10. Application of heteronuclear couplings to conformational analysis of oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, G. [Univ. of Maryland, College Park, MD (United States); Live, D. [Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Bax, A. [NIDDK National Institutes of Health, Bethesda, MD (United States)


    The value of vicinal coupling constants extracted from NMR spectra in deducing torsion angles for conformational analysis is well recognized. Due to the abundance of protons, their couplings have been mostly widely used. In many instances, couplings between protons and other nuclei may be a valuable complement to proton-proton couplings or, in some instances, may be the only coupling available to characterize the torsion angle about a bond. Recently, heteronuclear couplings have been used to great benefit in studies of isotopically enriched proteins, and this general approach has been extended to peptides at natural abundance. The possibility of using this approach to study oligonucleotides is also attractive but has not as yet been widely exploited. With the development of strategies for labeling such molecules, particularly RNAs, this may become an important component in conformational analysis. For DNA, labeling is less accessible, but sufficient quantities of unlabeled material are readily available for measuring these couplings at natural abundance. We chose several DNA systems to explore the usefulness of heteronuclear couplings in addressing the sugar conformation and the glycosidic torsion angle. Intensities of cross peaks in long-range HMQC experiments can be related to the couplings. Crosspeaks involving H1{prime} and C1{prime} atoms have been emphasized because of the superior shift dispersion at these positions between sugar protons and carbon atoms. Results will be shown for the self-complementary Dickerson duplex dodecamer sequence d(CGCGAATTCGCG) and for d(GGTCGG), which dimerizes to form a G-tetrad structure incorporating both syn and anti base orientations. The couplings provide a clear discrimination between presence of C3{prime}-endo and C2{prime}-endo conformations of the sugars and syn and anti bases arrangements.

  11. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides


    Pawel Bialk; Brett Sansbury; Natalia Rivera-Torres; Kevin Bloh; Dula Man; Kmiec, Eric B.


    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRIS...

  12. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides


    Pawel Bialk; Brett Sansbury; Natalia Rivera-Torres; Kevin Bloh; Dula Man; Kmiec, Eric B.


    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRIS...

  13. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide

    Directory of Open Access Journals (Sweden)

    Roh C


    Full Text Available Changhyun RohDivision of Biotechnology, Advanced Radiation Technology Institute (ARTI, Korea Atomic Energy Research Institute (KAERI, Jeongeup, Republic of KoreaAbstract: Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS, and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV nucleocapsid (N protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (--catechin gallate and (--gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (--catechin gallate and (--gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 µg mL–1, (--catechin gallate and (--gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.Keywords: SARS, RNA oligonucleotide, quantum dots, inhibitor, screening

  14. LNA-modified isothermal oligonucleotide microarray for differentiating bacilli of similar origin. (United States)

    Yan, Jing; Yuan, Ying; Mu, Runqing; Shang, Hong; Guan, Yifu


    Oligonucleotide microarray has been one of the most powerful tools in the 'Post-Genome Era' for its high sensitivity, high throughput and parallel processing capability. To achieve high detection specificity, we fabricated an isothermal microarray using locked nucleic acid (LNA)-modified oligonucleotide probes, since LNA has demonstrated the advanced ability to enhance the binding affinity toward their complementary nucleotides. After designing the nucleotide sequences of these oligonucleotide probes for gram-positive bacilli of similar origin (Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium and Bacillus circulans), we unified the melting temperatures of these oligonucleotide probes by modifying some nucleotides using LNA. Furthermore, we optimized the experimental procedures of hydrating microarray slides, blocking side surface as well as labelling the PCR products. Experimental results revealed that KOD Dash DNA polymerase could efficiently incorporate Cy3-dCTP into the PCR products, and the LNA-isothermal oligonucleotide microarray were able to distinguish the bacilli of similar origin with a high degree of accuracy and specificity under the optimized experimental condition.

  15. Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries. (United States)

    Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie


    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

  16. LNA-modified isothermal oligonucleotide microarray for differentiating bacilli of similar origin

    Indian Academy of Sciences (India)

    Jing Yan; Ying Yuan; Runqing Mu; Hong Shang; Yifu Guan


    Oligonucleotide microarray has been one of the most powerful tools in the ‘Post-Genome Era’ for its high sensitivity, high throughput and parallel processing capability. To achieve high detection specificity, we fabricated an isothermal microarray using locked nucleic acid (LNA)-modified oligonucleotide probes, since LNA has demonstrated the advanced ability to enhance the binding affinity toward their complementary nucleotides. After designing the nucleotide sequences of these oligonucleotide probes for gram-positive bacilli of similar origin (Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium and Bacillus circulans), we unified the melting temperatures of these oligonucleotide probes by modifying some nucleotides using LNA. Furthermore, we optimized the experimental procedures of hydrating microarray slides, blocking side surface as well as labelling the PCR products. Experimental results revealed that KOD Dash DNA polymerase could efficiently incorporate Cy3-dCTP into the PCR products, and the LNA-isothermal oligonucleotide microarray were able to distinguish the bacilli of similar origin with a high degree of accuracy and specificity under the optimized experimental condition.

  17. Surface modification of plasmonic nanostructured materials with thiolated oligonucleotides in 10 seconds using selective microwave heating

    Energy Technology Data Exchange (ETDEWEB)

    Abel, B.; Aslan, K. [Morgan State University, Department of Chemistry, 1700 East Cold Spring Lane, Baltimore, MD 21251 (United States)


    This study demonstrates the proof-of-principle of rapid surface modification of plasmonic nanostructured materials with oligonucleotides using low power microwave heating. Due to their interesting optical and electronic properties, silver nanoparticle films (SNFs, 2 nm thick) deposited onto glass slides were used as the model plasmonic nanostructured materials. Rapid surface modification of SNFs with oligonucleotides was carried out using two strategies (1) Strategy 1: for ss-oligonucleotides, surface hybridization and (2) Strategy 2: for ds-oligonucleotides, solution hybridization, where the samples were exposed to 10, 15, 30 and 60 seconds microwave heating. To assess the efficacy of our new rapid surface modification technique, identical experiments carried out without the microwave heating (i.e., conventional method), which requires 24 hours for the completion of the identical steps. It was found that SNFs can be modified with ss- and ds-oligonucleotides in 10 seconds, which typically requires several hours of incubation time for the chemisorption of thiol groups on to the planar metal surface using conventional techniques. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  18. Novel Efficient Cell-Penetrating, Peptide-Mediated Strategy for Enhancing Telomerase Inhibitor Oligonucleotides. (United States)

    Muñoz-Alarcón, Andrés; Eriksson, Jonas; Langel, Ülo


    At present, there are several therapeutic approaches for targeting telomerase in tumors. One in particular, currently undergoing clinical trials, is based on synthetic lipid-modified oligonucleotide antagonists aimed at inhibiting the ribonucleoprotein subunit of human telomerase. However, while enabling efficient uptake, the lipid modifications reduce the potency of the therapeutic oligonucleotides compared to nonmodified oligonucleotides. Moreover, lipid modification may increase oligonucleotide accumulation in the liver causing undesirable hepatotoxicity. Noncovalent complexation strategies for cell-penetrating peptide (CPP)-mediated delivery present an option to circumvent the need for potency-reducing modifications, while allowing for a highly efficient uptake, and could significantly improve the efficiency of telomerase-targeting cancer therapeutics. Delivery of a nonlipidated locked nucleic acid/2'-O-methyl mixmer significantly inhibits the telomerase activity in treated HeLa cells. The inhibitory effect was further improved through addition of a CPP. Furthermore, calculated IC50-values for the oligonucleotide delivered by CPPs into HeLa cells are more than 20 times lower than telomerase inhibitor Imetelstat, currently undergoing clinical trials. These results emphasize the potential of CPP-mediated delivery of future pharmaceuticals and provide means by which to enhance an already promising therapeutic strategy for cancer treatment.

  19. Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry. (United States)

    Owens, D R; Bothner, B; Phung, Q; Harris, K; Siuzdak, G


    Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.

  20. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. (United States)

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels


    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  1. Conjugates of Phthalocyanines With Oligonucleotides as Reagents for Sensitized or Catalytic DNA Modification

    Directory of Open Access Journals (Sweden)


    Full Text Available Several conjugates of metallophthalocyanines with deoxyribooligonucleotides were synthesized to investigate sequence-specific modification of DNA by them. Oligonucleotide parts of these conjugates were responsible for the recognition of selected complementary sequences on the DNA target. Metallophthalocyanines were able to induce the DNA modification: phthalocyanines of Zn(II and Al(III were active as photosensitizers in the generation of singlet oxygen 1 O 2 , while phthalocyanine of Co(II promoted DNA oxidation by molecular oxygen through the catalysis of formation of reactive oxygen species ( ⋅ O 2 − , O 2 H 2 , OH. Irradiation of the reaction mixture containing either Zn(II- or Al(III-tetracarboxyphthalocyanine conjugates of oligonucleotide pd(TCTTCCCA with light of > 340 nm wavelength (Hg lamp or He/Ne laser resulted in the modification of the 22-nucleotide target d(TGAATGGGAAGAGGGTCAGGTT. A conjugate of Co(II-tetracarboxyphthalocyanine with the oligonucleotide was found to modify the DNA target in the presence of O 2 and 2-mercaptoethanol or in the presence of O 2 H 2 . Under both sensitized and catalyzed conditions, the nucleotides G 13 – G 15 were mainly modified, providing evidence that the reaction proceeded in the double-stranded oligonucleotide. These results suggest the possible use of phthalocyanine-oligonucleotide conjugates as novel artificial regulators of gene expression and therapeutic agents for treatment of cancer.

  2. Oligonucleotide-induced alternative splicing of serotonin 2C receptor reduces food intake. (United States)

    Zhang, Zhaiyi; Shen, Manli; Gresch, Paul J; Ghamari-Langroudi, Masoud; Rabchevsky, Alexander G; Emeson, Ronald B; Stamm, Stefan


    The serotonin 2C receptor regulates food uptake, and its activity is regulated by alternative pre-mRNA splicing. Alternative exon skipping is predicted to generate a truncated receptor protein isoform, whose existence was confirmed with a new antiserum. The truncated receptor sequesters the full-length receptor in intracellular membranes. We developed an oligonucleotide that promotes exon inclusion, which increases the ratio of the full-length to truncated receptor protein. Decreasing the amount of truncated receptor results in the accumulation of full-length, constitutively active receptor at the cell surface. After injection into the third ventricle of mice, the oligonucleotide accumulates in the arcuate nucleus, where it changes alternative splicing of the serotonin 2C receptor and increases pro-opiomelanocortin expression. Oligonucleotide injection reduced food intake in both wild-type and ob/ob mice. Unexpectedly, the oligonucleotide crossed the blood-brain barrier and its systemic delivery reduced food intake in wild-type mice. The physiological effect of the oligonucleotide suggests that a truncated splice variant regulates the activity of the serotonin 2C receptor, indicating that therapies aimed to change pre-mRNA processing could be useful to treat hyperphagia, characteristic for disorders like Prader-Willi syndrome.

  3. Array-CGH联合NIPT进行染色体微缺失和重复产前检测的应用价值

    Institute of Scientific and Technical Information of China (English)

    池一婵; 符芳; 李茹; 黄海辉; 徐婉芳; 何怡; 林洋洋; 叶菀华; 隗伏冰


    目的 探讨无创产前检测技术(noninvasive prenatal testing,NIPT)联合基于微阵列芯片的比较基因杂交技术(array-based comparative genomic hybridization,Array-CGH)在染色体微缺失或重复中的产前检测价值.方法 选择孕12~24周高危孕妇,采集静脉血并提取循环胎儿游离DNA(cell-free fetal DNA,cffDNA),利用NIPT技术检测,结果为阳性者,适时进行羊膜腔或脐静脉穿刺并以Array-CGH 技术检测,以DECIPHER和OMIM数据库对检测数据进行分析,所有病例随访至胎儿出生.结果 NIPT检出10例阳性,Array-CGH检测和随访证实其中6例为染色体缺失或重复,但所检出的缺失或重复以及片段长度与NIPT不一样;4例Chr7长臂偏多为假阳性.另1例NIPT结果阴性但Array-CGH发现异常.结论 Array-CGH联合NIPT进行胎儿染色体微缺失和重复产前检测具有重要临床应用价值.

  4. Factor XI antisense oligonucleotide for prevention of venous thrombosis. (United States)

    Büller, Harry R; Bethune, Claudette; Bhanot, Sanjay; Gailani, David; Monia, Brett P; Raskob, Gary E; Segers, Annelise; Verhamme, Peter; Weitz, Jeffrey I


    Experimental data indicate that reducing factor XI levels attenuates thrombosis without causing bleeding, but the role of factor XI in the prevention of postoperative venous thrombosis in humans is unknown. FXI-ASO (ISIS 416858) is a second-generation antisense oligonucleotide that specifically reduces factor XI levels. We compared the efficacy and safety of FXI-ASO with those of enoxaparin in patients undergoing total knee arthroplasty. In this open-label, parallel-group study, we randomly assigned 300 patients who were undergoing elective primary unilateral total knee arthroplasty to receive one of two doses of FXI-ASO (200 mg or 300 mg) or 40 mg of enoxaparin once daily. The primary efficacy outcome was the incidence of venous thromboembolism (assessed by mandatory bilateral venography or report of symptomatic events). The principal safety outcome was major or clinically relevant nonmajor bleeding. Around the time of surgery, the mean (±SE) factor XI levels were 0.38±0.01 units per milliliter in the 200-mg FXI-ASO group, 0.20±0.01 units per milliliter in the 300-mg FXI-ASO group, and 0.93±0.02 units per milliliter in the enoxaparin group. The primary efficacy outcome occurred in 36 of 134 patients (27%) who received the 200-mg dose of FXI-ASO and in 3 of 71 patients (4%) who received the 300-mg dose of FXI-ASO, as compared with 21 of 69 patients (30%) who received enoxaparin. The 200-mg regimen was noninferior, and the 300-mg regimen was superior, to enoxaparin (P<0.001). Bleeding occurred in 3%, 3%, and 8% of the patients in the three study groups, respectively. This study showed that factor XI contributes to postoperative venous thromboembolism; reducing factor XI levels in patients undergoing elective primary unilateral total knee arthroplasty was an effective method for its prevention and appeared to be safe with respect to the risk of bleeding. (Funded by Isis Pharmaceuticals; FXI-ASO TKA number, NCT01713361.).

  5. Delivery of antisense oligonucleotide to the cornea by iontophoresis. (United States)

    Berdugo, M; Valamanesh, F; Andrieu, C; Klein, C; Benezra, D; Courtois, Y; Behar-Cohen, F


    We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the

  6. High-resolution genome-wide array-based comparative genome hybridization reveals cryptic chromosome changes in AML and MDS cases with trisomy 8 as the sole cytogenetic aberration. (United States)

    Paulsson, K; Heidenblad, M; Strömbeck, B; Staaf, J; Jönsson, G; Borg, A; Fioretos, T; Johansson, B


    Although trisomy 8 as the sole chromosome aberration is the most common numerical abnormality in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), little is known about its pathogenetic effects. Considering that +8 is a frequent secondary change in AML/MDS, cryptic--possibly primary--genetic aberrations may occur in cases with trisomy 8 as the apparently single anomaly. However, no such hidden anomalies have been reported. We performed a high-resolution genome-wide array-based comparative genome hybridization (array CGH) analysis of 10 AML/MDS cases with isolated +8, utilizing a 32K bacterial artificial chromosome array set, providing >98% coverage of the genome with a resolution of 100 kb. Array CGH revealed intrachromosomal imbalances, not corresponding to known genomic copy number polymorphisms, in 4/10 cases, comprising nine duplications and hemizygous deletions ranging in size from 0.5 to 2.2 Mb. A 1.8 Mb deletion at 7p14.1, which had occurred prior to the +8, was identified in MDS transforming to AML. Furthermore, a deletion including ETV6 was present in one case. The remaining seven imbalances involved more than 40 genes. The present results show that cryptic genetic abnormalities are frequent in trisomy 8-positive AML/MDS cases and that +8 as the sole cytogenetic aberration is not always the primary genetic event.

  7. Immunomodulatory oligonucleotide IMT504: Effects on mesenchymal stem cells as a first-in-class immunoprotective/immunoregenerative therapy (United States)

    Zorzopulos, Jorge; Opal, Steven M; Hernando-Insúa, Andrés; Rodriguez, Juan M; Elías, Fernanda; Fló, Juan; López, Ricardo A; Chasseing, Norma A; Lux-Lantos, Victoria A; Coronel, Maria F; Franco, Raul; Montaner, Alejandro D; Horn, David L


    The immune responses of humans and animals to insults (i.e., infections, traumas, tumoral transformation and radiation) are based on an intricate network of cells and chemical messengers. Abnormally high inflammation immediately after insult or abnormally prolonged pro-inflammatory stimuli bringing about chronic inflammation can lead to life-threatening or severely debilitating diseases. Mesenchymal stem cell (MSC) transplant has proved to be an effective therapy in preclinical studies which evaluated a vast diversity of inflammatory conditions. MSCs lead to resolution of inflammation, preparation for regeneration and actual regeneration, and then ultimate return to normal baseline or homeostasis. However, in clinical trials of transplanted MSCs, the expectations of great medical benefit have not yet been fulfilled. As a practical alternative to MSC transplant, a synthetic drug with the capacity to boost endogenous MSC expansion and/or activation may also be effective. Regarding this, IMT504, the prototype of a major class of immunomodulatory oligonucleotides, induces in vivo expansion of MSCs, resulting in a marked improvement in preclinical models of neuropathic pain, osteoporosis, diabetes and sepsis. IMT504 is easily manufactured and has an excellent preclinical safety record. In the small number of patients studied thus far, IMT504 has been well-tolerated, even at very high dosage. Further clinical investigation is necessary to demonstrate the utility of IMT504 for resolution of inflammation and regeneration in a broad array of human diseases that would likely benefit from an immunoprotective/immunoregenerative therapy.

  8. Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays. (United States)

    Kakiuchi-Kiyota, Satoko; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan


    Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity.

  9. Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides. (United States)

    Oehlke, J; Birth, P; Klauschenz, E; Wiesner, B; Beyermann, M; Oksche, A; Bienert, M


    The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

  10. Oligonucleotides conjugated with short chemically defined polyethylene glycol chains are efficient antisense agents. (United States)

    Shokrzadeh, Nasrin; Winkler, Anna-Maria; Dirin, Mehrdad; Winkler, Johannes


    Ligand conjugation is an attractive approach to rationally modify the poor pharmacokinetic behavior and cellular uptake properties of antisense oligonucleotides. Polyethylene glycol (PEG) attachment is a method to increase solubility of oligonucleotides and prevent the rapid elimination, thus increasing tissue distribution. On the other hand, the attachment of long PEG chains negatively influences the pharmacodynamic effect by reducing the hybridization efficiency. We examined the use of short PEG ligands on the in vitro effect of antisense agents. Circular dichroism showed that the tethering of PEG12-chains to phosphodiester and phosphorothioate oligonucleotides had no influence on their secondary structure and did not reduce the affinity to the counter strand. In an in vitro tumor model, a luciferase reporter assay indicated unchanged gene silencing activity compared to unmodified compounds, and even slightly superior target down regulation was found after treatment with a phosphorothioate modified conjugate. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Gene silencing by siRNAs and antisense oligonucleotides in the laboratory and the clinic (United States)

    Watts, Jonathan K.; Corey, David R.


    Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past twenty years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology. PMID:22069063

  12. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides (United States)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard


    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  13. ProbeMaker: an extensible framework for design of sets of oligonucleotide probes (United States)

    Stenberg, Johan; Nilsson, Mats; Landegren, Ulf


    Background Procedures for genetic analyses based on oligonucleotide probes are powerful tools that can allow highly parallel investigations of genetic material. Such procedures require the design of large sets of probes using application-specific design constraints. Results ProbeMaker is a software framework for computer-assisted design and analysis of sets of oligonucleotide probe sequences. The tool assists in the design of probes for sets of target sequences, incorporating sequence motifs for purposes such as amplification, visualization, or identification. An extension system allows the framework to be equipped with application-specific components for evaluation of probe sequences, and provides the possibility to include support for importing sequence data from a variety of file formats. Conclusion ProbeMaker is a suitable tool for many different oligonucleotide design and analysis tasks, including the design of probe sets for various types of parallel genetic analyses, experimental validation of design parameters, and in silico testing of probe sequence evaluation algorithms. PMID:16171527

  14. Streptavidin-coated gold nanoparticles: critical role of oligonucleotides on stability and fractal aggregation

    Directory of Open Access Journals (Sweden)

    Roberta D'Agata


    Full Text Available Gold nanoparticles (AuNPs exhibit unique properties that can be modulated through a tailored surface functionalization, enabling their targeted use in biochemical sensing and medical diagnostics. In particular, streptavidin-modified AuNPs are increasingly used for biosensing purposes. We report here a study of AuNPs surface-functionalized with streptavidin-biotinylated oligonucleotide, focussing on the role played by the oligonucleotide probes in the stabilization/destabilization of the functionalized nanoparticle dispersion. The behaviour of the modified AuNP dispersion as a consequence of the competitive displacement of the biotinylated oligonucleotide has been investigated and the critical role of displaced oligonucletides in triggering the quasi one-dimensional aggregation of nanoparticles is demonstrated for the first time. The thorough understanding of the fundamental properties of bioconjugated AuNPs is of great importance for the design of highly sensitive and reliable functionalized AuNP-based assays.

  15. Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study (United States)

    Sebastiani, F.; Longo, M.; Orecchini, A.; Comez, L.; De Francesco, A.; Muthmann, M.; Teixeira, S. C. M.; Petrillo, C.; Sacchetti, F.; Paciaroni, A.


    The dynamics of the human oligonucleotide AG3(T2AG3)3 has been investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in two different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calculated from the temperature-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This additional flexibility, which likely results from a significant relative chain-end contribution to the average chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides.

  16. Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study

    Energy Technology Data Exchange (ETDEWEB)

    Sebastiani, F.; Comez, L.; Sacchetti, F. [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); CNR, Istituto Officina dei Materiali, Unità di Perugia, c/o Dipartimento di Fisica e Geologia, Università di Perugia, 06123 Perugia (Italy); Longo, M. [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); Elettra—Sincrotrone Trieste, 34149 Basovizza, Trieste (Italy); Orecchini, A.; Petrillo, C.; Paciaroni, A., E-mail: [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); De Francesco, A. [CNR-IOM OGG c/o Institut Laue-Langevin, 71 Avenue des Martyrs, CS20156, 38042 Grenoble Cedex 9 (France); Muthmann, M. [Jülich Centre for Neutron Science, Forschungszentrum Jülich GmbH, Outstation at Heinz Maier-Leibnitz Zentrum, Lichtenbergstrasse 1, 85747 Garching (Germany); Teixeira, S. C. M. [EPSAM, Keele University, Staffordshire ST5 5BG (United Kingdom); Institut Laue–Langevin, 71 Avenue des Martyrs, CS20156, 38042 Grenoble Cedex 9 (France)


    The dynamics of the human oligonucleotide AG{sub 3}(T{sub 2}AG{sub 3}){sub 3} has been investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in two different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calculated from the temperature-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This additional flexibility, which likely results from a significant relative chain-end contribution to the average chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides.

  17. Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation

    DEFF Research Database (Denmark)

    Taskova, Maria; Madsen, Charlotte Stahl; Jensen, Knud Jørgen


    , most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides...... by the internal attachment of peptides but not by LNA alone. Independently of the peptide sequence, the conjugates are stable for up to 24 h in 90% human serum and duplexes of POCs with complementary DNA for up to 160 h in 90% human serum. Such excellent stability has not been previously reported for DNA...... and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media....

  18. Kinetic effects on signal normalization in oligonucleotide microchips with labeled immobilized probes. (United States)

    Pan'kov, S V; Chechetkin, V R; Somova, O G; Antonova, O V; Moiseeva, O V; Prokopenko, D V; Yurasov, R A; Gryadunov, D A; Chudinov, A V


    Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tuberculosis.

  19. Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique. (United States)

    Vasilkova, Daria V; Azhibek, Dulat M; Zatsepin, Timofei S; Naraikina, Yulia V; Prassolov, Vladimir S; Prokofjeva, Maria M; Zvereva, Maria I; Rubtsova, Maria P


    Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.

  20. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile. (United States)

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B


    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe

  1. Acute kidney injury during therapy with an antisense oligonucleotide directed against PCSK9. (United States)

    van Poelgeest, Eveline P; Swart, Reinout M; Betjes, Michiel G H; Moerland, Matthijs; Weening, Jan J; Tessier, Yann; Hodges, Michael R; Levin, Arthur A; Burggraaf, Jacobus


    Antisense oligonucleotides have been explored widely in clinical trials and generally are considered to be nontoxic for the kidney, even at high concentrations. We report a case of toxic acute tubular injury in a healthy 56-year-old female volunteer after a pharmacologically active dose of a locked nucleic acid antisense oligonucleotide was administered. The patient received 3 weekly subcutaneous doses of experimental drug SPC5001, an antisense oligonucleotide directed against PCSK9 (proprotein convertase subtilisin/kexin type 9) that is under investigation as an agent to reduce low-density lipoprotein cholesterol levels. Five days after the last dose, the patient's serum creatinine level increased from 0.81 mg/dL at baseline (corresponding to an estimated glomerular filtration rate [eGFR] of 78 mL/min/1.73 m(2)) to 2.67 mg/dL (eGFR, 20 mL/min/1.73 m(2)), and this increase coincided with the presence of white blood cells, granular casts, and minimal hematuria on urine microscopy. The patient's serum creatinine level peaked at 3.81 mg/dL (eGFR, 13 mL/min/1.73 m(2)) 1 week after the last oligonucleotide dose. Kidney biopsy showed multifocal tubular necrosis and signs of oligonucleotide accumulation. Upon conservative treatment, the patient's serum creatinine level gradually decreased and reached her baseline level 44 days after the last oligonucleotide was administered. The patient recovered fully and kidney function was normal at every follow-up visit. Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

  2. CGH microarray studies in idiopathic developmental/cognitive impairment: association of historical and clinical features and the De Vries Score

    Directory of Open Access Journals (Sweden)

    Promilla Perattur


    Full Text Available Objective. Studies have confirmed that copy number variations (CNV in the human genome contribute to the etiology of mental retardation/ development delay/ congenital anomalies. We sought to evaluate the use of a microarray in the context of a clinical genetics practice, to determine if there were any specific clinical findings that predict the discovery of a CNV. Patients and methods. 334 cases with idiopathic mental retardation/impairment/development delay/disability or a combination of these findings were studied using array comparative genomic hybridization (Signature Chip Version 4. The subjects had previously had a non diagnostic medical genetics evaluation. Clinical findings were collated by a chart review. Each patient was scored according to a previously published clinical checklist by de Vries and colleagues. Results. Of 334 patients, 8 were excluded due to a syndromic diagnosis being established by clinical and/or microarray testing. Out of the remaining 326 patients, 33 (10% showed CNVs, of which 5 were maternally inherited, 4 paternally inherited, 11 were de novo, and the origin of 13 remained unknown. The mean de Vries score was greater in the CNV group than in the non CNV group (4.17 and 3.95, respectively. No patient in the CNV group had a score of less than 3, while in the non CNV group, 12% of patients had scores less than 3. Conclusions. The De Vries clinical score was higher in CNV cases compared to those with no CNV (p=0.04 but this difference is unlikely to be clinically meaningful. Several features reached statistical significance of p<0.05 but we were unable to delineate patterns of features that might increase the yield of positive CNV results.

  3. Inherited Xq13.2-q21.31 duplication in a boy with recurrent seizures and pubertal gynecomastia: Clinical, chromosomal and aCGH characterization

    Directory of Open Access Journals (Sweden)

    Natália D. Linhares


    Full Text Available We report on a 16-year-old boy with a maternally inherited ~18.3 Mb Xq13.2-q21.31 duplication delimited by aCGH. As previously described in patients with similar duplications, his clinical features included intellectual disability, developmental delay, speech delay, generalized hypotonia, infantile feeding difficulties, self-injurious behavior, short stature and endocrine problems. As additional findings, he presented recurrent seizures and pubertal gynecomastia. His mother was phenotypically normal and had completely skewed inactivation of the duplicated X chromosome, as most female carriers of such duplications. Five previously reported patients with partial Xq duplications presented duplication breakpoints similar to those of our patient. One of them, a fetus with multiple congenital abnormalities, had the same cytogenetic duplication breakpoint. Three of the reported patients shared many features with our proband but the other had some clinical features of the Prader-Willi syndrome. It was suggested that ATRX overexpression could be involved in the major clinical features of patients with partial Xq duplications. We propose that this gene could also be involved with the obesity of the patient with the Prader-Willi-like phenotype. Additionally, we suggest that the PCDH11X gene could be a candidate for our patient's recurrent seizures. In males, the Xq13-q21 duplication should be considered in the differential diagnosis of Prader-Willi syndrome, as previously suggested, and neuromuscular diseases, particularly mitochondriopathies.

  4. Use of thiolated oligonucleotides as anti-fouling diluents in electrochemical peptide-based sensors. (United States)

    McQuistan, Adam; Zaitouna, Anita J; Echeverria, Elena; Lai, Rebecca Y


    We incorporated short thiolated oligonucleotides as passivating diluents in the fabrication of electrochemical peptide-based (E-PB) sensors, with the goal of creating a negatively charged layer capable of resisting non-specific adsorption of matrix contaminants. The E-PB HIV sensors fabricated using these diluents were found to be more specific and selective, while retaining attributes similar to the sensor fabricated without these diluents. Overall, these results highlight the advantages of using oligonucleotides as anti-fouling diluents in self-assembled monolayer-based sensors.

  5. Oligonucleotide-templated chemical reactions: pushing the boundaries of a nature-inspired process. (United States)

    Percivalle, Claudia; Bartolo, Jean-François; Ladame, Sylvain


    Widespread in nature, oligonucleotide-templated reactions of phosphodiester bond formation have inspired chemists who are now applying this elegant strategy to the catalysis of a broad range of otherwise inefficient reactions. This review highlights the increasing diversity of chemical reactions that can be efficiently catalysed by an oligonucleotide template, using Watson-Crick base-pairing to bring both reagents in close enough proximity to react, thus increasing significantly their effective molarity. The applications of this elegant concept for nucleic acid sensing and controlled organic synthesis will also be discussed.

  6. Transcriptional inhibition of the bacteriophage T7 early promoter region by oligonucleotide triple helix formation. (United States)

    Ross, C; Samuel, M; Broitman, S L


    We have identified a purine-rich triplex binding sequence overlapping a -35 transcriptional early promoter region of the bacteriophage T7. Triplex-forming oligonucleotide designed to bind this target was annealed to T7 templates and introduced into in vitro transcription systems under conditions favoring specific initiation from this promoter. These templates demonstrated significant transcriptional inhibition relative to naked genomic templates and templates mixed with non-triplex-forming oligonucleotide. It is suggested that triplex formation along this target interferes with transcriptional initiation, and this mechanism may hold potential to disrupt bacteriophage T7 early transcription in vivo.

  7. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes. (United States)

    Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O


    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.

  8. Customized oligonucleotide microchips that convert multiple genetic information to simple patterns, are portable and reusable (United States)

    Mirzabekov, Andrei; Guschin, Dmitry Y.; Chik, Valentine; Drobyshev, Aleksei; Fotin, Alexander; Yershov, Gennadiy; Lysov, Yuri


    This inv