Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas

Science.gov (United States)

Mohapatra, Gayatry; Engler, David A.; Starbuck, Kristen D.; Kim, James C.; Bernay, Derek C.; Scangas, George A.; Rousseau, Audrey; Batchelor, Tracy T.; Betensky, Rebecca A.; Louis, David N.

2010-01-01

Molecular genetic analysis of cancer is rapidly evolving as a result of improvement in genomic technologies and the growing applicability of such analyses to clinical oncology. Array based comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA), particularly in solid tumors, and has been applied to the study of malignant gliomas. In the clinical setting, however, gliomas are often sampled by small biopsies and thus formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis, especially for rare types of gliomas. Moreover, the biological basis for the marked intratumoral heterogeneity in gliomas is most readily addressed in FFPE material. Therefore, for gliomas, the ability to use DNA from FFPE tissue is essential for both clinical and research applications. In this study, we have constructed a custom bacterial artificial chromosome (BAC) array and show excellent sensitivity and specificity for detecting CNAs in a panel of paired frozen and FFPE glioma samples. Our study demonstrates a high concordance rate between CNAs detected in FFPE compared to frozen DNA. We have also developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. This labeling technique was applied to a panel of biphasic anaplastic oligoastrocytomas (AOA) to identify genetic changes unique to each component. Together, results from these studies suggest that BAC and oligonucleotide aCGH are sensitive tools for detecting CNAs in FFPE DNA, and can enable genome-wide analysis of rare, small and/or histologically heterogeneous gliomas. PMID:21080181

Application of high resolution SNP arrays in patients with congenital ...

Indian Academy of Sciences (India)

TING-YING LEI

lent oligonucleotide-based array-CGH to determine the exact breakpoints in 14 patients with partial deletions of chromo- some 13q21.1-qter. They were able to refine the smallest deletion region linked to cleft lip/palate (13q31.3–13q33.1). Except for the arrays that measure DNA copy number differ- ences only, SNP arrays, ...

CGHnormaliter: a bioconductor package for normalization of array CGH data with many CNAs.

NARCIS (Netherlands)

van Houte, B.P.P.; Binsl, T.W.; Hettling, H.; Heringa, J.

2010-01-01

Summary: CGHnormaliter is a package for normalization of array comparative genomic hybridization (aCGH) data. It uses an iterative procedure that effectively eliminates the influence of imbalanced copy numbers. This leads to a more reliable assessment of copy number alterations (CNAs). CGHnormaliter

Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion.

Science.gov (United States)

Zhang, Shulin; Li, Fang-Yuan; Bass, Harold N; Pursley, Amber; Schmitt, Eric S; Brown, Blaire L; Brundage, Ellen K; Mardach, Rebecca; Wong, Lee-Jun

2010-01-01

Thymidine kinase 2 (TK2), encoded by the TK2 gene on chromosome 16q22, is one of the deoxyribonucleoside kinases responsible for the maintenance of mitochondrial deoxyribonucleotide pools. Defects in TK2 mainly cause a myopathic form of the mitochondrial DNA depletion syndrome (MDDS). Currently, only point mutations and small insertions and deletions have been reported in TK2 gene; gross rearrangements of TK2 gene and possible hepatic involvement in patients with TK2 mutations have not been described. We report a non-consanguineous Jordanian family with three deceased siblings due to mtDNA depletion. Sequence analysis of the father detected a heterozygous c.761T>A (p.I254N) mutation in his TK2 gene; however, point mutations in the mother were not detected. Subsequent gene dosage analysis using oligonucleotide array CGH identified an intragenic approximately 5.8-kb deletion encompassing the 5'UTR to intron 2 of her TK2 gene. Sequence analysis confirmed that the deletion spans c.1-495 to c.283-2899 of the TK2 gene (nucleotide 65,136,256-65,142,086 of chromosome 16). Analysis of liver and muscle specimens from one of the deceased infants in this family revealed compound heterozygosity for the paternal point mutation and maternal intragenic deletion. In addition, a significant reduction of the mtDNA content in liver and muscle was detected (10% and 20% of age- and tissue-matched controls, respectively). Prenatal diagnosis was performed in the third pregnancy. The fetus was found to carry both the point mutation and the deletion. This child died 6months after birth due to myopathy. A serum specimen demonstrated elevated liver transaminases in two of the infants from whom results were available. This report expands the mutation spectrum associated with TK2 deficiency. While the myopathic form of MDDS appears to be the main phenotype of TK2 mutations, liver dysfunction may also be a part of the mitochondrial depletion syndrome caused by TK2 gene defects.

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

2012-02-01

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.

Array-CGH Analysis in a Cohort of Phenotypically Well-Characterized Individuals with "Essential" Autism Spectrum Disorders

Science.gov (United States)

Napoli, Eleonora; Russo, Serena; Casula, Laura; Alesi, Viola; Amendola, Filomena Alessandra; Angioni, Adriano; Novelli, Antonio; Valeri, Giovanni; Menghini, Deny; Vicari, Stefano

2018-01-01

Copy-number variants (CNVs) are associated with susceptibility to autism spectrum disorder (ASD). To detect the presence of CNVs, we conducted an array-comparative genomic hybridization (array-CGH) analysis in 133 children with "essential" ASD phenotype. Genetic analyses documented that 12 children had causative CNVs (C-CNVs), 29…

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

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Oga Atsunori

2008-12-01

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.

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

International Nuclear Information System (INIS)

Furuya, Tomoko; Uchiyama, Tetsuji; Adachi, Atsushi; Okada, Takae; Nakao, Motonao; Oga, Atsunori; Yang, Song-Ju; Kawauchi, Shigeto; Sasaki, Kohsuke

2008-01-01

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. 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. 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. These results suggest that the mini-array makes it possible to determine an optimal treatment for each of the patients with gastric adenocarcinoma

Identification of clinically relevant viridans streptococci by an oligonucleotide array.

Science.gov (United States)

Chen, Chao Chien; Teng, Lee Jene; Kaiung, Seng; Chang, Tsung Chain

2005-04-01

Viridans streptococci (VS) are common etiologic agents of subacute infective endocarditis and are capable of causing a variety of pyogenic infections. Many species of VS are difficult to differentiate by phenotypic traits. An oligonucleotide array based on 16S-23S rRNA gene intergenic spacer (ITS) sequences was developed to identify 11 clinically relevant VS. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The method consisted of PCR amplification of the ITS regions by using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species-specific oligonucleotides immobilized on a nylon membrane. After 120 strains of the 11 species of VG and 91 strains of other bacteria were tested, the sensitivity and specificity of the oligonucleotide array were found to be 100% (120 of 120 strains) and 95.6% (87 of 91 strains), respectively. S. pneumoniae cross-hybridized to the probes used for the identification of S. mitis, and simple biochemical tests such as optochin susceptibility or bile solubility should be used to differentiate S. pneumoniae from S. mitis. In conclusion, identification of species of VS by use of the present oligonucleotide array is accurate and could be used as an alternative reliable method for species identification of strains of VS.

Genotype-phenotype analysis of recombinant chromosome 4 syndrome: an array-CGH study and literature review.

Science.gov (United States)

Hemmat, Morteza; Hemmat, Omid; Anguiano, Arturo; Boyar, Fatih Z; El Naggar, Mohammed; Wang, Jia-Chi; Wang, Borris T; Sahoo, Trilochan; Owen, Renius; Haddadin, Mary

2013-05-02

Recombinant chromosome 4, a rare constitutional rearrangement arising from pericentric inversion, comprises a duplicated segment of 4p13~p15→4pter and a deleted segment of 4q35→4qter. To date, 10 cases of recombinant chromosome 4 have been reported. We describe the second case in which array-CGH was used to characterize recombinant chromosome 4 syndrome. The patient was a one-year old boy with consistent clinical features. Conventional cytogenetics and FISH documented a recombinant chromosome 4, derived from a paternal pericentric inversion, leading to partial trisomy 4p and partial monosomy of 4q. Array-CGH, performed to further characterize the rearranged chromosome 4 and delineate the breakpoints, documented a small (4.36 Mb) 4q35.1 terminal deletion and a large (23.81 Mb) 4p15.1 terminal duplication. Genotype-phenotype analysis of 10 previously reported cases and the present case indicated relatively consistent clinical features and breakpoints. This consistency was more evident in our case and another characterized by array-CGH, where both showed the common breakpoints of p15.1 and q35.1. A genotype-phenotype correlation study between rec(4), dup(4p), and del(4q) syndromes revealed that urogenital and cardiac defects are probably due to the deletion of 4q whereas the other clinical features are likely due to 4p duplication. Our findings support that the clinical features of patients with rec(4) are relatively consistent and specific to the regions of duplication or deletion. Recombinant chromosome 4 syndrome thus appears to be a discrete entity that can be suspected on the basis of clinical features or specific deleted and duplicated chromosomal regions.

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)

Becher, Naja; Gjørup, Vibike; Christensen, Rikke

2012-01-01

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...

Combined use of expression and CGH arrays pinpoints novel candidate genes in Ewing sarcoma family of tumors

International Nuclear Information System (INIS)

Savola, Suvi; Knuutila, Sakari; Klami, Arto; Tripathi, Abhishek; Niini, Tarja; Serra, Massimo; Picci, Piero; Kaski, Samuel; Zambelli, Diana; Scotlandi, Katia

2009-01-01

Ewing sarcoma family of tumors (ESFT), characterized by t(11;22)(q24;q12), is one of the most common tumors of bone in children and young adults. In addition to EWS/FLI1 gene fusion, copy number changes are known to be significant for the underlying neoplastic development of ESFT and for patient outcome. Our genome-wide high-resolution analysis aspired to pinpoint genomic regions of highest interest and possible target genes in these areas. Array comparative genomic hybridization (CGH) and expression arrays were used to screen for copy number alterations and expression changes in ESFT patient samples. A total of 31 ESFT samples were analyzed by aCGH and in 16 patients DNA and RNA level data, created by expression arrays, was integrated. Time of the follow-up of these patients was 5–192 months. Clinical outcome was statistically evaluated by Kaplan-Meier/Logrank methods and RT-PCR was applied on 42 patient samples to study the gene of the highest interest. Copy number changes were detected in 87% of the cases. The most recurrent copy number changes were gains at 1q, 2, 8, and 12, and losses at 9p and 16q. Cumulative event free survival (ESFT) and overall survival (OS) were significantly better (P < 0.05) for primary tumors with three or less copy number changes than for tumors with higher number of copy number aberrations. In three samples copy number imbalances were detected in chromosomes 11 and 22 affecting the FLI1 and EWSR1 loci, suggesting that an unbalanced t(11;22) and subsequent duplication of the derivative chromosome harboring fusion gene is a common event in ESFT. Further, amplifications on chromosomes 20 and 22 seen in one patient sample suggest a novel translocation type between EWSR1 and an unidentified fusion partner at 20q. In total 20 novel ESFT associated putative oncogenes and tumor suppressor genes were found in the integration analysis of array CGH and expression data. Quantitative RT-PCR to study the expression levels of the most interesting

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

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Siggberg Linda

2012-09-01

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.

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

International Nuclear Information System (INIS)

Samuelson, Emma; Karlsson, Sara; Partheen, Karolina; Nilsson, Staffan; Szpirer, Claude; Behboudi, Afrouz

2012-01-01

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. 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. 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. 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 genes with a critical role in mammary tumor development. Genetic

Spatially Defined Oligonucleotide Arrays. Technical Report for Phase II; FINAL

International Nuclear Information System (INIS)

None

2000-01-01

The goal of the Human Genome Project is to sequence all 3 billion base pairs of the human genome. Progress in this has been rapid; GenBank(reg s ign) finished 1994 with 286 million bases of sequence and grew by 2470 in the first quarter of 1995. The challenge to the scientific community is to understand the biological relevance of this genetic information. In most cases the sequence being generated for any single region of the genome represents the genotype of a single individual. A complete understanding of the function of specific genes and other regions of the genome and their role in human disease and development will only become apparent when the sequence of many more individuals is known. Access to genetic information is ultimately limited by the ability to screen DNA sequence. Although the pioneering sequencing methods of Sanger et al. (15) and Maxam and Gilbert (11) have become standard in virtually all molecular biology laboratories, the basic protocols remain largely unchanged. The throughput of this sequencing technology is now becoming the rate-limiting step in both large-scale sequencing projects such as the Human Genome Project and the subsequent efforts to understand genetic diversity. This has inspired the development of advanced DNA sequencing technologies (9), Incremental improvements to Sanger sequencing have been made in DNA labeling and detection. High-speed electrophoresis methods using ultrathin gels or capillary arrays are now being more widely employed. However, these methods are throughput-limited by their sequential nature and the speed and resolution of separations. This limitation will become more pronounced as the need to rapidly screen newly discovered genes for biologically relevant polymorphisms increases. An alternative to gel-based sequencing is to use high-density oligonucleotide probe arrays. Oligonucleotide probe arrays display specific oligonucleotide probes at precise locations in a high density, information-rich format (5

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.

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

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Mark A. van de Wiel

2007-01-01

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.

Recurrence, submicroscopic complexity, and potential clinical relevance of copy gains detected by array CGH that are shown to be unbalanced insertions by FISH.

Science.gov (United States)

Neill, Nicholas J; Ballif, Blake C; Lamb, Allen N; Parikh, Sumit; Ravnan, J Britt; Schultz, Roger A; Torchia, Beth S; Rosenfeld, Jill A; Shaffer, Lisa G

2011-04-01

Insertions occur when a segment of one chromosome is translocated and inserted into a new region of the same chromosome or a non-homologous chromosome. We report 71 cases with unbalanced insertions identified using array CGH and FISH in 4909 cases referred to our laboratory for array CGH and found to have copy-number abnormalities. Although the majority of insertions were non-recurrent, several recurrent unbalanced insertions were detected, including three der(Y)ins(Y;18)(q?11.2;p11.32p11.32)pat inherited from parents carrying an unbalanced insertion. The clinical significance of these recurrent rearrangements is unclear, although the small size, limited gene content, and inheritance pattern of each suggests that the phenotypic consequences may be benign. Cryptic, submicroscopic duplications were observed at or near the insertion sites in two patients, further confounding the clinical interpretation of these insertions. Using FISH, linear amplification, and array CGH, we identified a 126-kb duplicated region from 19p13.3 inserted into MECP2 at Xq28 in a patient with symptoms of Rett syndrome. Our results demonstrate that although the interpretation of most non-recurrent insertions is unclear without high-resolution insertion site characterization, the potential for an otherwise benign duplication to result in a clinically relevant outcome through the disruption of a gene necessitates the use of FISH to determine whether copy-number gains detected by array CGH represent tandem duplications or unbalanced insertions. Further follow-up testing using techniques such as linear amplification or sequencing should be used to determine gene involvement at the insertion site after FISH has identified the presence of an insertion.

High-resolution array CGH clarifies events occurring on 8p in carcinogenesis

International Nuclear Information System (INIS)

Cooke, Susanna L; Pole, Jessica CM; Chin, Suet-Feung; Ellis, Ian O; Caldas, Carlos; Edwards, Paul AW

2008-01-01

Rearrangement of the short arm of chromosome 8 (8p) is very common in epithelial cancers such as breast cancer. Usually there is an unbalanced translocation breakpoint in 8p12 (29.7 Mb – 38.5 Mb) with loss of distal 8p, sometimes with proximal amplification of 8p11-12. Rearrangements in 8p11-12 have been investigated using high-resolution array CGH, but the first 30 Mb of 8p are less well characterised, although this region contains several proposed tumour suppressor genes. We analysed the whole of 8p by array CGH at tiling-path BAC resolution in 32 breast and six pancreatic cancer cell lines. Regions of recurrent rearrangement distal to 8p12 were further characterised, using regional fosmid arrays. FISH, and quantitative RT-PCR on over 60 breast tumours validated the existence of similar events in primary material. We confirmed that 8p is usually lost up to at least 30 Mb, but a few lines showed focal loss or copy number steps within this region. Three regions showed rearrangements common to at least two cases: two regions of recurrent loss and one region of amplification. Loss within 8p23.3 (0 Mb – 2.2 Mb) was found in six cell lines. Of the genes always affected, ARHGEF10 showed a point mutation of the remaining normal copies in the DU4475 cell line. Deletions within 12.7 Mb – 19.1 Mb in 8p22, in two cases, affected TUSC3. A novel amplicon was found within 8p21.3 (19.1 Mb – 23.4 Mb) in two lines and one of 98 tumours. The pattern of rearrangements seen on 8p may be a consequence of the high density of potential targets on this chromosome arm, and ARHGEF10 may be a new candidate tumour suppressor gene

Oligo-based High-resolution aCGH Analysis Enhances Routine Cytogenetic Diagnostics in Haematological Malignancies.

Science.gov (United States)

Kjeldsen, Eigil

2015-01-01

The purpose of the present study was to evaluate the detection rate of genomic aberrations in haematological malignancies using oligobased array-CGH (oaCGH) analysis in combination with karyotyping and fluorescence in situ hybridization (FISH) analyses, and its feasibility in a clinical pragmatic approach. The 4x180K Cancer Cytochip array was applied in 96 patients with various haematological malignancies in a prospective setting and in 41 acute myeloid leukemia (AML) patients retrospectively. Combined use of oaCGH analysis and karyotyping improved the overall detection rate in comparison to karyotyping-alone and vice versa. In cases with normal karyotypes oaCGH analysis detected genomic aberrations in 66% (39/60) of cases. In the group of simple karyotypes oaCGH analysis extended karyotypic findings in 39% (12/31) while oaCGH analysis extended the karyotypic findings in 89% (39/44) of cases with complex karyotypes. In 7% (5/75) of cases oaCGH analysis failed in detecting the observed abnormalities by karyotyping. oaCGH analysis is a valuable asset in routine cytogenetics of haematological malignancies. Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

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

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Quimsiyeh Mazin

2008-12-01

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.

Different Array CGH profiles within hereditary breast cancer tumors associated to BRCA1 expression and overall survival

International Nuclear Information System (INIS)

Alvarez, Carolina; Aravena, Andrés; Tapia, Teresa; Rozenblum, Ester; Solís, Luisa; Corvalán, Alejandro; Camus, Mauricio; Alvarez, Manuel; Munroe, David; Maass, Alejandro; Carvallo, Pilar

2016-01-01

Array CGH analysis of breast tumors has contributed to the identification of different genomic profiles in these tumors. Loss of DNA repair by BRCA1 functional deficiency in breast cancer has been proposed as a relevant contribution to breast cancer progression for tumors with no germline mutation. Identifying the genomic alterations taking place in BRCA1 not expressing tumors will lead us to a better understanding of the cellular functions affected in this heterogeneous disease. Moreover, specific genomic alterations may contribute to the identification of potential therapeutic targets and offer a more personalized treatment to breast cancer patients. Forty seven tumors from hereditary breast cancer cases, previously analyzed for BRCA1 expression, and screened for germline BRCA1 and 2 mutations, were analyzed by Array based Comparative Genomic Hybridization (aCGH) using Agilent 4x44K arrays. Overall survival was established for tumors in different clusters using Log-rank (Mantel-Cox) Test. Gene lists obtained from aCGH analysis were analyzed for Gene Ontology enrichment using GOrilla and DAVID tools. Genomic profiling of the tumors showed specific alterations associated to BRCA1 or 2 mutation status, and BRCA1 expression in the tumors, affecting relevant cellular processes. Similar cellular functions were found affected in BRCA1 not expressing and BRCA1 or 2 mutated tumors. Hierarchical clustering classified hereditary breast tumors in four major, groups according to the type and amount of genomic alterations, showing one group with a significantly poor overall survival (p = 0.0221). Within this cluster, deletion of PLEKHO1, GDF11, DARC, DAG1 and CD63 may be associated to the worse outcome of the patients. These results support the fact that BRCA1 lack of expression in tumors should be used as a marker for BRCAness and to select these patients for synthetic lethality approaches such as treatment with PARP inhibitors. In addition, the identification of specific

Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species.

Science.gov (United States)

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

2013-01-01

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.

Array CGH Analysis and Developmental Delay: A Diagnostic Tool for Neurologists.

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Cameron, F; Xu, J; Jung, J; Prasad, C

2013-11-01

Developmental delay occurs in 1-3% of the population, with unknown etiology in approximately 50% of cases. Initial genetic work up for developmental delay previously included chromosome analysis and subtelomeric FISH (fluorescent in situ hybridization). Array Comparative Genomic Hybridization (aCGH) has emerged as a tool to detect genetic copy number changes and uniparental disomy and is the most sensitive test in providing etiological diagnosis in developmental delay. aCGH allows for the provision of prognosis and recurrence risks, improves access to resources, helps limit further investigations and may alter medical management in many cases. aCGH has led to the delineation of novel genetic syndromes associated with developmental delay. An illustrative case of a 31-year-old man with long standing global developmental delay and recently diagnosed 4q21 deletion syndrome with a deletion of 20.8 Mb genomic interval is provided. aCGH is now recommended as a first line test in children and adults with undiagnosed developmental delay and congenital anomalies. Puce d'hybridation génomique comparative et retard de développement : un outil diagnostic pour les neurologues. Le retard de développement survient chez 1 à 3% de la population et son étiologie est inconnue chez à peu près 50% des cas. L'évaluation génétique initiale pour un retard de développement incluait antérieurement une analyse chromosomique et une analyse par FISH (hybridation in situ en fluorescence) de régions subtélomériques. La puce d'hybridation génomique comparative (CGHa) est devenue un outil de détection des changements du nombre de copies géniques ainsi que de la disomie uniparentale et elle est le test le plus sensible pour fournir un diagnostic étiologique dans le retard de développement. Le CGHa permet d'offrir un pronostic et un risque de récurrence, améliore l'accès aux ressources, aide à limiter les évaluations et peut modifier le traitement médical dans bien des cas

Analysis of oligonucleotide array experiments with repeated measures using mixed models

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Getchell Thomas V

2004-12-01

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.

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

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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.

Combining gene expression data from different generations of oligonucleotide arrays

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Kong Sek

2004-10-01

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

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

2012-05-01

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.

MYC and MYCN amplification can be reliably assessed by aCGH in medulloblastoma.

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Bourdeaut, Franck; Grison, Camille; Maurage, Claude-Alain; Laquerriere, Annie; Vasiljevic, Alexandre; Delisle, Marie-Bernadette; Michalak, Sophie; Figarella-Branger, Dominique; Doz, François; Richer, Wilfrid; Pierron, Gaelle; Miquel, Catherine; Delattre, Olivier; Couturier, Jérôme

2013-04-01

As prognostic factors, MYC and MYCN amplifications are routinely assessed in medulloblastomas. Fluorescence in situ hybridization (FISH) is currently considered as the technique of reference. Recently, array comparative genomic hybridization (aCGH) has been developed as an alternative technique to evaluate genomic abnormalities in other tumor types; however, this technique has not been widely adopted as a replacement for FISH in medulloblastoma. In this study, 34 tumors were screened by both FISH and aCGH. In all cases showing amplification by FISH, aCGH also unambiguously revealed the abnormality. The aCGH technique was also performed on tumors showing no amplification by FISH, and the absence of amplification was confirmed in all cases. Interestingly, one tumor showed a subclonal MYC amplification by FISH. This subclonal amplification was observed in approximately 20% of tumor cells and was clearly evident on aCGH. In conclusion, our analysis confirms that aCGH is as safe as FISH for the detection of MYC/MYCN gene amplification. Given its cost efficiency in comparison to two FISH tests and the global genomic information additionally provided by an aCGH experiment, this reproducible technique can be safely retained as an alternative to FISH for routine investigation of medulloblastoma. Copyright © 2013 Elsevier Inc. All rights reserved.

Efficient oligonucleotide probe selection for pan-genomic tiling arrays

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Zhang Wei

2009-09-01

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

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.

Correction of Spatial Bias in Oligonucleotide Array Data

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Philippe Serhal

2013-01-01

Full Text Available Background. Oligonucleotide microarrays allow for high-throughput gene expression profiling assays. The technology relies on the fundamental assumption that observed hybridization signal intensities (HSIs for each intended target, on average, correlate with their target’s true concentration in the sample. However, systematic, nonbiological variation from several sources undermines this hypothesis. Background hybridization signal has been previously identified as one such important source, one manifestation of which appears in the form of spatial autocorrelation. Results. We propose an algorithm, pyn, for the elimination of spatial autocorrelation in HSIs, exploiting the duality of desirable mutual information shared by probes in a common probe set and undesirable mutual information shared by spatially proximate probes. We show that this correction procedure reduces spatial autocorrelation in HSIs; increases HSI reproducibility across replicate arrays; increases differentially expressed gene detection power; and performs better than previously published methods. Conclusions. The proposed algorithm increases both precision and accuracy, while requiring virtually no changes to users’ current analysis pipelines: the correction consists merely of a transformation of raw HSIs (e.g., CEL files for Affymetrix arrays. A free, open-source implementation is provided as an R package, compatible with standard Bioconductor tools. The approach may also be tailored to other platform types and other sources of bias.

Correction of Spatial Bias in Oligonucleotide Array Data

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Lemieux, Sébastien

2013-01-01

Background. Oligonucleotide microarrays allow for high-throughput gene expression profiling assays. The technology relies on the fundamental assumption that observed hybridization signal intensities (HSIs) for each intended target, on average, correlate with their target's true concentration in the sample. However, systematic, nonbiological variation from several sources undermines this hypothesis. Background hybridization signal has been previously identified as one such important source, one manifestation of which appears in the form of spatial autocorrelation. Results. We propose an algorithm, pyn, for the elimination of spatial autocorrelation in HSIs, exploiting the duality of desirable mutual information shared by probes in a common probe set and undesirable mutual information shared by spatially proximate probes. We show that this correction procedure reduces spatial autocorrelation in HSIs; increases HSI reproducibility across replicate arrays; increases differentially expressed gene detection power; and performs better than previously published methods. Conclusions. The proposed algorithm increases both precision and accuracy, while requiring virtually no changes to users' current analysis pipelines: the correction consists merely of a transformation of raw HSIs (e.g., CEL files for Affymetrix arrays). A free, open-source implementation is provided as an R package, compatible with standard Bioconductor tools. The approach may also be tailored to other platform types and other sources of bias. PMID:23573083

Whole genome DNA copy number changes identified by high density oligonucleotide arrays

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Huang Jing

2004-05-01

Full Text Available Abstract Changes in DNA copy number are one of the hallmarks of the genetic instability common to most human cancers. Previous micro-array-based methods have been used to identify chromosomal gains and losses; however, they are unable to genotype alleles at the level of single nucleotide polymorphisms (SNPs. Here we describe a novel algorithm that uses a recently developed high-density oligonucleotide array-based SNP genotyping method, whole genome sampling analysis (WGSA, to identify genome-wide chromosomal gains and losses at high resolution. WGSA simultaneously genotypes over 10,000 SNPs by allele-specific hybridisation to perfect match (PM and mismatch (MM probes synthesised on a single array. The copy number algorithm jointly uses PM intensity and discrimination ratios between paired PM and MM intensity values to identify and estimate genetic copy number changes. Values from an experimental sample are compared with SNP-specific distributions derived from a reference set containing over 100 normal individuals to gain statistical power. Genomic regions with statistically significant copy number changes can be identified using both single point analysis and contiguous point analysis of SNP intensities. We identified multiple regions of amplification and deletion using a panel of human breast cancer cell lines. We verified these results using an independent method based on quantitative polymerase chain reaction and found that our approach is both sensitive and specific and can tolerate samples which contain a mixture of both tumour and normal DNA. In addition, by using known allele frequencies from the reference set, statistically significant genomic intervals can be identified containing contiguous stretches of homozygous markers, potentially allowing the detection of regions undergoing loss of heterozygosity (LOH without the need for a matched normal control sample. The coupling of LOH analysis, via SNP genotyping, with copy number

New insights in the interpretation of array-CGH: autism spectrum disorder and positive family history for intellectual disability predict the detection of pathogenic variants.

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Cappuccio, Gerarda; Vitiello, Francesco; Casertano, Alberto; Fontana, Paolo; Genesio, Rita; Bruzzese, Dario; Ginocchio, Virginia Maria; Mormile, Angela; Nitsch, Lucio; Andria, Generoso; Melis, Daniela

2016-04-12

Array-CGH (aCGH) is presently used into routine clinical practice for diagnosis of patients with intellectual disability (ID), multiple congenital anomalies (MCA), and autism spectrum disorder (ASD). ACGH could detect small chromosomal imbalances, copy number variations (CNVs), and closely define their size and gene content. ACGH detects pathogenic imbalances in 14-20 % of patients with ID. The aims of this study were: to establish clinical clues potentially associated with pathogenic CNVs and to identify cytogenetic indicators to predict the pathogenicity of the variants of uncertain significance (VOUS) in a large cohort of paediatric patients. We enrolled 214 patients referred for either: ID, and/or ASD and/or MCA to genetic services at the Federico II University of Naples, Department of Translational Medicine. For each patient we collected clinical and imaging data. All the patients were tested with aCGH or as first-tier test or as part of a wider diagnostic work-up. Pathologic data were detected in 65 individuals (30 %) and 46 CNVs revealed a known syndrome. The pathological CNVs were usually deletions showing the highest gene-dosage content. The positive family history for ID/ASD/MCA and ASD were good indicators for detecting pathological chromosomal rearrangements. Other clinical features as eyes anomalies, hearing loss, neurological signs, cutaneous dyscromia and endocrinological problems seem to be potential predictors of pathological CNVs. Among patients carrying VOUS we analyzed genetic features including CNVs size, presence of deletion or duplication, genic density, multiple CNVs, to clinical features. Higher gene density was found in patients affected by ID. This result suggest that higher gene content has more chances to include pathogenic gene involved and causing ID in these patients. Our study suggest the use of aCGH as first-tier test in patients with neurdevelopmental phenotypes. The inferred results have been used for building a flow-chart to be

Carcinoma ex-pleomorphic adenoma derived from recurrent pleomorphic adenoma shows important difference by array CGH compared to recurrent pleomorphic adenoma without malignant transformation

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Fernanda Viviane Mariano

Full Text Available Abstract Introduction: A key step of cancer development is the progressive accumulation of genomic changes resulting in disruption of several biological mechanisms. Carcinoma ex-pleomorphic adenoma (CXPA is an aggressive neoplasm that arises from a pleomorphic adenoma. CXPA derived from a recurrent PA (RPA has been rarely reported, and the genomic changes associated with these tumors have not yet been studied. Objective: We analyzed CXPA from RPAs and RPAs without malignant transformation using array-comparative genomic hybridization (array-CGH to identify somatic copy number alterations and affected genes. Methods: DNA samples extracted from FFPE tumors were submitted to array-CGH investigation, and data was analyzed by Nexus Copy Number Discovery Edition v.7. Results: No somatic copy number alterations were found in RPAs without malignant transformation. As for CXPA from RPA, although genomic profiles were unique for each case, we detected some chromosomal regions that appear to be preferentially affected by copy number alterations. The first case of CXPA-RPA (frankly invasive myoepithelial carcinoma showed copy number alterations affecting 1p36.33p13, 5p and chromosomes 3 and 8. The second case of CXPA-RPA (frankly invasive epithelial-myoepithelial carcinoma showed several alterations at chromosomes 3, 8, and 16, with two amplifications at 8p12p11.21 and 12q14.3q21.2. The third case of CXPA-RPA (minimally invasive epithelial-myoepithelial carcinoma exhibited amplifications at 12q13.3q14.1, 12q14.3, and 12q15. Conclusion: The occurrence of gains at chromosomes 3 and 8 and genomic amplifications at 8p and 12q, mainly those encompassing the HMGA2, MDM2, WIF1, WHSC1L1, LIRG3, CDK4 in CXAP from RPA can be a significant promotional factor in malignant transformation.

Novel applications of array comparative genomic hybridization in molecular diagnostics.

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Cheung, Sau W; Bi, Weimin

2018-05-31

In 2004, the implementation of array comparative genomic hybridization (array comparative genome hybridization [CGH]) into clinical practice marked a new milestone for genetic diagnosis. Array CGH and single-nucleotide polymorphism (SNP) arrays enable genome-wide detection of copy number changes in a high resolution, and therefore microarray has been recognized as the first-tier test for patients with intellectual disability or multiple congenital anomalies, and has also been applied prenatally for detection of clinically relevant copy number variations in the fetus. Area covered: In this review, the authors summarize the evolution of array CGH technology from their diagnostic laboratory, highlighting exonic SNP arrays developed in the past decade which detect small intragenic copy number changes as well as large DNA segments for the region of heterozygosity. The applications of array CGH to human diseases with different modes of inheritance with the emphasis on autosomal recessive disorders are discussed. Expert commentary: An exonic array is a powerful and most efficient clinical tool in detecting genome wide small copy number variants in both dominant and recessive disorders. However, whole-genome sequencing may become the single integrated platform for detection of copy number changes, single-nucleotide changes as well as balanced chromosomal rearrangements in the near future.

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH.

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Guillaud-Bataille, Marine; Valent, Alexander; Soularue, Pascal; Perot, Christine; Inda, Maria Mar; Receveur, Aline; Smaïli, Sadek; Roest Crollius, Hugues; Bénard, Jean; Bernheim, Alain; Gidrol, Xavier; Danglot, Gisèle

2004-07-29

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.

Genomic analysis of CD8+ NK/T cell line, ‘SRIK-NKL’, with array-based CGH (aCGH), SKY/FISH and molecular mapping

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Rossi, Michael; LaDuca, Jeff; Cowell, John; Srivastava, Bejai I.S.; Matsui, Sei-ichi

2010-01-01

We performed aCGH, SKY /FISH, molecular mapping and expression analyses on a permanent CD8+ NK/T cell line, ‘SRIK-NKL’ established from a lymphoma (ALL) patient, in attempt to define the fundamental genetic profile of its unique NK phenotypes. aCGH revealed hemizygous deletion of 6p containing genes responsible for hematopoietic functions. The SKY demonstrated that a constitutive reciprocal translocation, rcpt(5;14)(p13.2;q11) is a stable marker. Using somatic hybrids containing der(5) derived from SRIK-NKL, we found that the breakpoint in one homologue of no. 5 is located upstream of IL7R and also that the breakpoint in no. 14 is located within TRA@. The FISH analysis using BAC which contains TRA@ and its flanking region further revealed a ~231 kb deletion within 14q11 in the der(5) but not in the normal homologue of no. 14. The RT-PCR analysis detected mRNA for TRA@ transcripts which were extending across, but not including, the deleted region. IL7R was detected at least at mRNA levels. These findings were consistent with the immunological findings that TRA@ and IL7R are both expressed at mRNA levels and TRA@ at cytoplasmic protein levels in SRIK-NKL cells. In addition to rept(5;14), aCGH identified novel copy number abnormalities suggesting that the unique phenotype of the SRIK-NKL cell line is not solely due to the TRA@ rearrangement. These findings provide supportive evidence for the notion that SRIK-NKL cells may be useful for studying not only the function of NK cells but also genetic deregulations associated with leukemiogenesis. PMID:17640729

Template-Directed Ligation of Peptides to Oligonucleotides

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

1996-01-01

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.

Optical alignment using a CGH and an autostigmatic microscope

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Parks, Robert E.

2017-08-01

We show how custom computer generated holograms (CGH) are used along with an autostigmatic microscope (ASM) to align both optical and mechanical components relative to the CGH. The patterns in the CGHs define points and lines in space when interrogated with the focus of the ASM. Once the ASM is aligned to the CGH, an optical or mechanical component such as a lens, a well-polished ball or a cylinder can be aligned to the ASM in 3 or 4 degrees of freedom and thus to the CGH. In this case we show how a CGH is used to make a fixture for cementing a doublet lens without the need for a rotary table or a precision vertical stage.

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

2005-11-01

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 http://affymetrix.arabidopsis.info/xspecies/ and may be used to facilitate transcriptomic analyses of a wide range of plant and animal

Systematic validation and atomic force microscopy of non-covalent short oligonucleotide barcode microarrays.

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Michael A Cook

Full Text Available BACKGROUND: Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20-60 base unique sequence tags, or "barcodes", associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5'-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM, we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface. CONCLUSIONS/SIGNIFICANCE: These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis.

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

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

2003-01-01

with an overview of these parameters. We present here a flexible tool named OligoWiz for designing oligonucleotides for multiple purposes. OligoWiz presents a set of parameter scores in a graphical interface to facilitate an overview for the user. Additional custom parameter scores can easily be added......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...... to the program to extend the default parameters: homology, DeltaTm, low-complexity, position and GATC-only. Furthermore we present an analysis of the limitations in designing oligonucleotide sets that can detect transcripts from multiple organisms. OligoWiz is available at www.cbs.dtu.dk/services/OligoWiz/....

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

2008-11-01

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

Prevention of Ovarian High-Grade Serous Carcinoma by Elucidating Its Early Change

Science.gov (United States)

2013-10-01

the Affymetrix 6.0 SNP array instead of the oligonucleotide Agilent 1M CGH array for somatic copy number alterations, due to familiarity and reliable...epithelium. As a result, a significant effort has been placed on determining the menstrual status of samples collected – this has included reviewing the...HRT at time of surgery o Unknown menstrual cycle status at time of surgery The tissue microarray includes the following permutations (2 cores per

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

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Aakra Ågot

2009-03-01

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.

Clinical Utility of Array Comparative Genomic Hybridization for Detection of Chromosomal Abnormalities in Pediatric Acute Lymphoblastic Leukemia

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Rabin, Karen R.; Man, Tsz-Kwong; Yu, Alexander; Folsom, Matthew R.; Zhao, Yi-Jue; Rao, Pulivarthi H.; Plon, Sharon E.; Naeem, Rizwan C.

2014-01-01

Background Accurate detection of recurrent chromosomal abnormalities is critical to assign patients to risk-based therapeutic regimens for pediatric acute lymphoblastic leukemia (ALL). Procedure We investigated the utility of array comparative genomic hybridization (aCGH) for detection of chromosomal abnormalities compared to standard clinical evaluation with karyotype and fluorescent in-situ hybridization (FISH). Fifty pediatric ALL diagnostic bone marrows were analyzed by bacterial artificial chromosome (BAC) array, and findings compared to standard clinical evaluation. Results Sensitivity of aCGH was 79% to detect karyotypic findings other than balanced translocations, which cannot be detected by aCGH because they involve no copy number change. aCGH also missed abnormalities occurring in subclones constituting less than 25% of cells. aCGH detected 44 additional abnormalities undetected or misidentified by karyotype, 21 subsequently validated by FISH, including abnormalities in 4 of 10 cases with uninformative cytogenetics. aCGH detected concurrent terminal deletions of both 9p and 20q in three cases, in two of which the 20q deletion was undetected by karyotype. A narrow region of loss at 7p21 was detected in two cases. Conclusions An array with increased BAC density over regions important in ALL, combined with PCR for fusion products of balanced translocations, could minimize labor- and time-intensive cytogenetic assays and provide key prognostic information in the approximately 35% of cases with uninformative cytogenetics. PMID:18253961

Analysis of aCGH Integrating Different Sources of Information by Means of a CBR

OpenAIRE

Zato Domínguez, Carolina; de Paz Santana, Juan F.; Bajo Pérez, Javier; Corchado Rodríguez, Juan M.

2011-01-01

Knowledge management is a key element in medical environments. Arrays CGH make possible the realization of tests on patients for the detection of mutations in chromosomal regions. The detection of the regions with mutations associated to different pathologies is an important step for the selection of relevant genes, proteins or diseases. The corresponding information of the mutations and genes is distributed in dif- ferent public sources and databases, so it is necessary the use of systems th...

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

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Ramón Díaz-Uriarte

2007-01-01

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 (http://www.asterias.info 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.

Sparsity-based fast CGH generation using layer-based approach for 3D point cloud model

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Kim, Hak Gu; Jeong, Hyunwook; Ro, Yong Man

2017-03-01

Computer generated hologram (CGH) is becoming increasingly important for a 3-D display in various applications including virtual reality. In the CGH, holographic fringe patterns are generated by numerically calculating them on computer simulation systems. However, a heavy computational cost is required to calculate the complex amplitude on CGH plane for all points of 3D objects. This paper proposes a new fast CGH generation based on the sparsity of CGH for 3D point cloud model. The aim of the proposed method is to significantly reduce computational complexity while maintaining the quality of the holographic fringe patterns. To that end, we present a new layer-based approach for calculating the complex amplitude distribution on the CGH plane by using sparse FFT (sFFT). We observe the CGH of a layer of 3D objects is sparse so that dominant CGH is rapidly generated from a small set of signals by sFFT. Experimental results have shown that the proposed method is one order of magnitude faster than recently reported fast CGH generation.

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

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Qin, Li-Xuan; Beyer, Richard P; Hudson, Francesca N; Linford, Nancy J; Morris, Daryl E; Kerr, Kathleen F

2006-01-17

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. 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 using mismatch probes for low-intensity genes

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

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Morris Daryl E

2006-01-01

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

High resolution array-based comparative genomic hybridisation of medulloblastomas and supra-tentorial primitive neuroectodermal tumours

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McCabe, Martin Gerard; Ichimura, Koichi; Liu, Lu; Plant, Karen; Bäcklund, L Magnus; Pearson, Danita M; Collins, Vincent Peter

2010-01-01

Medulloblastomas and supratentorial primitive neuroectodermal tumours are aggressive childhood tumours. We report our findings using array comparative genomic hybridisation (CGH) on a whole-genome BAC/PAC/cosmid array with a median clone separation of 0.97Mb to study 34 medulloblastomas and 7 supratentorial primitive neuroectodermal tumours. Array CGH allowed identification and mapping of numerous novel small regions of copy number change to genomic sequence, in addition to the large regions already known from previous studies. Novel amplifications were identified, some encompassing oncogenes, MYCL1, PDGFRA, KIT and MYB, not previously reported to show amplification in these tumours. In addition, one supratentorial primitive neuroectodermal tumour had lost both copies of the tumour suppressor genes CDKN2A & CDKN2B. Ten medulloblastomas had findings suggestive of isochromosome 17q. In contrast to previous reports using conventional CGH, array CGH identified three distinct breakpoints in these cases: Ch 17: 17940393-19251679 (17p11.2, n=6), Ch 17: 20111990-23308272 (17p11.2-17q11.2, n=4) and Ch 17: 38425359-39091575 (17q21.31, n=1). Significant differences were found in the patterns of copy number change between medulloblastomas and supratentorial primitive neuroectodermal tumours, providing further evidence that these tumours are genetically distinct despite their morphological and behavioural similarities. PMID:16783165

Massively parallel sequencing, aCGH, and RNA-Seq technologies provide a comprehensive molecular diagnosis of Fanconi anemia.

Science.gov (United States)

Chandrasekharappa, Settara C; Lach, Francis P; Kimble, Danielle C; Kamat, Aparna; Teer, Jamie K; Donovan, Frank X; Flynn, Elizabeth; Sen, Shurjo K; Thongthip, Supawat; Sanborn, Erica; Smogorzewska, Agata; Auerbach, Arleen D; Ostrander, Elaine A

2013-05-30

Current methods for detecting mutations in Fanconi anemia (FA)-suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom molecular inversion probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at The Rockefeller University. DNA sequencing was complemented with custom array comparative genomic hybridization (aCGH) and RNA sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 different FA genes. RNA-seq analysis revealed lack of allele specific expression associated with a deletion and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq-targeted capture, aCGH, and RNA-seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2), and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA.

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

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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.

Comparative cytogenetic characterization of primary canine melanocytic lesions using array CGH and fluorescence in situ hybridization.

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Poorman, Kelsey; Borst, Luke; Moroff, Scott; Roy, Siddharth; Labelle, Philippe; Motsinger-Reif, Alison; Breen, Matthew

2015-06-01

Melanocytic lesions originating from the oral mucosa or cutaneous epithelium are common in the general dog population, with up to 100,000 diagnoses each year in the USA. Oral melanoma is the most frequent canine neoplasm of the oral cavity, exhibiting a highly aggressive course. Cutaneous melanocytomas occur frequently, but rarely develop into a malignant form. Despite the differential prognosis, it has been assumed that subtypes of melanocytic lesions represent the same disease. To address the relative paucity of information about their genomic status, molecular cytogenetic analysis was performed on the three recognized subtypes of canine melanocytic lesions. Using array comparative genomic hybridization (aCGH) analysis, highly aberrant distinct copy number status across the tumor genome for both of the malignant melanoma subtypes was revealed. The most frequent aberrations included gain of dog chromosome (CFA) 13 and 17 and loss of CFA 22. Melanocytomas possessed fewer genome wide aberrations, yet showed a recurrent gain of CFA 20q15.3-17. A distinctive copy number profile, evident only in oral melanomas, displayed a sigmoidal pattern of copy number loss followed immediately by a gain, around CFA 30q14. Moreover, when assessed by fluorescence in situ hybridization (FISH), copy number aberrations of targeted genes, such as gain of c-MYC (80 % of cases) and loss of CDKN2A (68 % of cases), were observed. This study suggests that in concordance with what is known for human melanomas, canine melanomas of the oral mucosa and cutaneous epithelium are discrete and initiated by different molecular pathways.

Rapid and accurate synthesis of TALE genes from synthetic oligonucleotides.

Science.gov (United States)

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

2016-01-01

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.

[Analysis of clinical outcomes of different embryo stage biopsy in array comparative genomic hybridization based preimplantation genetic diagnosis and screening].

Science.gov (United States)

Shen, J D; Wu, W; Shu, L; Cai, L L; Xie, J Z; Ma, L; Sun, X P; Cui, Y G; Liu, J Y

2017-12-25

Objective: To evaluate the efficiency of the application of array comparative genomic hybridization (array-CGH) in preimplantation genetic diagnosis or screening (PGD/PGS), and compare the clinical outcomes of different stage embryo biopsy. Methods: The outcomes of 381 PGD/PGS cycles referred in the First Affiliated Hospital of Nanjing Medical University from July 2011 to August 2015 were retrospectively analyzed. There were 320 PGD cycles with 156 cleavage-stage-biopsy cycles and 164 trophectoderm-biopsy cycles, 61 PGS cycles with 23 cleavage-stage-biopsy cycles and 38 trophectoderm-biopsy cycles. Chromosomal analysis was performed by array-CGH technology combined with whole genome amplification. Single embryo transfer was performed in all transfer cycles. Live birth rate was calculated as the main clinical outcomes. Results: The embryo diagnosis rate of PGD/PGS by array-CGH were 96.9%-99.1%. In PGD biopsy cycles, the live birth rate per embryo transfer cycle and live birth rate per embryo biopsy cycle were 50.0%(58/116) and 37.2%(58/156) in cleavage-stage-biopsy group, 67.5%(85/126) and 51.8%(85/164) in trophectoderm-biopsy group (both P 0.05). Conclusions: High diagnosis rate and idea live birth rate are achieved in PGD/PGS cycles based on array-CGH technology. The live birth rate of trophectoderm-biopsy group is significantly higher than that of cleavage-stage-biopsy group in PGD cycles; the efficiency of trophectoderm-biopsy is better.

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.

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

Science.gov (United States)

Liao, Can; Fu, Fang; Yang, Xin; Sun, Yi-Min; Li, Dong-Zhi

2011-06-01

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. 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. 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. 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.

Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics.

Science.gov (United States)

Marlowe, Jennifer L; Akopian, Violetta; Karmali, Priya; Kornbrust, Douglas; Lockridge, Jennifer; Semple, Sean

2017-08-01

The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG). The members of the Formulated Oligonucleotide Subcommittee of the OSWG have utilized their collective experience working with a variety of formulations and their associated oligonucleotide payloads, as well as their insights into regulatory considerations and expectations, to generate a series of consensus recommendations for the pharmacokinetic characterization and nonclinical safety assessment of this unique class of therapeutics. It should be noted that the focus of Subcommittee discussions was on lipid nanoparticle and other types of particulate formulations of therapeutic oligonucleotides and not on conjugates or other types of modifications of oligonucleotide structure intended to facilitate delivery.

Analysis of human HPRT- deletion mutants by the microarray-CGH (comparative genomic hybridization)

International Nuclear Information System (INIS)

Kodaira, M.; Sasaki, K.; Tagawa, H.; Omine, H.; Kushiro, J.; Takahashi, N.; Katayama, H.

2003-01-01

We are trying to evaluate genetic effects of radiation on human using mutation frequency as an indicator. For the efficient detection of mutations, it is important to understand the mechanism and the characteristics of radiation-induced mutations. We have started the analysis of hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutants induced by X-ray in order to clarify the deletion size and the mutation-distribution. We analyzed 39 human X-ray induced HPRT-deletion mutants by using the microarray-CGH. The array for this analysis contains 57 BAC clones covering as much as possible of the 4Mb of the 5' side and 10Mb of the 3' side of the HPRT gene based on the NCBI genome database. DNA from parent strain and each HPRT-mutant strain are labeled with Cy5 and Cy3 respectively, and were mixed and hybridized on the array. Fluorescent intensity ratio of the obtained spots was analyzed using software we developed to identify clones corresponding to the deletion region. The deletion in these strains ranged up to 3.5 Mb on the 5' side and 6 Mb on the 3' side of the HPRT gene. Deletions in 13 strains ended around BAC clones located at about 3 Mb on the 5' side. On the 3' side, deletions extended up to the specific clones located at 1.5 Mb in 11 strains. The mutations seem to be complex on the 3' end of deletion; some accompanied duplications with deletions and others could not be explained by one mutation event. We need to confirm these results, taking into account the experimental reproducibility and the accuracy of the published genetic map. The results of the research using the microarray-CGH help us to search the regions where deletions are easily induced and to identify the factors affecting the range of deletions

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

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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: http://embio.yonsei.ac.kr/~Park/mgvd.php.

Cross-platform array comparative genomic hybridization meta-analysis separates hematopoietic and mesenchymal from epithelial tumors

NARCIS (Netherlands)

Jong, C.; Marchiori, E.; van der Vaart, A.W.; Chin, S.F.; Carvalho, B; Tijssen, M.; Eijk, P.P.; van den IJssel, P.; Grabsch, H.; Quirke, P.; Oudejans, J.J.; Meijer, G.J.; Caldas, C.; Ylstra, B.

2007-01-01

A series of studies have been published that evaluate the chromosomal copy number changes of different tumor classes using array comparative genomic hybridization (array CGH); however, the chromosomal aberrations that distinguish the different tumor classes have not been fully characterized.

Microarray-based ultra-high resolution discovery of genomic deletion mutations

Science.gov (United States)

2014-01-01

Background Oligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low. Results Here we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size. Conclusions Our findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence. PMID:24655320

Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization.

Science.gov (United States)

Yang, Wen-Xu; Pan, Hong; Li, Lin; Wu, Hai-Rong; Wang, Song-Tao; Bao, Xin-Hua; Jiang, Yu-Wu; Qi, Yu

2016-03-20

Wolf-Hirschhorn syndrome (WHS) is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH). Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype.

Array-based gene expression, CGH and tissue data defines a 12q24 gain in neuroblastic tumors with prognostic implication

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Kilpinen Sami

2010-05-01

Full Text Available Abstract Background Neuroblastoma has successfully served as a model system for the identification of neuroectoderm-derived oncogenes. However, in spite of various efforts, only a few clinically useful prognostic markers have been found. Here, we present a framework, which integrates DNA, RNA and tissue data to identify and prioritize genetic events that represent clinically relevant new therapeutic targets and prognostic biomarkers for neuroblastoma. Methods A single-gene resolution aCGH profiling was integrated with microarray-based gene expression profiling data to distinguish genetic copy number alterations that were strongly associated with transcriptional changes in two neuroblastoma cell lines. FISH analysis using a hotspot tumor tissue microarray of 37 paraffin-embedded neuroblastoma samples and in silico data mining for gene expression information obtained from previously published studies including up to 445 healthy nervous system samples and 123 neuroblastoma samples were used to evaluate the clinical significance and transcriptional consequences of the detected alterations and to identify subsequently activated gene(s. Results In addition to the anticipated high-level amplification and subsequent overexpression of MYCN, MEIS1, CDK4 and MDM2 oncogenes, the aCGH analysis revealed numerous other genetic alterations, including microamplifications at 2p and 12q24.11. Most interestingly, we identified and investigated the clinical relevance of a previously poorly characterized amplicon at 12q24.31. FISH analysis showed low-level gain of 12q24.31 in 14 of 33 (42% neuroblastomas. Patients with the low-level gain had an intermediate prognosis in comparison to patients with MYCN amplification (poor prognosis and to those with no MYCN amplification or 12q24.31 gain (good prognosis (P = 0.001. Using the in silico data mining approach, we identified elevated expression of five genes located at the 12q24.31 amplicon in neuroblastoma (DIABLO, ZCCHC

Bridging the gap from prenatal karyotyping to whole-genome array comparative genomic hybridization in Hong Kong: survey on knowledge and acceptance of health-care providers and pregnant women.

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Cheng, Hiu Yee Heidi; Kan, Anita Sik-Yau; Hui, Pui Wah; Lee, Chin Peng; Tang, Mary Hoi Yin

2017-12-01

The use of array comparative genomic hybridization (aCGH) has been increasingly widespread. The challenge of integration of this technology into prenatal diagnosis was the interpretation of results and communicating findings of unclear clinical significance. This study assesses the knowledge and acceptance of prenatal aCGH in Hong Kong obstetricians and pregnant women. The aim is to identify the needs and gaps before implementing the replacement of karyotyping with aCGH. Questionnaires with aCGH information in the form of pamphlets were sent by post to obstetrics and gynecology doctors. For the pregnant women group, a video presentation, pamphlets on aCGH and a self-administered questionnaire were provided at the antenatal clinic. The perception of aCGH between doctors and pregnant women was similar. Doctors not choosing aCGH were more concerned about the difficulty in counseling of variants of unknown significance and adult-onset disease in pregnant women, whereas pregnant women not choosing aCGH were more concerned about the increased waiting time leading to increased anxiety. Prenatal aCGH is perceived as a better test by both doctors and patients. Counseling support, training, and better understanding and communication of findings of unclear clinical significance are necessary to improve doctor-patient experience.

Direct microcontact printing of oligonucleotides for biochip applications

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Trévisiol E

2005-07-01

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

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

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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.

2004-01-01

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

CGH Celebrates Take Your Child To Work Day 2015

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Shady Grove celebrated Take Your Child To Work Day this year with a variety of activities and sessions aimed at inspiring school-aged children to explore career paths in science and public service. CGH hosted its inaugural Take Your Child To Work Day session: An Introduction to Global Health.

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

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Romania, Paolo; Castellano, Aurora; Surace, Cecilia; Citti, Arianna; De Ioris, Maria Antonietta; Sirleto, Pietro; De Mariano, Marilena; Longo, Luca; Boldrini, Renata; Angioni, Adriano; Locatelli, Franco; Fruci, Doriana

2013-01-01

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.

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.

Salivary gland carcinosarcoma: oligonucleotide array CGH reveals similar genomic profiles in epithelial and mesenchymal components

NARCIS (Netherlands)

Vékony, H.; Leemans, C.R.; Ylstra, B.; Meijer, G.A.; van der Waal, I.; Bloemena, E.

2009-01-01

In this study, we present a case of parotid gland de novo carcinosarcoma. Salivary gland carcinosarcoma (or true malignant mixed tumor) is a rare biphasic neoplasm, composed of both malignant epithelial and malignant mesenchymal components. It is yet unclear whether these two phenotypes occur by

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

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Jee Justin

2008-12-01

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.

CGH Supports World Cancer Day Every Day

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We celebrate World Cancer Day every year on February 4th. This year the theme “We can. I can.” invites us to think not only about how we can work with one another to reduce the global burden of cancer, but how we as individuals can make a difference. Every day the staff at CGH work to establish and build upon programs that are aimed at improving the lives of people affected by cancer.

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

2011-01-01

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...

Functionalization and Self-Assembly of DNA Bidimensional Arrays

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Ramon Eritja

2011-09-01

Full Text Available Oligonucleotides carrying amino, thiol groups, as well as fluorescein, c-myc peptide sequence and nanogold at internal positions were prepared and used for the assembly of bidimensional DNA arrays.

The Medicinal Chemistry of Therapeutic Oligonucleotides.

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Wan, W Brad; Seth, Punit P

2016-11-10

Oligonucleotide-based therapeutics have made rapid progress in the clinic for treatment of a variety of disease indications. Unmodified oligonucleotides are polyanionic macromolecules with poor drug-like properties. Over the past two decades, medicinal chemists have identified a number of chemical modification and conjugation strategies which can improve the nuclease stability, RNA-binding affinity, and pharmacokinetic properties of oligonucleotides for therapeutic applications. In this perspective, we present a summary of the most commonly used nucleobase, sugar and backbone modification, and conjugation strategies used in oligonucleotide medicinal chemistry.

Genome-Wide DNA Copy Number Analysis of Acute Lymphoblastic Leukemia Identifies New Genetic Markers Associated with Clinical Outcome.

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Maribel Forero-Castro

Full Text Available Identifying additional genetic alterations associated with poor prognosis in acute lymphoblastic leukemia (ALL is still a challenge.To characterize the presence of additional DNA copy number alterations (CNAs in children and adults with ALL by whole-genome oligonucleotide array (aCGH analysis, and to identify their associations with clinical features and outcome. Array-CGH was carried out in 265 newly diagnosed ALLs (142 children and 123 adults. The NimbleGen CGH 12x135K array (Roche was used to analyze genetic gains and losses. CNAs were analyzed with GISTIC and aCGHweb software. Clinical and biological variables were analyzed. Three of the patients showed chromothripsis (cth6, cth14q and cth15q. CNAs were associated with age, phenotype, genetic subtype and overall survival (OS. In the whole cohort of children, the losses on 14q32.33 (p = 0.019 and 15q13.2 (p = 0.04 were related to shorter OS. In the group of children without good- or poor-risk cytogenetics, the gain on 1p36.11 was a prognostic marker independently associated with shorter OS. In adults, the gains on 19q13.2 (p = 0.001 and Xp21.1 (p = 0.029, and the loss of 17p (p = 0.014 were independent markers of poor prognosis with respect to OS. In summary, CNAs are frequent in ALL and are associated with clinical parameters and survival. Genome-wide DNA copy number analysis allows the identification of genetic markers that predict clinical outcome, suggesting that detection of these genetic lesions will be useful in the management of patients newly diagnosed with ALL.

A web server for mining Comparative Genomic Hybridization (CGH) data

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Liu, Jun; Ranka, Sanjay; Kahveci, Tamer

2007-11-01

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.

An application of CART algorithm in genetics: IGFs and cGH polymorphisms in Japanese quail

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Kaplan, Selçuk

2017-04-01

The avian insulin-like growth factor-1 (IGFs) and avian growth hormone (cGH) genes are the most important genes that can affect bird performance traits because of its important function in growth and metabolism. Understanding the molecular genetic basis of variation in growth-related traits is of importance for continued improvement and increased rates of genetic gain. The objective of the present study was to identify polymorphisms of cGH and IGFs genes in Japanese quail using conventional least square method (LSM) and CART algorithm. Therefore, this study was aimed to demonstrate at determining the polymorphisms of two genes related growth characteristics via CART algorithm. A simulated data set was generated to analyze by adhering the results of some poultry genetic studies which it includes live weights at 5 weeks of age, 3 alleles and 6 genotypes of cGH and 2 alleles and 3 genotypes of IGFs. As a result, it has been determined that the CART algorithm has some advantages as for that LSM.

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

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

2005-01-01

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. PMID:16437687

Array Comparative Genomic Hybridization as the First-line Investigation for Neonates with Congenital Heart Disease: Experience in a Single Tertiary Center.

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Choi, Bo Geum; Hwang, Su Kyung; Kwon, Jung Eun; Kim, Yeo Hyang

2018-03-01

The purpose of the present study was to investigate the advantages and disadvantages of verifying genetic abnormalities using array comparative genomic hybridization (a-CGH) immediately after diagnosis of congenital heart disease (CHD). Among neonates under the age of 28 days who underwent echocardiography from January 1, 2014 to April 30, 2016, neonates whose chromosomal and genomic abnormalities were tested using a-CGH in cases of an abnormal finding on echocardiography were enrolled. Of the 166 patients diagnosed with CHD, 81 underwent a-CGH and 11 patients (11/81, 13.5%) had abnormal findings on a-CGH. 22q11.2 deletion syndrome was the most common (4/11, 36.4%). On the first a-CGH, 4 patients were negative (4/81, 5%). Three of them were finally diagnosed with Williams syndrome using fluorescent in situ hybridization (FISH), 1 patient was diagnosed with Noonan syndrome through exome sequencing. All of them exhibited diffuse pulmonary artery branch hypoplasia, as well as increased velocity of blood flow, on repeated echocardiography. Five patients started rehabilitation therapy at mean 6 months old age in outpatient clinics and epilepsy was diagnosed in 2 patients. Parents of 2 patients (22q11.2 deletion syndrome and Patau syndrome) refused treatment due to the anticipated prognosis. Screening tests for genetic abnormalities using a-CGH in neonates with CHD has the advantage of early diagnosis of genetic abnormality during the neonatal period in which there is no obvious symptom of genetic abnormality. However, there are disadvantages that some genetic abnormalities cannot be identified on a-CGH. Copyright © 2018. The Korean Society of Cardiology.

Preimplantation genetic diagnosis for chromosomal rearrangements with the use of array comparative genomic hybridization at the blastocyst stage.

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Christodoulou, Christodoulos; Dheedene, Annelies; Heindryckx, Björn; van Nieuwerburgh, Filip; Deforce, Dieter; De Sutter, Petra; Menten, Björn; Van den Abbeel, Etienne

2017-01-01

To establish the value of array comparative genomic hybridization (CGH) for preimplantation genetic diagnosis (PGD) in embryos of translocation carriers in combination with vitrification and frozen embryo transfer in nonstimulated cycles. Retrospective data analysis study. Academic centers for reproductive medicine and genetics. Thirty-four couples undergoing PGD for chromosomal rearrangements from October 2013 to December 2015. Trophectoderm biopsy at day 5 or day 6 of embryo development and subsequently whole genome amplification and array CGH were performed. This approach revealed a high occurrence of aneuploidies and structural rearrangements unrelated to the parental rearrangement. Nevertheless, we observed a benefit in pregnancy rates of these couples. We detected chromosomal abnormalities in 133/207 embryos (64.2% of successfully amplified), and 74 showed a normal microarray profile (35.7%). In 48 of the 133 abnormal embryos (36.1%), an unbalanced rearrangement originating from the parental translocation was identified. Interestingly, 34.6% of the abnormal embryos (46/133) harbored chromosome rearrangements that were not directly linked to the parental translocation in question. We also detected a combination of unbalanced parental-derived rearrangements and aneuploidies in 27 of the 133 abnormal embryos (20.3%). The use of trophectoderm biopsy at the blastocyst stage is less detrimental to the survival of the embryo and leads to a more reliable estimate of the genomic content of the embryo than cleavage-stage biopsy. In this small cohort PGD study, we describe the successful implementation of array CGH analysis of blastocysts in patients with a chromosomal rearrangement to identify euploid embryos for transfer. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Analysis of genomic alterations in neuroblastoma by multiplex ligation-dependent probe amplification and array comparative genomic hybridization: a comparison of results.

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Combaret, Valérie; Iacono, Isabelle; Bréjon, Stéphanie; Schleiermacher, Gudrun; Pierron, Gäelle; Couturier, Jérôme; Bergeron, Christophe; Blay, Jean-Yves

2012-12-01

In cases of neuroblastoma, recurring genetic alterations--losses of the 1p, 3p, 4p, and 11q and/or gains of 1q, 2p, and 17q chromosome arms--are currently used to define the therapeutic strategy in therapeutic protocols for low- and intermediate-risk patients. Different genome-wide analysis techniques, such as array comparative genomic hybridization (aCGH) or multiplex ligation-dependent probe amplification (MLPA), have been suggested for detecting chromosome segmental abnormalities. In this study, we compared the results of the two technologies in the analyses of the DNA of tumor samples from 91 neuroblastoma patients. Similar results were obtained with the two techniques for 75 samples (82%). In five cases (5.5%), the MLPA results were not interpretable. Discrepancies between the aCGH and MLPA results were observed in 11 cases (12%). Among the discrepancies, a 18q21.2-qter gain and 16p11.2 and 11q14.1-q14.3 losses were detected only by aCGH. The MLPA results showed that the 7p, 7q, and 14q chromosome arms were affected in six cases, while in two cases, 2p and 17q gains were observed; these results were confirmed by neither aCGH nor fluorescence in situ hybridization (FISH) analysis. Because of the higher sensitivity and specificity of genome-wide information, reasonable cost, and shorter time of aCGH analysis, we recommend the aCGH procedure for the analysis of genomic alterations in neuroblastoma. Copyright © 2012 Elsevier Inc. All rights reserved.

Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

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Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M; Högberg, Björn

2013-07-01

Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

Electronic Structures of LNA Phosphorothioate Oligonucleotides

DEFF Research Database (Denmark)

Bohr, Henrik G.; Shim, Irene; Stein, Cy

2017-01-01

Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces...

Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

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Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Högberg, Björn

2013-01-01

Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides. PMID:23727986

Chromosomal minimal critical regions in therapy-related leukemia appear different from those of de novo leukemia by high-resolution aCGH.

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Nathalie Itzhar

Full Text Available Therapy-related acute leukemia (t-AML, is a severe complication of cytotoxic therapy used for primary cancer treatment. The outcome of these patients is poor, compared to people who develop de novo acute leukemia (p-AML. Cytogenetic abnormalities in t-AML are similar to those found in p-AML but present more frequent unfavorable karyotypes depending on the inducting agent. Losses of chromosome 5 or 7 are observed after alkylating agents while balanced translocations are found after topoisomerase II inhibitors. This study compared t-AML to p-AML using high resolution array CGH in order to find copy number abnormalities (CNA at a higher resolution than conventional cytogenetics. More CNAs were observed in 30 t-AML than in 36 p-AML: 104 CNAs were observed with 63 losses and 41 gains (mean number 3.46 per case in t-AML, while in p-AML, 69 CNAs were observed with 32 losses and 37 gains (mean number of 1.9 per case. In primary leukemia with a previously "normal" karyotype, 18% exhibited a previously undetected CNA, whereas in the (few t-AML with a normal karyotype, the rate was 50%. Several minimal critical regions (MCRs were found in t-AML and p-AML. No common MCRs were found in the two groups. In t-AML a 40 kb deleted MCR pointed to RUNX1 on 21q22, a gene coding for a transcription factor implicated in frequent rearrangements in leukemia and in familial thrombocytopenia. In de novo AML, a 1 Mb MCR harboring ERG and ETS2 was observed from patients with complex aCGH profiles. High resolution cytogenomics obtained by aCGH and similar techniques already published allowed us to characterize numerous non random chromosome abnormalities. This work supports the hypothesis that they can be classified into several categories: abnormalities common to all AML; those more frequently found in t-AML and those specifically found in p-AML.

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

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Fairbanks Benjamin D

2006-04-01

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

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

2009-10-01

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

Radio-marking and in vivo imagery of oligonucleotides

International Nuclear Information System (INIS)

Kuehnast, Bertrand

2000-01-01

This research thesis is part of activities aimed at the development of new molecules like oligonucleotides. Its first objective was the development and validation of a marking method with fluorine-18 of oligonucleotides for their in-vivo pharmacological assessment with positron emission tomography (PET). Further investigations addressed the use of iodine-125 for oligonucleotide marking purpose. This radio-marking, and in vivo and ex vivo imagery techniques are described, and their potential is highlighted for the pharmacological assessment of different oligonucleotides

Computer holography: 3D digital art based on high-definition CGH

International Nuclear Information System (INIS)

Matsushima, K; Arima, Y; Nishi, H; Yamashita, H; Yoshizaki, Y; Ogawa, K; Nakahara, S

2013-01-01

Our recent works of high-definition computer-generated holograms (CGH) and the techniques used for the creation, such as the polygon-based method, silhouette method and digitized holography, are summarized and reviewed in this paper. The concept of computer holography is proposed in terms of integrating and crystalizing the techniques into novel digital art.

Genome-wide detection of copy number variations among diverse horse breeds by array CGH.

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Wei Wang

Full Text Available 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.

Peptide-LNA oligonucleotide conjugates

DEFF Research Database (Denmark)

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

2013-01-01

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......(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...

Oligonucleotide-based theranostic nanoparticles in cancer therapy

Science.gov (United States)

Shahbazi, Reza; Ozpolat, Bulent; Ulubayram, Kezban

2016-01-01

Theranostic approaches, combining the functionality of both therapy and imaging, have shown potential in cancer nanomedicine. Oligonucleotides such as small interfering RNA and microRNA, which are powerful therapeutic agents, have been effectively employed in theranostic systems against various cancers. Nanoparticles are used to deliver oligonucleotides into tumors by passive or active targeting while protecting the oligonucleotides from nucleases in the extracellular environment. The use of quantum dots, iron oxide nanoparticles and gold nanoparticles and tagging with contrast agents, like fluorescent dyes, optical or magnetic agents and various radioisotopes, has facilitated early detection of tumors and evaluation of therapeutic efficacy. In this article, we review the advantages of theranostic applications in cancer therapy and imaging, with special attention to oligonucleotide-based therapeutics. PMID:27102380

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.

Science.gov (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

2014-08-01

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. © 2014 Wiley Periodicals, Inc.

Severe intellectual disability, omphalocele, hypospadia and high blood pressure associated to a deletion at 2q22.1q22.3: case report

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Mulatinho Milene

2012-06-01

Full Text Available Abstract Background Recently, array-comparative genomic hybridization (aCGH platforms have significantly improved the resolution of chromosomal analysis allowing the identification of genomic copy number gains and losses smaller than 5 Mb. Here we report on a young man with unexplained severe mental retardation, autism spectrum disorder, congenital malformations comprising hypospadia and omphalocele, and episodes of high blood pressure. An ~ 6 Mb interstitial deletion that includes the causative genes is identified by oligonucleotide-based aCGH. Results Our index case exhibited a de novo chromosomal abnormality at 2q22 [del(2(q22.1q22.3dn] which was not visible at the 550 haploid band level. The deleted region includes eight genes: HNMT, SPOPL, NXPH2, LOC64702, LRP1B, KYNU, ARHGAP15 and GTDC1. Discussion aCGH revealed an ~ 6 Mb deletion in 2q22.1 to 2q22.3 in an as-yet unique clinical case associated with intellectual disability, congenital malformations and autism spectrum disorder. Interestingly, the deletion is co-localized with a fragile site (FRA2K, which could be involved in the formation of this chromosomal aberration. Further studies are needed to determine if deletions of 2q22.1 to 2q22.3 define a new microdeletion syndrome.

High Quality Genomic Copy Number Data from Archival Formalin-Fixed Paraffin-Embedded Leiomyosarcoma: Optimisation of Universal Linkage System Labelling

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Salawu, Abdulazeez; Ul-Hassan, Aliya; Hammond, David; Fernando, Malee; Reed, Malcolm; Sisley, Karen

2012-01-01

Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment. PMID:23209738

Array-based techniques for fingerprinting medicinal herbs

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Xue Charlie

2011-05-01

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.

Regulation of Gene Expression with Double-Stranded Phosphorothioate Oligonucleotides

Science.gov (United States)

Bielinska, Anna; Shivdasani, Ramesh A.; Zhang, Liquan; Nabel, Gary J.

1990-11-01

Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins is necessary for determining how these factors participate in cellular differentiation. The functions of these proteins can be antagonized by several methods, each with specific limitations. Inhibition of sequence-specific DNA-binding proteins was achieved with double-stranded (ds) phosphorothioate oligonucleotides that contained octamer or kappaB consensus sequences. The phosphorothioate oligonucleotides specifically bound either octamer transcription factor or nuclear factor (NF)-kappaB. The modified oligonucleotides accumulated in cells more effectively than standard ds oligonucleotides and modulated gene expression in a specific manner. Octamer-dependent activation of a reporter plasmid or NF-kappaB-dependent activation of the human immunodeficiency virus (HIV) enhancer was inhibited when the appropriate phosphorothioate oligonucleotide was added to a transiently transfected B cell line. Addition of phosphorothioate oligonucleotides that contained the octamer consensus to Jurkat T leukemia cells inhibited interleukin-2 (IL-2) secretion to a degree similar to that observed with a mutated octamer site in the IL-2 enhancer. The ds phosphorothioate oligonucleotides probably compete for binding of specific transcription factors and may provide anti-viral, immunosuppressive, or other therapeutic effects.

A French multicenter study of over 700 patients with 22q11 deletions diagnosed using FISH or aCGH.

Science.gov (United States)

Poirsier, Céline; Besseau-Ayasse, Justine; Schluth-Bolard, Caroline; Toutain, Jérôme; Missirian, Chantal; Le Caignec, Cédric; Bazin, Anne; de Blois, Marie Christine; Kuentz, Paul; Catty, Marie; Choiset, Agnès; Plessis, Ghislaine; Basinko, Audrey; Letard, Pascaline; Flori, Elisabeth; Jimenez, Mélanie; Valduga, Mylène; Landais, Emilie; Lallaoui, Hakima; Cartault, François; Lespinasse, James; Martin-Coignard, Dominique; Callier, Patrick; Pebrel-Richard, Céline; Portnoi, Marie-France; Busa, Tiffany; Receveur, Aline; Amblard, Florence; Yardin, Catherine; Harbuz, Radu; Prieur, Fabienne; Le Meur, Nathalie; Pipiras, Eva; Kleinfinger, Pascale; Vialard, François; Doco-Fenzy, Martine

2016-06-01

Although 22q11.2 deletion syndrome (22q11.2DS) is the most recurrent human microdeletion syndrome associated with a highly variable phenotype, little is known about the condition's true incidence and the phenotype at diagnosis. We performed a multicenter, retrospective analysis of postnatally diagnosed patients recruited by members of the Association des Cytogénéticiens de Langue Française (the French-Speaking Cytogeneticists Association). Clinical and cytogenetic data on 749 cases diagnosed between 1995 and 2013 were collected by 31 French cytogenetics laboratories. The most frequent reasons for referral of postnatally diagnosed cases were a congenital heart defect (CHD, 48.6%), facial dysmorphism (49.7%) and developmental delay (40.7%). Since 2007 (the year in which array comparative genomic hybridization (aCGH) was introduced for the routine screening of patients with intellectual disability), almost all cases have been diagnosed using FISH (96.1%). Only 15 cases (all with an atypical phenotype) were diagnosed with aCGH; the deletion size ranged from 745 to 2904 kb. The deletion was inherited in 15.0% of cases and was of maternal origin in 85.5% of the latter. This is the largest yet documented cohort of patients with 22q11.2DS (the most commonly diagnosed microdeletion) from the same population. French cytogenetics laboratories diagnosed at least 108 affected patients (including fetuses) per year from among a national population of ∼66 million. As observed for prenatal diagnoses, CHDs were the most frequently detected malformation in postnatal diagnoses. The most common CHD in postnatal diagnoses was an isolated septal defect.

Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization

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Wen-Xu Yang

2016-01-01

Conclusions: The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype.

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

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Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M

2015-11-17

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.

Electronic Structures of LNA Phosphorothioate Oligonucleotides

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Henrik G. Bohr

2017-09-01

Full Text Available Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM calculations and chromatography experiments on locked nucleic acid (LNA phosphorothioate (PS oligonucleotides. iso-potential electrostatic surfaces are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules. The QM calculations give details of the electronic structures in terms of e.g., energy and bonding, which make them distinguish or differentiate between the individual PS diastereoisomers determined by the position of sulfur atoms. Rules are derived from the electronic calculations of these molecules and include the effects of the phosphorothioate chirality and formation of electrostatic potential surfaces. Physical and electrochemical descriptors of the PS oligonucleotides are compared to the experiments in which chiral states on these molecules can be distinguished. The calculations demonstrate that electronic structure, electrostatic potential, and topology are highly sensitive to single PS configuration changes and can give a lead to understanding the activity of the molecules. Keywords: LNA phosphorothioate, DNA/LNA oligonucleotide, diastereoisomers, Hartree-Fock calculations, iso-potential surface, anion chromatograms

Sequence-dependent theory of oligonucleotide hybridization kinetics

International Nuclear Information System (INIS)

Marimuthu, Karthikeyan; Chakrabarti, Raj

2014-01-01

A theoretical approach to the prediction of the sequence and temperature-dependent rate constants for oligonucleotide hybridization reactions has been developed based on the theory of relaxation kinetics. One-sided and two-sided melting reaction mechanisms for oligonucleotide hybridization reactions have been considered, analyzed, modified, and compared to select a physically consistent as well as robust model for prediction of the relaxation times of DNA hybridization reactions that agrees with the experimental evidence. The temperature- and sequence-dependent parameters of the proposed model have been estimated using available experimental data. The relaxation time model that we developed has been combined with the nearest neighbor model of hybridization thermodynamics to estimate the temperature- and sequence-dependent rate constants of an oligonucleotide hybridization reaction. The model-predicted rate constants are compared to experimentally determined rate constants for the same oligonucleotide hybridization reactions. Finally, we consider a few important applications of kinetically controlled DNA hybridization reactions

ACE-it: a tool for genome-wide integration of gene dosage and RNA expression data

NARCIS (Netherlands)

van Wieringen, W.N.; Belien, J.A.M.; Vosse, S.; Achame, E.M.; Ylstra, B.

2006-01-01

Summary: We describe a tool, called ACE-it (Array CGH Expression integration tool). ACE-it links the chromosomal position of the gene dosage measured by array CGH to the genes measured by the expression array. ACE-it uses this link to statistically test whether gene dosage affects RNA expression. ©

A pilot study of transcription unit analysis in rice using oligonucleotide tiling-path microarray

DEFF Research Database (Denmark)

Stolc, Viktor; Li, Lei; Wang, Xiangfeng

2005-01-01

As the international efforts to sequence the rice genome are completed, an immediate challenge and opportunity is to comprehensively and accurately define all transcription units in the rice genome. Here we describe a strategy of using high-density oligonucleotide tiling-path microarrays to map...... transcription of the japonica rice genome. In a pilot experiment to test this approach, one array representing the reverse strand of the last 11.2 Mb sequence of chromosome 10 was analyzed in detail based on a mathematical model developed in this study. Analysis of the array data detected 77% of the reference...... gene models in a mixture of four RNA populations. Moreover, significant transcriptional activities were found in many of the previously annotated intergenic regions. These preliminary results demonstrate the utility of genome tiling microarrays in evaluating annotated rice gene models...

Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel.

Science.gov (United States)

Tang, Weizhong; Zhou, Huafu; Li, Wei

2015-01-01

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.

A comparative analysis of measles virus RNA by oligonucleotide fingerprinting

International Nuclear Information System (INIS)

Stephenson, J.R.; Meulen, V. ter

1982-01-01

Isolates from two cases of acute measles, one case of acute measles encephalitis and three patients with subacute sclerosing panencephalitis were compared. This comparison was based upon the electrophoretic analysis of T 1 oligonucleotides from single-stranded, full-length RNA isolated from cytoplasmic nucleocapsids. Although all viruses have oligonucleotides in common, each isolate generated a unique pattern of oligonucleotides. However, no group of oligonucleotides was observed which would allow differentiation between viruses isolated from acute infections and those isolated from CNS diseases; indicating that probably all measles viruses differ in their nucleotide sequence, regardless of origin. (Author)

Subclassification and Detection of New Markers for the Discrimination of Primary Liver Tumors by Gene Expression Analysis Using Oligonucleotide Arrays.

Science.gov (United States)

Hass, Holger G; Vogel, Ulrich; Scheurlen, Michael; Jobst, Jürgen

2017-12-26

The failure to correctly differentiate between intrahepatic cholangiocarcinoma [CC] and hepatocellular carcinoma [HCC] is a significant clinical problem, particularly in terms of the different treatment goals for both cancers. In this study a specific gene expression profile to discriminate these two subgroups of liver cancer was established and potential diagnostic markers for clinical use were analyzed. To evaluate the gene expression profiles of HCC and intrahepatic CC, Oligonucleotide arrays ( Affymetrix U133A) were used. Overexpressed genes were checked for their potential use as new markers for discrimination and their expression values were validated by reverse transcription polymerase chain reaction and immunohistochemistry analyses. 695 genes/expressed sequence tags (ESTs) in HCC (245 up-/450 down-regulated) and 552 genes/ESTs in CC (221 up-/331 down-regulated) were significantly dysregulated (p〈0.05, fold change >2, ≥70%). Using a supervised learning method, and one-way analysis of variance a specific 270-gene expression profile that enabled rapid, reproducible differentiation between both tumors and non-malignant liver tissues was established. A panel of 12 genes (e.g. HSP90β, ERG1, GPC3, TKT, ACLY, and NME1 for HCC; SPT2, T4S3, CNX43, TTD1, HBD01 for CC) were detected and partly described for the first time as potential discrimination markers. A specific gene expression profile for discrimination of primary liver cancer was identified and potential marker genes with feasible clinical impact were described.

Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases

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Wupeng Liao

2017-01-01

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.

Unexpected structural complexity of supernumerary marker chromosomes characterized by microarray comparative genomic hybridization

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Hing Anne V

2008-04-01

Full Text Available Abstract Background Supernumerary marker chromosomes (SMCs are structurally abnormal extra chromosomes that cannot be unambiguously identified by conventional banding techniques. In the past, SMCs have been characterized using a variety of different molecular cytogenetic techniques. Although these techniques can sometimes identify the chromosome of origin of SMCs, they are cumbersome to perform and are not available in many clinical cytogenetic laboratories. Furthermore, they cannot precisely determine the region or breakpoints of the chromosome(s involved. In this study, we describe four patients who possess one or more SMCs (a total of eight SMCs in all four patients that were characterized by microarray comparative genomic hybridization (array CGH. Results In at least one SMC from all four patients, array CGH uncovered unexpected complexity, in the form of complex rearrangements, that could have gone undetected using other molecular cytogenetic techniques. Although array CGH accurately defined the chromosome content of all but two minute SMCs, fluorescence in situ hybridization was necessary to determine the structure of the markers. Conclusion The increasing use of array CGH in clinical cytogenetic laboratories will provide an efficient method for more comprehensive characterization of SMCs. Improved SMC characterization, facilitated by array CGH, will allow for more accurate SMC/phenotype correlation.

Application of Microarray-Based Comparative Genomic Hybridization in Prenatal and Postnatal Settings: Three Case Reports

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Jing Liu

2011-01-01

Full Text Available Microarray-based comparative genomic hybridization (array CGH is a newly emerged molecular cytogenetic technique for rapid evaluation of the entire genome with sub-megabase resolution. It allows for the comprehensive investigation of thousands and millions of genomic loci at once and therefore enables the efficient detection of DNA copy number variations (a.k.a, cryptic genomic imbalances. The development and the clinical application of array CGH have revolutionized the diagnostic process in patients and has provided a clue to many unidentified or unexplained diseases which are suspected to have a genetic cause. In this paper, we present three clinical cases in both prenatal and postnatal settings. Among all, array CGH played a major discovery role to reveal the cryptic and/or complex nature of chromosome arrangements. By identifying the genetic causes responsible for the clinical observation in patients, array CGH has provided accurate diagnosis and appropriate clinical management in a timely and efficient manner.

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

2013-01-01

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...

Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis

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Christoph Niemietz

2015-09-01

Full Text Available The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR, expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP. Misfolded variants of TTR are linked to the establishment of extracellular protein deposition in various tissues, including the heart and the peripheral nervous system. Recent progress in the chemistry and formulation of antisense (ASO and small interfering RNA (siRNA designed for a knockdown of TTR mRNA in the liver has allowed to address the issue of gene-specific molecular therapy in a clinical setting of FAP. The two therapeutic oligonucleotides bind to RNA in a sequence specific manner but exploit different mechanisms. Here we describe major developments that have led to the advent of therapeutic oligonucleotides for treatment of TTR-related disease.

Explorative data analysis of MCL reveals gene expression networks implicated in survival and prognosis supported by explorative CGH analysis

International Nuclear Information System (INIS)

Blenk, Steffen; Engelmann, Julia C; Pinkert, Stefan; Weniger, Markus; Schultz, Jörg; Rosenwald, Andreas; Müller-Hermelink, Hans K; Müller, Tobias; Dandekar, Thomas

2008-01-01

Mantle cell lymphoma (MCL) is an incurable B cell lymphoma and accounts for 6% of all non-Hodgkin's lymphomas. On the genetic level, MCL is characterized by the hallmark translocation t(11;14) that is present in most cases with few exceptions. Both gene expression and comparative genomic hybridization (CGH) data vary considerably between patients with implications for their prognosis. We compare patients over and below the median of survival. Exploratory principal component analysis of gene expression data showed that the second principal component correlates well with patient survival. Explorative analysis of CGH data shows the same correlation. On chromosome 7 and 9 specific genes and bands are delineated which improve prognosis prediction independent of the previously described proliferation signature. We identify a compact survival predictor of seven genes for MCL patients. After extensive re-annotation using GEPAT, we established protein networks correlating with prognosis. Well known genes (CDC2, CCND1) and further proliferation markers (WEE1, CDC25, aurora kinases, BUB1, PCNA, E2F1) form a tight interaction network, but also non-proliferative genes (SOCS1, TUBA1B CEBPB) are shown to be associated with prognosis. Furthermore we show that aggressive MCL implicates a gene network shift to higher expressed genes in late cell cycle states and refine the set of non-proliferative genes implicated with bad prognosis in MCL. The results from explorative data analysis of gene expression and CGH data are complementary to each other. Including further tests such as Wilcoxon rank test we point both to proliferative and non-proliferative gene networks implicated in inferior prognosis of MCL and identify suitable markers both in gene expression and CGH data

Comparative Genomic Hybridization (CGH) reveals a neo-X chromosome and biased gene movement in stalk-eyed flies (genus Teleopsis).

Science.gov (United States)

Baker, Richard H; Wilkinson, Gerald S

2010-09-16

Chromosomal location has a significant effect on the evolutionary dynamics of genes involved in sexual dimorphism, impacting both the pattern of sex-specific gene expression and the rate of duplication and protein evolution for these genes. For nearly all non-model organisms, however, knowledge of chromosomal gene content is minimal and difficult to obtain on a genomic scale. In this study, we utilized Comparative Genomic Hybridization (CGH), using probes designed from EST sequence, to identify genes located on the X chromosome of four species in the stalk-eyed fly genus Teleopsis. Analysis of log(2) ratio values of female-to-male hybridization intensities from the CGH microarrays for over 3,400 genes reveals a strongly bimodal distribution that clearly differentiates autosomal from X-linked genes for all four species. Genotyping of 33 and linkage mapping of 28 of these genes in Teleopsis dalmanni indicate the CGH results correctly identified chromosomal location in all cases. Syntenic comparison with Drosophila indicates that 90% of the X-linked genes in Teleopsis are homologous to genes located on chromosome 2L in Drosophila melanogaster, suggesting the formation of a nearly complete neo-X chromosome from Muller element B in the dipteran lineage leading to Teleopsis. Analysis of gene movement both relative to Drosophila and within Teleopsis indicates that gene movement is significantly associated with 1) rates of protein evolution, 2) the pattern of gene duplication, and 3) the evolution of eyespan sexual dimorphism. Overall, this study reveals that diopsids are a critical group for understanding the evolution of sex chromosomes within Diptera. In addition, we demonstrate that CGH is a useful technique for identifying chromosomal sex-linkage and should be applicable to other organisms with EST or partial genomic information.

Comparative Genomic Hybridization (CGH reveals a neo-X chromosome and biased gene movement in stalk-eyed flies (genus Teleopsis.

Directory of Open Access Journals (Sweden)

Richard H Baker

2010-09-01

Full Text Available Chromosomal location has a significant effect on the evolutionary dynamics of genes involved in sexual dimorphism, impacting both the pattern of sex-specific gene expression and the rate of duplication and protein evolution for these genes. For nearly all non-model organisms, however, knowledge of chromosomal gene content is minimal and difficult to obtain on a genomic scale. In this study, we utilized Comparative Genomic Hybridization (CGH, using probes designed from EST sequence, to identify genes located on the X chromosome of four species in the stalk-eyed fly genus Teleopsis. Analysis of log(2 ratio values of female-to-male hybridization intensities from the CGH microarrays for over 3,400 genes reveals a strongly bimodal distribution that clearly differentiates autosomal from X-linked genes for all four species. Genotyping of 33 and linkage mapping of 28 of these genes in Teleopsis dalmanni indicate the CGH results correctly identified chromosomal location in all cases. Syntenic comparison with Drosophila indicates that 90% of the X-linked genes in Teleopsis are homologous to genes located on chromosome 2L in Drosophila melanogaster, suggesting the formation of a nearly complete neo-X chromosome from Muller element B in the dipteran lineage leading to Teleopsis. Analysis of gene movement both relative to Drosophila and within Teleopsis indicates that gene movement is significantly associated with 1 rates of protein evolution, 2 the pattern of gene duplication, and 3 the evolution of eyespan sexual dimorphism. Overall, this study reveals that diopsids are a critical group for understanding the evolution of sex chromosomes within Diptera. In addition, we demonstrate that CGH is a useful technique for identifying chromosomal sex-linkage and should be applicable to other organisms with EST or partial genomic information.

Modern methods for the synthesis of peptide-oligonucleotide conjugates

International Nuclear Information System (INIS)

Zubin, Evgenii M; Oretskaya, Tat'yana S; Romanova, Elena A

2002-01-01

The published data on the methods of chemical solution and solid-phase synthesis of peptide-oligonucleotide conjugates are reviewed. The known methods are systematised and their advantages and disadvantages are considered. The approaches to the solution synthesis of peptide-oligonucleotide conjugates are systematised according to the type of chemical bonds between the fragments, whereas those to the solid-phase synthesis are classified according to the procedure used for the preparation of conjugates, viz., stepwise elongation of oligonucleotide and peptide chains on the same polymeric support or solid-phase condensation of two presynthesised fragments. The bibliography includes 141 references.

Prognostic Impact of Array-based Genomic Profiles in Esophageal Squamous Cell Cancer

International Nuclear Information System (INIS)

Carneiro, Ana; Isinger, Anna; Karlsson, Anna; Johansson, Jan; Jönsson, Göran; Bendahl, Pär-Ola; Falkenback, Dan; Halvarsson, Britta; Nilbert, Mef

2008-01-01

Esophageal squamous cell carcinoma (ESCC) is a genetically complex tumor type and a major cause of cancer related mortality. Although distinct genetic alterations have been linked to ESCC development and prognosis, the genetic alterations have not gained clinical applicability. We applied array-based comparative genomic hybridization (aCGH) to obtain a whole genome copy number profile relevant for identifying deranged pathways and clinically applicable markers. A 32 k aCGH platform was used for high resolution mapping of copy number changes in 30 stage I-IV ESCC. Potential interdependent alterations and deranged pathways were identified and copy number changes were correlated to stage, differentiation and survival. Copy number alterations affected median 19% of the genome and included recurrent gains of chromosome regions 5p, 7p, 7q, 8q, 10q, 11q, 12p, 14q, 16p, 17p, 19p, 19q, and 20q and losses of 3p, 5q, 8p, 9p and 11q. High-level amplifications were observed in 30 regions and recurrently involved 7p11 (EGFR), 11q13 (MYEOV, CCND1, FGF4, FGF3, PPFIA, FAD, TMEM16A, CTTS and SHANK2) and 11q22 (PDFG). Gain of 7p22.3 predicted nodal metastases and gains of 1p36.32 and 19p13.3 independently predicted poor survival in multivariate analysis. aCGH profiling verified genetic complexity in ESCC and herein identified imbalances of multiple central tumorigenic pathways. Distinct gains correlate with clinicopathological variables and independently predict survival, suggesting clinical applicability of genomic profiling in ESCC

SIGMA: A System for Integrative Genomic Microarray Analysis of Cancer Genomes

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Davies Jonathan J

2006-12-01

Full Text Available Abstract Background The prevalence of high resolution profiling of genomes has created a need for the integrative analysis of information generated from multiple methodologies and platforms. Although the majority of data in the public domain are gene expression profiles, and expression analysis software are available, the increase of array CGH studies has enabled integration of high throughput genomic and gene expression datasets. However, tools for direct mining and analysis of array CGH data are limited. Hence, there is a great need for analytical and display software tailored to cross platform integrative analysis of cancer genomes. Results We have created a user-friendly java application to facilitate sophisticated visualization and analysis such as cross-tumor and cross-platform comparisons. To demonstrate the utility of this software, we assembled array CGH data representing Affymetrix SNP chip, Stanford cDNA arrays and whole genome tiling path array platforms for cross comparison. This cancer genome database contains 267 profiles from commonly used cancer cell lines representing 14 different tissue types. Conclusion In this study we have developed an application for the visualization and analysis of data from high resolution array CGH platforms that can be adapted for analysis of multiple types of high throughput genomic datasets. Furthermore, we invite researchers using array CGH technology to deposit both their raw and processed data, as this will be a continually expanding database of cancer genomes. This publicly available resource, the System for Integrative Genomic Microarray Analysis (SIGMA of cancer genomes, can be accessed at http://sigma.bccrc.ca.

Transcriptional landscape estimation from tiling array data using a model of signal shift and drift

DEFF Research Database (Denmark)

Rasmussen, Simon; Jarmer, Hanne Østergaard; Nicolas, P

2009-01-01

MOTIVATION: High-density oligonucleotide tiling array technology holds the promise of a better description of the complexity and the dynamics of transcriptional landscapes. In organisms such as bacteria and yeasts, transcription can be measured on a genome-wide scale with a resolution >25 bp...

Optical Characterization of Oligonucleotide DNA Influenced by Magnetic Fields

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Seyedeh Maryam Banihashemian

2013-09-01

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.

[Analysis of genomic copy number variations in two sisters with primary amenorrhea and hyperandrogenism].

Science.gov (United States)

Zhang, Yanliang; Xu, Qiuyue; Cai, Xuemei; Li, Yixun; Song, Guibo; Wang, Juan; Zhang, Rongchen; Dai, Yong; Duan, Yong

2015-12-01

To analyze genomic copy number variations (CNVs) in two sisters with primary amenorrhea and hyperandrogenism. G-banding was performed for karyotype analysis. The whole genome of the two sisters were scanned and analyzed by array-based comparative genomic hybridization (array-CGH). The results were confirmed with real-time quantitative PCR (RT-qPCR). No abnormality was found by conventional G-banded chromosome analysis. Array-CGH has identified 11 identical CNVs from the sisters which, however, overlapped with CNVs reported by the Database of Genomic Variants (http://projects.tcag.ca/variation/). Therefore, they are likely to be benign. In addition, a -8.44 Mb 9p11.1-p13.1 duplication (38,561,587-47,002,387 bp, hg18) and a -80.9 kb 4q13.2 deletion (70,183,990-70,264,889 bp, hg18) were also detected in the elder and younger sister, respectively. The relationship between such CNVs and primary amenorrhea and hyperandrogenism was however uncertain. RT-qPCR results were in accordance with array-CGH. Two CNVs were detected in two sisters by array-CGH, for which further studies are needed to clarify their correlation with primary amenorrhea and hyperandrogenism.

Fully automated parallel oligonucleotide synthesizer

Czech Academy of Sciences Publication Activity Database

Lebl, M.; Burger, Ch.; Ellman, B.; Heiner, D.; Ibrahim, G.; Jones, A.; Nibbe, M.; Thompson, J.; Mudra, Petr; Pokorný, Vít; Poncar, Pavel; Ženíšek, Karel

2001-01-01

Roč. 66, č. 8 (2001), s. 1299-1314 ISSN 0010-0765 Institutional research plan: CEZ:AV0Z4055905 Keywords : automated oligonucleotide synthesizer Subject RIV: CC - Organic Chemistry Impact factor: 0.778, year: 2001

Synthesis of 3'-, or 5'-, or internal methacrylamido-modified oligonucleotides

Science.gov (United States)

Golova, Julia B.; Chernov, Boris K.

2010-04-27

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.

Effects of Atelocollagen Formulation Containing Oligonucleotide on Endothelial Permeability

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Koji Hanai

2012-01-01

Full Text Available Atelocollagen is a major animal protein that is used as a highly biocompatible biomaterial. To date, atelocollagen has been used as an effective drug delivery technology to sustain the release of antitumor proteins and to enhance the antitumor activity of oligonucleotides in in vivo models. However, the biological effects of this technology are not fully understood. In the present study, we investigated the effects of atelocollagen on endothelial paracellular barrier function. An atelocollagen formulation containing oligonucleotides specifically increased the permeability of two types of endothelial cells, and the change was dependent on the molecular size, structure of the oligonucleotides used and the concentrations of the oligonucleotide and atelocollagen in the formulation. An immunohistochemical examination revealed that the formulation had effects on the cellular skeleton and intercellular structure although it did not affect the expression of adherens junction or tight junction proteins. These changes were induced through p38 MAP kinase signaling. It is important to elucidate the biological functions of atelocollagen in order to be able to exploit its drug delivery properties.

Degradation product characterization of therapeutic oligonucleotides using liquid chromatography mass spectrometry.

Science.gov (United States)

Elzahar, N M; Magdy, N; El-Kosasy, Amira M; Bartlett, Michael G

2018-05-01

Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90 °C. The main degradation products were observed to be losses of nucleotide moieties from the 3'- and 5'-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover, the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences. Graphical abstract Identification of degradation products across several generations of oligonucleotide therapeutics using LC-MS.

Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

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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.

Biominetic High Density Lipoproteins for the Delivery of Therapeutic Oligonucleotides

Science.gov (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

Oligonucleotide-based biosensors for in vitro diagnostics and environmental hazard detection.

Science.gov (United States)

Jung, Il Young; Lee, Eun Hee; Suh, Ah Young; Lee, Seung Jin; Lee, Hyukjin

2016-04-01

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.

Functionalization of PVC membrane with ss oligonucleotides for a potentiometric biosensor.

Science.gov (United States)

Shishkanova, T V; Volf, R; Krondak, M; Král, V

2007-05-15

A novel application of a single stranded (ss) oligonucleotide as an active component of polymeric membrane in an ion-selective electrode (ISE) is described. The original oligonucleotides, oligo(dA)(15), modified by cholesterol, triphenylmethyl and hexadecyl derivatives, were immobilized into poly(vinyl chloride) (PVC) membrane using extraction protocol. In parallel, the adsorption protocol was used to immobilize unmodified oligo(dA)(15) on the PVC membrane based on tridodecylmethyammonium chloride (TDDMA(+)Cl(-)). Immobilization of ss oligonucleotide probe through spacer was more effective for the potentiometric detection of the hybridization between complementary oligonucleotides. It was found that cholesterol-oligo(dA)(15) modified membranes were sensitive toward complementary oligo(dT)(15) in the concentration range 2-80 nM at pH 7. An explanation for the detection mechanism is proposed.

A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

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Chen Feng

2010-10-01

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

Synthetic Method for Oligonucleotide Block by Using Alkyl-Chain-Soluble Support.

Science.gov (United States)

Matsuno, Yuki; Shoji, Takao; Kim, Shokaku; Chiba, Kazuhiro

2016-02-19

A straightforward method for the synthesis of oligonucleotide blocks using a Cbz-type alkyl-chain-soluble support (Z-ACSS) attached to the 3'-OH group of 3'-terminal nucleosides was developed. The Z-ACSS allowed for the preparation of fully protected deoxyribo- and ribo-oligonucleotides without chromatographic purification and released dimer- to tetramer-size oligonucleotide blocks via hydrogenation using a Pd/C catalyst without significant loss or migration of protective groups such as 5'-end 4,4'-dimethoxtrityl, 2-cyanoethyl on internucleotide bonds, or 2'-TBS.

Thermodynamics of Oligonucleotide Duplex Melting

Science.gov (United States)

Schreiber-Gosche, Sherrie; Edwards, Robert A.

2009-01-01

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…

Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation

DEFF Research Database (Denmark)

Taskova, Maria; Madsen, Charlotte S.; Jensen, Knud J.

2017-01-01

Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, m...... and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.......Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide......, 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...

Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

International Nuclear Information System (INIS)

Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

2016-01-01

Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer. - Highlights: • Core/shell type Iron oxide nanoparticles were used as a novel and efficient method. • This method increases exogenous DNA uptake by rooster spermatozoa. • Static magnetic field decreased DNA uptake by rooster spermatozoa.

Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

Energy Technology Data Exchange (ETDEWEB)

Katebi, Samira; Esmaeili, Abolghasem, E-mail: aesmaeili@sci.ui.ac.ir; Ghaedi, Kamran

2016-03-15

Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer. - Highlights: • Core/shell type Iron oxide nanoparticles were used as a novel and efficient method. • This method increases exogenous DNA uptake by rooster spermatozoa. • Static magnetic field decreased DNA uptake by rooster spermatozoa.

New Tools for Embryo Selection: Comprehensive Chromosome Screening by Array Comparative Genomic Hybridization

Directory of Open Access Journals (Sweden)

Lorena Rodrigo

2014-01-01

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.

Oligonucleotide-based pharmaceuticals: Non-clinical and clinical safety signals and non-clinical testing strategies.

Science.gov (United States)

Mustonen, Enni-Kaisa; Palomäki, Tiina; Pasanen, Markku

2017-11-01

Antisense oligonucleotides, short interfering RNAs (siRNAs) and aptamers are oligonucleotide-based pharmaceuticals with a promising role in targeted therapies. Currently, five oligonucleotide-based pharmaceuticals have achieved marketing authorization in Europe or USA and many more are undergoing clinical testing. However, several safety concerns have been raised in non-clinical and clinical studies. Oligonucleotides share properties with both chemical and biological pharmaceuticals and therefore they pose challenges also from the regulatory point of view. We have analyzed the safety data of oligonucleotides and evaluated the applicability of current non-clinical toxicological guidelines for assessing the safety of oligonucleotide-based pharmaceuticals. Oligonucleotide-based pharmaceuticals display a similar toxicological profile, exerting adverse effects on liver and kidney, evoking hematological alterations, as well as causing immunostimulation and prolonging the coagulation time. It is possible to extrapolate some of these effects from non-clinical studies to humans. However, evaluation strategies for genotoxicity testing of "non-natural" oligonucleotides should be revised. Additionally, the selective use of surrogates and prediction of clinical endpoints for non-clinically observed immunostimulation is complicated by its multiple potential manifestations, demanding improvements in the testing strategies. Utilizing more relevant and mechanistic-based approaches and taking better account of species differences, could possibly improve the prediction of relevant immunological/proinflammatory effects in humans. Copyright © 2017 Elsevier Inc. All rights reserved.

Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

Science.gov (United States)

Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

2016-03-01

Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (Prooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer.

Quantitation of ultraviolet-induced single-strand breaks using oligonucleotide chip

International Nuclear Information System (INIS)

Pal, Sukdeb; Kim, Min Jung; Choo, Jaebum; Kang, Seong Ho; Lee, Kyeong-Hee; Song, Joon Myong

2008-01-01

A simple, accurate and robust methodology was established for the direct quantification of ultraviolet (UV)-induced single-strand break (SSB) using oligonucleotide chip. Oligonucleotide chips were fabricated by covalently anchoring the fluorescent-labeled ssDNAs onto silicon dioxide chip surfaces. Assuming that the possibility of more than one UV-induced SSB to be generated in a small oligonucleotide is extremely low, SSB formation was investigated quantifying the endpoint probe density by fluorescence measurement upon UV irradiation. The SSB yields obtained based on the highly sensitive laser-induced fluorometric determination of fluorophore-labeled oligonucleotides were found to coincide well with that predicted from a theoretical extrapolation of the results obtained for plasmid DNAs using conventional agarose gel electrophoresis. The developed method has the potential to serve as a high throughput, sample-thrifty, and time saving tool to realize more realistic, and direct quantification of radiation and chemical-induced strand breaks. It will be especially useful for determining the frequency of SSBs or lesions convertible to SSBs by specific cleaving reagents or enzymes

Chromosome copy analysis by single-cell comparative genomic hybridization technique based on primer extension preamplification and degenerate oligonucleotide primed-PCR%引物延伸预扩增结合简并引物PCR在单细胞比较基因组杂交分析染色体异常中的应用

Institute of Scientific and Technical Information of China (English)

谭珂; 狄玉芬; 程德华; 徐芳; 卢光绣; 谭跃球

2010-01-01

Objective To establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD). Methods Twelve single cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR)amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed. Results The amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR,and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods. Conclusion PEP-DOP-PCR can effectively amplify the whole genome DNA of single cell.Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneousty, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.%目的 建立一种可信的单细胞全基因组扩增(whole genome amplification.WGA)技术,结合比较基因组杂交(comparative genomic hybridization,CGH

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

2017-01-01

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......, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12nm) in solution was revealed to be independent of concentration. The topological folding...

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

Science.gov (United States)

Liebert, Ann D.; Bicknell, Brian

2017-02-01

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.

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

International Nuclear Information System (INIS)

Baudis, Michael

2007-01-01

Chromosomal abnormalities have been associated with most human malignancies, with gains and losses on some genomic regions associated with particular entities. 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. 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). 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

Thermal Stability of Modified i-Motif Oligonucleotides with Naphthalimide Intercalating Nucleic Acids

DEFF Research Database (Denmark)

El-Sayed, Ahmed Ali; Pedersen, Erik B.; Khaireldin, Nahid Y.

2016-01-01

In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion of naphtha......In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion...... of naphthalimide (1H-benzo[de]isoquinoline-1,3(2H)-dione) as the intercalating nucleic acid. The stabilities of i-motif structures with inserted naphthalimide intercalating nucleotides were studied using UV melting temperatures (Tm) and circular dichroism spectra at different pH values and conditions (crowding...

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

Science.gov (United States)

2010-01-01

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. Despite the small

Cellular Internalization of Therapeutic Oligonucleotides by Peptide Amphiphile Nanofibers and Nanospheres.

Science.gov (United States)

Mumcuoglu, Didem; Sardan Ekiz, Melis; Gunay, Gokhan; Tekinay, Turgay; Tekinay, Ayse B; Guler, Mustafa O

2016-05-11

Oligonucleotides are promising drug candidates due to the exceptionally high specificity they exhibit toward their target DNA and RNA sequences. However, their poor pharmacokinetic and pharmacodynamic properties, in conjunction with problems associated with their internalization by cells, necessitates their delivery through specialized carrier systems for efficient therapy. Here, we investigate the effects of carrier morphology on the cellular internalization mechanisms of oligonucleotides by using self-assembled fibrous or spherical peptide nanostructures. Size and geometry were both found to be important parameters for the oligonucleotide internalization process; direct penetration was determined to be the major mechanism for the internalization of nanosphere carriers, whereas nanofibers were internalized by clathrin- and dynamin-dependent endocytosis pathways. We further showed that glucose conjugation to carrier nanosystems improved cellular internalization in cancer cells due to the enhanced glucose metabolism associated with oncogenesis, and the internalization of the glucose-conjugated peptide/oligonucleotide complexes was found to be dependent on glucose transporters present on the surface of the cell membrane.

Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

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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.

Oligonucleotide assisted light-emitting Alq3 microrods: energy transfer effect with fluorescent dyes.

Science.gov (United States)

Cui, Chunzhi; Park, Dong Hyuk; Kim, Jeongyong; Joo, Jinsoo; Ahn, Dong June

2013-06-14

Oligonucleotide assisted tri(8-hydroxyquinoline) aluminium (Alq3) microrods were prepared for the first time. When hybridized with oligonucleotide labeled by Cy3 fluorescent dye, a significant photoluminescence variation of the Alq3 microrods was observed due to Förster resonance energy transfer, unlike when Cy5-oligonucleotide was used. Versatile nucleotide manipulation would open up wider applications of Alq3-based materials, based on this fundamental observation.

Dielectric properties of DNA oligonucleotides on the surface of silicon nanostructures

Energy Technology Data Exchange (ETDEWEB)

Bagraev, N. T., E-mail: bagraev@mail.ioffe.ru [St. Petersburg Polytechnic University (Russian Federation); Chernev, A. L. [Russian Academy of Sciences, St. Petersburg Academic University—Nanotechnology Research and Education Center (Russian Federation); Klyachkin, L. E. [St. Petersburg Polytechnic University (Russian Federation); Malyarenko, A. M. [Russian Academy of Sciences, Ioffe Physical–Technical Institute (Russian Federation); Emel’yanov, A. K.; Dubina, M. V. [Russian Academy of Sciences, St. Petersburg Academic University—Nanotechnology Research and Education Center (Russian Federation)

2016-10-15

Planar silicon nanostructures that are formed as a very narrow silicon quantum well confined by δ barriers heavily doped with boron are used to study the dielectric properties of DNA oligonucleotides deposited onto the surface of the nanostructures. The capacitance characteristics of the silicon nanostructures with oligonucleotides deposited onto their surface are determined by recording the local tunneling current–voltage characteristics by means of scanning tunneling microscopy. The results show the possibility of identifying the local dielectric properties of DNA oligonucleotide segments consisting of repeating G–C pairs. These properties apparently give grounds to correlate the segments with polymer molecules exhibiting the properties of multiferroics.

Comparison of gene coverage of mouse oligonucleotide microarray platforms

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Medrano Juan F

2006-03-01

Full Text Available Abstract Background The increasing use of DNA microarrays for genetical genomics studies generates a need for platforms with complete coverage of the genome. We have compared the effective gene coverage in the mouse genome of different commercial and noncommercial oligonucleotide microarray platforms by performing an in-house gene annotation of probes. We only used information about probes that is available from vendors and followed a process that any researcher may take to find the gene targeted by a given probe. In order to make consistent comparisons between platforms, probes in each microarray were annotated with an Entrez Gene id and the chromosomal position for each gene was obtained from the UCSC Genome Browser Database. Gene coverage was estimated as the percentage of Entrez Genes with a unique position in the UCSC Genome database that is tested by a given microarray platform. Results A MySQL relational database was created to store the mapping information for 25,416 mouse genes and for the probes in five microarray platforms (gene coverage level in parenthesis: Affymetrix430 2.0 (75.6%, ABI Genome Survey (81.24%, Agilent (79.33%, Codelink (78.09%, Sentrix (90.47%; and four array-ready oligosets: Sigma (47.95%, Operon v.3 (69.89%, Operon v.4 (84.03%, and MEEBO (84.03%. The differences in coverage between platforms were highly conserved across chromosomes. Differences in the number of redundant and unspecific probes were also found among arrays. The database can be queried to compare specific genomic regions using a web interface. The software used to create, update and query the database is freely available as a toolbox named ArrayGene. Conclusion The software developed here allows researchers to create updated custom databases by using public or proprietary information on genes for any organisms. ArrayGene allows easy comparisons of gene coverage between microarray platforms for any region of the genome. The comparison presented here

Chromosomes of Iberian Leuciscinae (Cyprinidae) Revisited: Evidence of Genome Restructuring in Homoploid Hybrids Using Dual-Color FISH and CGH

Czech Academy of Sciences Publication Activity Database

Pereira, C. S.; Ráb, Petr; Collares-Pereira, M. J.

2013-01-01

Roč. 141, 2/3 (2013), s. 143-152 ISSN 1424-8581 R&D Projects: GA ČR GA13-37277S Institutional support: RVO:67985904 Keywords : CGH/GISH * Chondrostoma s.I. * genome reshuffling hybridization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.905, year: 2013

Challenges to oligonucleotides-based therapeutics for Duchenne muscular dystrophy

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Goyenvalle Aurélie

2011-02-01

Full Text Available Abstract Antisense oligonucleotides are short nucleic acids designed to bind to specific messenger RNAs in order to modulate splicing patterns or inhibit protein translation. As such, they represent promising therapeutic tools for many disorders and have been actively developed for more than 20 years as a form of molecular medicine. Although significant progress has been made in developing these agents as drugs, they are yet not recognized as effective therapeutics and several hurdles remain to be overcome. Within the last few years, however, the prospect of successful oligonucleotides-based therapies has moved a step closer, in particular for Duchenne muscular dystrophy. Clinical trials have recently been conducted for this myopathy, where exon skipping is being used to achieve therapeutic outcomes. In this review, the recent developments and clinical trials using antisense oligonucleotides for Duchenne muscular dystrophy are discussed, with emphasis on the challenges ahead for this type of therapy, especially with regards to delivery and regulatory issues.

G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents.

Science.gov (United States)

Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela

2015-09-22

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.

A study of glasses-type color CGH using a color filter considering reduction of blurring

Science.gov (United States)

Iwami, Saki; Sakamoto, Yuji

2009-02-01

We have developed a glasses-type color computer generated hologram (CGH) by using a color filter. The proposed glasses consist of two "lenses" made of overlapping holograms and color filters. The holograms, which are calculated to reconstruct images in each primary color, are divided to small areas, which we called cells, and superimposed on one hologram. In the same way, colors of the filter correspond to the hologram cells. We can configure it very simply without a complex optical system, and the configuration yields a small and light weight system suitable for glasses. When the cell is small enough, the colors are mixed and reconstructed color images are observed. In addition, color expression of reconstruction images improves, too. However, using small cells blurrs reconstructed images because of the following reasons: (1) interference between cells because of the correlation with the cells, and (2) reduction of resolution caused by the size of the cell hologram. We are investigating in order to make a hologram that has high resolution reconstructed color images without ghost images. In this paper, we discuss (1) the details of the proposed glasses-type color CGH, (2) appropriate cell size for an eye system, (3) effects of cell shape on the reconstructed images, and (4) a new method to reduce the blurring of the images.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide insert].

Science.gov (United States)

Kupriushkin, M S; Pyshnyĭ, D V

2012-01-01

Non-nucleotide phosporamidites were synthetized, having branched backbone with different position of functional groups. Obtained phosphoramidite monomers contain intercalator moiety--6-chloro-2-methoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with tert-butyldimethylsilyl group for post-synthetic modification. Synthesized oligothymidilates contain one or more modified units in different positions of sequence. Melting temperature and thermodynamic parameters of formation of complementary duplexes formed by modified oligonucleotides was defined (change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide conjugate significantly changes upon hybridization with DNA.

Triplex-forming ability of modified oligonucleotides

DEFF Research Database (Denmark)

Højland, Torben; Babu, Bolle Ravindra; Bryld, Torsten

2007-01-01

We present our studies on the ability of several different nucleotide analogs as triplex-forming oligonucleotides. The modifications tested include 4'-C-hydroxymethyl, LNA, 2'-amino-LNA and N2'-functionalized 2'-amino-LNA. Triplexes containing monomers of N2'-glycyl-functionalized 2'-amino-LNA ar...

Binding of Dumbbell Oligonucleotides to MoMuLV Reverse Transcriptase: Inhibitory Properties of RNase H Activity

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Ajay Kumar

2010-01-01

Full Text Available Dumbbell oligonucleotides with loops of various chemistry were synthesized. Incubation of dumbbell oligonucleotides containing phosphorothioate bonds or trimethylene phosphate linkages in loops with S1 nuclease did not result in significant cleavage under conditions which led to the degradation of dumbbell oligonucleotide containing phophodiester bonds in the loops. The binding of reverse transcriptase of Moloney Murine Leukemia Virus (MoMuLV was evaluated with all the five oligonucleotides. The protein binds to all the dumbbell oligonucleotides with similar affinity. The dissociation constants evaluated using PAGE band mobility shift assays were of the order of 10-7. The inhibitory properties of the retroviral RNase H activity was evaluated using 3H –UTP-labeled RNA:RNA-DNA hybrid. It was found that the best dumbbell oligonucleotide, inhibitor contained phosphorothioate residues in both the loops. Our value studies demonstrated that this particularly designed oligonucleotide displays an IC50 of 18 nM in its inhibition on the reverse transcriptase RNase H activity, a magnitude lower than that of first nucleotide reverse transcriptase of HIV-1, tenofovir, introduced by Gilead Science in the market.

Array based characterization of a terminal deletion involving chromosome subband 15q26.2: an emerging syndrome associated with growth retardation, cardiac defects and developmental delay

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Björkhem Gudrun

2008-01-01

Full Text Available Abstract Background Subtelomeric regions are gene rich and deletions in these chromosomal segments have been demonstrated to account for approximately 2.5% of patients displaying mental retardation with or without association of dysmorphic features. However, cases that report de novo terminal deletions on chromosome arm 15q are rare. Methods In this study we present the first example of a detailed molecular genetic mapping of a de novo deletion in involving 15q26.2-qter, caused by the formation of a dicentric chromosome 15, using metaphase FISH and tiling resolution (32 k genome-wide array-based comparative genomic hybridization (CGH. Results After an initial characterization of the dicentric chromosome by metaphase FISH, array CGH analysis mapped the terminal deletion to encompass a 6.48 megabase (Mb region, ranging from 93.86–100.34 Mb on chromosome 15. Conclusion In conclusion, we present an additional case to the growing family of reported cases with 15q26-deletion, thoroughly characterized at the molecular cytogenetic level. In the deleted regions, four candidate genes responsible for the phenotype of the patient could be delineated: IGFR1, MEF2A, CHSY1, and TM2D3. Further characterization of additional patients harboring similar 15q-aberrations might hopefully in the future lead to the description of a clear cut clinically recognizable syndrome.

Associating Oligonucleotides with Positively Charged Liposomes

Czech Academy of Sciences Publication Activity Database

Jurkiewicz, P.; Okruszek, A.; Hof, Martin; Langner, M.

2003-01-01

Roč. 8, č. 1 (2003), s. 77-84 ISSN 1425-8153 R&D Projects: GA MŠk LN00A032 Institutional research plan: CEZ:AV0Z4040901 Keywords : oligonucleotides * fluorescence correlation spectroscopy * DOTAP Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 0.455, year: 2003

THERAPEUTIC ANTISENSE OLIGONUCLEOTIDES AGAINST CANCER: HURDLING TO THE CLINIC

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Pedro Miguel Duarte Moreno

2014-10-01

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.

Peroxide-mediated desulfurization of phosphorothioate oligonucleotides and its prevention.

Science.gov (United States)

Krotz, Achim H; Mehta, Rahul C; Hardee, Gregory E

2005-02-01

Desulfurization at the internucleotide phosphorothioate linkage of antisense oligonucleotides (ASOs) in dermatological formulations has been investigated using strong ion exchange chromatography and mass spectroscopy. The formation of phosphate diester linkages appeared to arise from a reaction between the phosphorothioate oligonucleotide and a potent oxidizing agent. Screening of excipients used in the formulation indicated that the cause of desulfurization was related to the presence of polyethylene glycol-derived nonionic surfactants MYRJ 52 or BRIJ 58. Autoxidation of the polyethylene glycol chain is suggested as the probable origin for the observed incompatibility. The ability of various antioxidants to prevent oxidative degradation of ASO-1 in simple test systems and in oil-in-water emulsions is described. It is found that in test systems both lipophilic and hydrophilic antioxidants are effective. However, in cream formulation (oil-in-water emulsions) of ASO-1 the addition of hydrophilic antioxidants L-cysteine or DL-alpha-lipoic acid has been shown to be superior in protecting the oligonucleotide from desulfurization upon storage. Copyright 2004 Wiley-Liss, Inc.

High-density rhesus macaque oligonucleotide microarray design using early-stage rhesus genome sequence information and human genome annotations

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Magness Charles L

2007-01-01

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

A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent

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Lykke Pedersen

2014-01-01

Full Text Available Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

Copy number alterations in small intestinal neuroendocrine tumors determined by array comparative genomic hybridization

International Nuclear Information System (INIS)

Hashemi, Jamileh; Fotouhi, Omid; Sulaiman, Luqman; Kjellman, Magnus; Höög, Anders; Zedenius, Jan; Larsson, Catharina

2013-01-01

Small intestinal neuroendocrine tumors (SI-NETs) are typically slow-growing tumors that have metastasized already at the time of diagnosis. The purpose of the present study was to further refine and define regions of recurrent copy number (CN) alterations (CNA) in SI-NETs. Genome-wide CNAs was determined by applying array CGH (a-CGH) on SI-NETs including 18 primary tumors and 12 metastases. Quantitative PCR analysis (qPCR) was used to confirm CNAs detected by a-CGH as well as to detect CNAs in an extended panel of SI-NETs. Unsupervised hierarchical clustering was used to detect tumor groups with similar patterns of chromosomal alterations based on recurrent regions of CN loss or gain. The log rank test was used to calculate overall survival. Mann–Whitney U test or Fisher’s exact test were used to evaluate associations between tumor groups and recurrent CNAs or clinical parameters. The most frequent abnormality was loss of chromosome 18 observed in 70% of the cases. CN losses were also frequently found of chromosomes 11 (23%), 16 (20%), and 9 (20%), with regions of recurrent CN loss identified in 11q23.1-qter, 16q12.2-qter, 9pter-p13.2 and 9p13.1-11.2. Gains were most frequently detected in chromosomes 14 (43%), 20 (37%), 4 (27%), and 5 (23%) with recurrent regions of CN gain located to 14q11.2, 14q32.2-32.31, 20pter-p11.21, 20q11.1-11.21, 20q12-qter, 4 and 5. qPCR analysis confirmed most CNAs detected by a-CGH as well as revealed CNAs in an extended panel of SI-NETs. Unsupervised hierarchical clustering of recurrent regions of CNAs revealed two separate tumor groups and 5 chromosomal clusters. Loss of chromosomes 18, 16 and 11 and again of chromosome 20 were found in both tumor groups. Tumor group II was enriched for alterations in chromosome cluster-d, including gain of chromosomes 4, 5, 7, 14 and gain of 20 in chromosome cluster-b. Gain in 20pter-p11.21 was associated with short survival. Statistically significant differences were observed between primary

Application of the micro-array comparative genomic hybridization technology in preimplantation genetic diagnosis%Array-CGH技术在胚胎植入前遗传学诊断中的应用进展

Institute of Scientific and Technical Information of China (English)

韩丹; 陈大蔚; 曹云霞; 周平

2015-01-01

As a new kind high-throughput genomics technology, micro array-based comparative genomic hybridization (aCGH) has brought the huge change for molecular biology and medical research. Because of the detection range covers the whole genome, high efficiency, easy operation etc, aCGH has been widely used in many areas of human genetic disease diagnosis, tumor genomics, systems biology and prenatal diagnosis. Human preimplantation genetic diagnosis (PGD) is an important part of assisted reproductive technology, with the development of molecular genetics technology, its application range is continuously widening. Based on aCGH technology in PGD for embryonic whole genome screening for aneuploidy and structural abnormalities, human PGD/human preimplantation genetic screening (PGS) implantation rate and clinical pregnancy rate have improved significantly. In this article, we discussed the advantages, disadvantages and prospects of aCGH in prenatal diagnosis.%微阵列比较基因组杂交（aCGH）作为一种新兴的高通量检测技术，给分子生物学及医学研究带来了巨大变化，因其检测范围覆盖全基因组、高效率、操作简便等特点，在人类遗传疾病诊断，肿瘤基因组学，系统生物学研究及产前诊断中已有了广泛应用。植入前遗传学诊断（PGD）是辅助生殖技术的重要组成部分，随着分子遗传学技术的发展，其应用范围也不断拓宽。基于aCGH技术在PGD中对胚胎全染色体组非整倍体及结构异常的筛查，PGD/植入前遗传学筛查（PGS）胚胎植入率和临床妊娠率均有显著提高，本文就aCGH技术在胚胎植入前遗传学诊断中的应用进行综述。

Versatile functionalization of nanoelectrodes by oligonucleotides via pyrrole electrochemistry.

Science.gov (United States)

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

2010-11-15

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.

Comparative genomic hybridization (CGH) analysis of stage 4 neuroblastoma reveals high frequency of 11q deletion in tumors lacking MYCN amplification

NARCIS (Netherlands)

Plantaz, D.; Vandesompele, J.; van Roy, N.; Lastowska, M.; Bown, N.; Combaret, V.; Favrot, M. C.; Delattre, O.; Michon, J.; Bénard, J.; Hartmann, O.; Nicholson, J. C.; Ross, F. M.; Brinkschmidt, C.; Laureys, G.; Caron, H.; Matthay, K. K.; Feuerstein, B. G.; Speleman, F.

2001-01-01

We have studied the occurrence and association of 11q deletions with other chromosomal imbalances in Stage 4 neuroblastomas. To this purpose we have performed comparative genomic hybridization (CGH) analysis on 50 Stage 4 neuroblastomas and these data were analyzed together with those from 33

Cross-species comparison of aCGH data from mouse and human BRCA1- and BRCA2-mutated breast cancers

International Nuclear Information System (INIS)

Holstege, Henne; Wessels, Lodewyk FA; Nederlof, Petra M; Jonkers, Jos; Beers, Erik van; Velds, Arno; Liu, Xiaoling; Joosse, Simon A; Klarenbeek, Sjoerd; Schut, Eva; Kerkhoven, Ron; Klijn, Christiaan N

2010-01-01

Genomic gains and losses are a result of genomic instability in many types of cancers. BRCA1- and BRCA2-mutated breast cancers are associated with increased amounts of chromosomal aberrations, presumably due their functions in genome repair. Some of these genomic aberrations may harbor genes whose absence or overexpression may give rise to cellular growth advantage. So far, it has not been easy to identify the driver genes underlying gains and losses. A powerful approach to identify these driver genes could be a cross-species comparison of array comparative genomic hybridization (aCGH) data from cognate mouse and human tumors. Orthologous regions of mouse and human tumors that are commonly gained or lost might represent essential genomic regions selected for gain or loss during tumor development. To identify genomic regions that are associated with BRCA1- and BRCA2-mutated breast cancers we compared aCGH data from 130 mouse Brca1 Δ/Δ ;p53 Δ/Δ , Brca2 Δ/Δ ;p53 Δ/Δ and p53 Δ/Δ mammary tumor groups with 103 human BRCA1-mutated, BRCA2-mutated and non-hereditary breast cancers. Our genome-wide cross-species analysis yielded a complete collection of loci and genes that are commonly gained or lost in mouse and human breast cancer. Principal common CNAs were the well known MYC-associated gain and RB1/INTS6-associated loss that occurred in all mouse and human tumor groups, and the AURKA-associated gain occurred in BRCA2-related tumors from both species. However, there were also important differences between tumor profiles of both species, such as the prominent gain on chromosome 10 in mouse Brca2 Δ/Δ ;p53 Δ/Δ tumors and the PIK3CA associated 3q gain in human BRCA1-mutated tumors, which occurred in tumors from one species but not in tumors from the other species. This disparity in recurrent aberrations in mouse and human tumors might be due to differences in tumor cell type or genomic organization between both species. The selection of the oncogenome during

Review on investigations of antisense oligonucleotides with the use of mass spectrometry.

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Studzińska, Sylwia

2018-01-01

Antisense oligonucleotides have been investigated as potential drugs for years. They inhibit target gene or protein expression. The present review summarizes their modifications, modes of action, and applications of liquid chromatography coupled with mass spectrometry for qualitative and quantitative analysis of these compounds. The most recent reports on a given topic were given prominence, while some early studies were reviewed in order to provide a theoretical background. The present review covers the issues of using ion-exchange chromatography, ion-pair reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography for the separation of antisense oligonucleotides. The application of mass spectrometry was described with regard to the ionization type used for the determination of these potential therapeutics. Moreover, the current approaches and applications of mass spectrometry for quantitative analysis of antisense oligonucleotides and their metabolites as well as their impurities during in vitro and in vivo studies were discussed. Finally, certain conclusions and perspectives on the determination of therapeutic oligonucleotides in various samples were briefly described. Copyright © 2017 Elsevier B.V. All rights reserved.

Non-invasive preimplantation genetic screening using array comparative genomic hybridization on spent culture media: a proof-of-concept pilot study.

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Feichtinger, Michael; Vaccari, Enrico; Carli, Luca; Wallner, Elisabeth; Mädel, Ulrike; Figl, Katharina; Palini, Simone; Feichtinger, Wilfried

2017-06-01

The aim of this pilot study was to assess if array comparative genomic hybridization (aCGH), non-invasive preimplantation genetic screening (PGS) on blastocyst culture media is feasible. Therefore, aCGH analysis was carried out on 22 spent blastocyst culture media samples after polar body PGS because of advanced maternal age. All oocytes were fertilized by intracytoplasmic sperm injection and all embryos underwent assisted hatching. Concordance of polar body analysis and culture media genetic results was assessed. Thirteen out of 18 samples (72.2%) revealed general concordance of ploidy status (euploid or aneuploid). At least one chromosomal aberration was found concordant in 10 out of 15 embryos found to be aneuploid by both polar body and culture media analysis. Overall, 17 out of 35 (48.6%) single chromosomal aneuploidies were concordant between the culture media and polar body analysis. By analysing negative controls (oocytes with fertilization failure), notable maternal contamination was observed. Therefore, non-invasive PGS could serve as a second matrix after polar body or cleavage stage PGS; however, in euploid results, maternal contamination needs to be considered and results interpreted with caution. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Detection of DNA oligonucleotides with base mutations by terahertz spectroscopy and microstructures.

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Mingjie Tang

Full Text Available DNA oligonucleotides with a 5-base mutation at the 3'-terminus were investigated by terahertz (THz spectroscopy in a marker-free manner. The four single-stranded oligonucleotides with 17nt have been detected with specificity on a microfluidic chip, and corroborated by spectral measurements with split-ring resonators. The number of hydrogen bonds formed between the oligonucleotide and its surrounding water molecules, deemed a key contribution to the THz absorption of biological solutions, was explored by molecular dynamics simulations to explain the experimental findings. Our work underlies the feasibility of THz spectroscopy combined with microstructures for marker-free detection of DNA, which may form the basis of a prospective diagnostic tool for studying genic mutation.

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides.

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Ito, Yuko; Sone, Yumiko; Mizutani, Takaharu

2004-03-01

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non

CGI: Java software for mapping and visualizing data from array-based comparative genomic hybridization and expression profiling.

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Gu, Joyce Xiuweu-Xu; Wei, Michael Yang; Rao, Pulivarthi H; Lau, Ching C; Behl, Sanjiv; Man, Tsz-Kwong

2007-10-06

With the increasing application of various genomic technologies in biomedical research, there is a need to integrate these data to correlate candidate genes/regions that are identified by different genomic platforms. Although there are tools that can analyze data from individual platforms, essential software for integration of genomic data is still lacking. Here, we present a novel Java-based program called CGI (Cytogenetics-Genomics Integrator) that matches the BAC clones from array-based comparative genomic hybridization (aCGH) to genes from RNA expression profiling datasets. The matching is computed via a fast, backend MySQL database containing UCSC Genome Browser annotations. This program also provides an easy-to-use graphical user interface for visualizing and summarizing the correlation of DNA copy number changes and RNA expression patterns from a set of experiments. In addition, CGI uses a Java applet to display the copy number values of a specific BAC clone in aCGH experiments side by side with the expression levels of genes that are mapped back to that BAC clone from the microarray experiments. The CGI program is built on top of extensible, reusable graphic components specifically designed for biologists. It is cross-platform compatible and the source code is freely available under the General Public License.

CGI: Java Software for Mapping and Visualizing Data from Array-based Comparative Genomic Hybridization and Expression Profiling

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Joyce Xiuweu-Xu Gu

2007-01-01

Full Text Available With the increasing application of various genomic technologies in biomedical research, there is a need to integrate these data to correlate candidate genes/regions that are identified by different genomic platforms. Although there are tools that can analyze data from individual platforms, essential software for integration of genomic data is still lacking. Here, we present a novel Java-based program called CGI (Cytogenetics-Genomics Integrator that matches the BAC clones from array-based comparative genomic hybridization (aCGH to genes from RNA expression profiling datasets. The matching is computed via a fast, backend MySQL database containing UCSC Genome Browser annotations. This program also provides an easy-to-use graphical user interface for visualizing and summarizing the correlation of DNA copy number changes and RNA expression patterns from a set of experiments. In addition, CGI uses a Java applet to display the copy number values of a specifi c BAC clone in aCGH experiments side by side with the expression levels of genes that are mapped back to that BAC clone from the microarray experiments. The CGI program is built on top of extensible, reusable graphic components specifically designed for biologists. It is cross-platform compatible and the source code is freely available under the General Public License.

NAA-modified DNA oligonucleotides with zwitterionic backbones: stereoselective synthesis of A-T phosphoramidite building blocks.

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Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

2015-01-01

Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the NAA-linkage at T-T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X-T motifs (X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A-T phosphoramidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide analogues.

Nucleobase-modified antisense oligonucleotides containing 5-(phenyltriazol)-2′-deoxyuridine nucleotides induce exon-skipping

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Le, Bao T.; Hornum, Mick; Sharma, Pawan K.

2017-01-01

Chemically-modified antisense oligonucleotide-mediated exon-skipping has been validated as a therapeutic strategy for tackling several disease pathologies, particularly duchenne muscular dystrophy. To date, only sugar-modified and internucleotide linkage-modified oligonucleotide chemistries have...

Fabrication of DNA nanotubes with an array of exterior magnetic nanoparticles.

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Rafati, Adele; Zarrabi, Ali; Gill, Pooria

2017-10-01

Described here a methodology for arraying of magnetic nanoparticles (MNPs) on the surface of DNA nanotubes (DNTs). Positioning of magnetic nanoparticles at exterior surface of DNTs were shaped after self-assembling of oligonucleotide staples within an M13mp18 DNA scaffold via an origami process. The staples were partially labeled with biotin to be arrayed at the surface of DNTs. Gel retardation assay of the DNTs carrying magnetic nanoparticles indicated a reversely behavioral electrophoretic movement in comparison to the nanotubes have been demonstrated previously. Also, high resolution transmission electron microscopy confirmed positioning magnetic nanoparticles at the exterior surface of DNTs, correctly. Ultrastructural characteristics of these DNA nanotubes using atomic force microscopy demonstrated topographic heights on their surfaces formed through positioning of magnetic nanoparticles outside the tubules. This nanoarchitecture would be potential for multiple arraying of nanoparticles that those be useful as functionalized chimeric nanocarriers for developing novel nanodrugs and nanobiosensors. Copyright © 2017. Published by Elsevier B.V.

Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance.

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Hayashi, Junsuke; Samezawa, Yusuke; Ochi, Yosuke; Wada, Shun-Ichi; Urata, Hidehito

2017-07-15

We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named "Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides" were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability. Copyright © 2017 Elsevier Ltd. All rights reserved.

Generation of a genomic tiling array of the human Major Histocompatibility Complex (MHC and its application for DNA methylation analysis

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Ottaviani Diego

2008-05-01

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.

Functional regulation of RNA-induced silencing complex by photoreactive oligonucleotides.

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Matsuyama, Yohei; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

2014-02-01

We developed a novel method for regulation of RISC function by photoreactive oligonucleotides (Ps-Oligo) containing 2'-O-psoralenylmethoxyethyl adenosine (Aps). We observed that inhibitory effects of Ps-Oligos on RISC function were enhanced by UV-irradiation compared with 2'-O-methyl-oligonucleotide without Aps. These results suggest Ps-Oligo inhibited RISC function by cross-linking effect, and we propose that the concept described in this report may be promising and applicable one to regulate the small RNA-mediated post-transcriptional regulation. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

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

2008-07-01

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

Clinical array-based karyotyping of breast cancer with equivocal HER2 status resolves gene copy number and reveals chromosome 17 complexity

International Nuclear Information System (INIS)

Gunn, Shelly; Gorre, Mercedes; Mohammed, Mansoor; Yeh, I-Tien; Lytvak, Irina; Tirtorahardjo, Budi; Dzidic, Natasha; Zadeh, Soheila; Kim, Jaeweon; McCaskill, Chris; Lim, Lony

2010-01-01

HER2 gene copy status, and concomitant administration of trastuzumab (Herceptin), remains one of the best examples of targeted cancer therapy based on understanding the genomic etiology of disease. However, newly diagnosed breast cancer cases with equivocal HER2 results present a challenge for the oncologist who must make treatment decisions despite the patient's unresolved HER2 status. In some cases both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are reported as equivocal, whereas in other cases IHC results and FISH are discordant for positive versus negative results. The recent validation of array-based, molecular karyotyping for clinical oncology testing provides an alternative method for determination of HER2 gene copy number status in cases remaining unresolved by traditional methods. In the current study, DNA extracted from 20 formalin fixed paraffin embedded (FFPE) tissue samples from newly diagnosed cases of invasive ductal carcinoma referred to our laboratory with unresolved HER2 status, were analyzed using a clinically validated genomic array containing 127 probes covering the HER2 amplicon, the pericentromeric regions, and both chromosome 17 arms. Array-based comparative genomic hybridization (array CGH) analysis of chromosome 17 resolved HER2 gene status in [20/20] (100%) of cases and revealed additional chromosome 17 copy number changes in [18/20] (90%) of cases. Array CGH analysis also revealed two false positives and one false negative by FISH due to 'ratio skewing' caused by chromosomal gains and losses in the centromeric region. All cases with complex rearrangements of chromosome 17 showed genome-wide chromosomal instability. These results illustrate the analytical power of array-based genomic analysis as a clinical laboratory technique for resolution of HER2 status in breast cancer cases with equivocal results. The frequency of complex chromosome 17 abnormalities in these cases suggests that the two

Correlation test to assess low-level processing of high-density oligonucleotide microarray data

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Bergh Jonas

2005-03-01

Full Text Available Abstract Background There are currently a number of competing techniques for low-level processing of oligonucleotide array data. The choice of technique has a profound effect on subsequent statistical analyses, but there is no method to assess whether a particular technique is appropriate for a specific data set, without reference to external data. Results We analyzed coregulation between genes in order to detect insufficient normalization between arrays, where coregulation is measured in terms of statistical correlation. In a large collection of genes, a random pair of genes should have on average zero correlation, hence allowing a correlation test. For all data sets that we evaluated, and the three most commonly used low-level processing procedures including MAS5, RMA and MBEI, the housekeeping-gene normalization failed the test. For a real clinical data set, RMA and MBEI showed significant correlation for absent genes. We also found that a second round of normalization on the probe set level improved normalization significantly throughout. Conclusion Previous evaluation of low-level processing in the literature has been limited to artificial spike-in and mixture data sets. In the absence of a known gold-standard, the correlation criterion allows us to assess the appropriateness of low-level processing of a specific data set and the success of normalization for subsets of genes.

DNA suspension arrays: silencing discrete artifacts for high-sensitivity applications.

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Matthew S Lalonde

2010-11-01

Full Text Available Detection of low frequency single nucleotide polymorphisms (SNPs has important implications in early screening for tumorgenesis, genetic disorders and pathogen drug resistance. Nucleic acid arrays are a powerful tool for genome-scale SNP analysis, but detection of low-frequency SNPs in a mixed population on an array is problematic. We demonstrate a model assay for HIV-1 drug resistance mutations, wherein ligase discrimination products are collected on a suspension array. In developing this system, we discovered that signal from multiple polymorphisms was obscured by two discrete hybridization artifacts. Specifically: 1 tethering of unligated probes on the template DNA elicited false signal and 2 unpredictable probe secondary structures impaired probe capture and suppressed legitimate signal from the array. Two sets of oligonucleotides were used to disrupt these structures; one to displace unligated reporter labels from the bead-bound species and another to occupy sequences which interfered with array hybridization. This artifact silencing system resulted in a mean 21-fold increased sensitivity for 29 minority variants of 17 codons in our model assay for mutations most commonly associated with HIV-1 drug resistance. Furthermore, since the artifacts we characterized are not unique to our system, their specific inhibition might improve the quality of data from solid-state microarrays as well as from the growing number of multiple analyte suspension arrays relying on sequence-specific nucleic acid target capture.

Short G-rich oligonucleotides as a potential therapeutic for Huntington's Disease

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Parekh-Olmedo Hetal

2006-10-01

Full Text Available Abstract Background Huntington's Disease (HD is an inherited autosomal dominant genetic disorder in which neuronal tissue degenerates. The pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant HD protein. Mutant huntingtin (Htt is produced by HD genes that contain an increased number of glutamine codons within the first exon and this expansion leads to the production of a protein that misfolds. Recent studies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions. Results As such, efforts to find a therapy for HD have focused on agents that disrupt or block the mutant Htt aggregation pathway. Here, we report that short guanosine monotonic oligonucleotides capable of adopting a G-quartet structure, are effective inhibitors of aggregation. By utilizing a biochemical/immunoblotting assay as an initial screen, we identified a 20-mer, all G-oligonucleotide (HDG as an active molecule. Subsequent testing in a cell-based assay revealed that HDG was an effective inhibitor of aggregation of a fusion protein, comprised of a mutant Htt fragment and green fluorescent protein (eGFP. Taken together, our results suggest that a monotonic G-oligonucleotide, capable of adopting a G-quartet conformation is an effective inhibitor of aggregation. This oligonucleotide can also enable cell survival in PC12 cells overexpressing a mutant Htt fragment fusion gene. Conclusion Single-stranded DNA oligonucleotides capable of forming stable G-quartets can inhibit aggregation of the mutant Htt fragment protein. This activity maybe an important part of the pathogenecity of Huntington's Disease. Our results reveal a new class of agents that could be developed as a therapeutic approach for Huntington's Disease.

Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

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Elela Sherif

2006-01-01

Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

Selective Detection of Peptide-Oligonucleotide Heteroconjugates Utilizing Capillary HPLC-ICPMS

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Catron, Brittany; Caruso, Joseph A.; Limbach, Patrick A.

2012-06-01

A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as 31P+, the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1-2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.

i-Genome: A database to summarize oligonucleotide data in genomes

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Chang Yu-Chung

2004-10-01

Full Text Available Abstract Background Information on the occurrence of sequence features in genomes is crucial to comparative genomics, evolutionary analysis, the analyses of regulatory sequences and the quantitative evaluation of sequences. Computing the frequencies and the occurrences of a pattern in complete genomes is time-consuming. Results The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase. The database supports various complete genomes including human, yeast, worm, and 128 microbial genomes. Conclusions This investigation presents and implements an efficiently computational approach to accumulate the occurrences of the oligonucleotides or patterns in complete genomes. A database is established to maintain the information of the sequence features, including the distributions of oligonucleotide, the gene distribution, the distribution of repetitive elements in genomes and the occurrences of the oligonucleotides. The database can provide more effective and efficient way to access the repetitive features in genomes.

In vitro transcription in the presence of DNA oligonucleotides can generate strong anomalous initiation sites.

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Chow, C W; Clark, M P; Rinaldo, J E; Chalkley, R

1996-03-01

In the present study, we have explored an unexpected observation in transcription initiation that is mediated by single-stranded oligonucleotides. Initially, our goal was to understand the function of different upstream regulatory elements/initiation sites in the rat xanthine dehydrogenase/oxidase (XDH/XO) promoter. We performed in vitro transcription with HeLa nuclear extracts in the presence of different double-stranded oligonucleotides against upstream elements as competitors. A new and unusual transcription initiation site was detected by primer extension. This new initiation site maps to the downstream region of the corresponding competitor. Subsequent analyses have indicated that the induction of a new transcription initiation site is anomalous which is due to the presence of a small amount of single-stranded oligonucleotide in the competitor. We found that this anomalous initiation site is insensitive to the orientation of the promoter and requires only a small amount of single-stranded oligonucleotide (< 2-fold molar excess relative to template). We surmise that a complementary interaction between the single-stranded oligonucleotide and transiently denatured promoter template may be responsible for this sequence-specific transcription initiation artifact. To study the regulation of transcription initiation by in vitro transcription approaches, we propose that one should probe the effect of removing transacting factors by adding an excess of a cognate oligonucleotide which does not bear exact sequence identity to the template.

Characterization of canine osteosarcoma by array comparative genomic hybridization and RT-qPCR: signatures of genomic imbalance in canine osteosarcoma parallel the human counterpart.

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Angstadt, Andrea Y; Motsinger-Reif, Alison; Thomas, Rachael; Kisseberth, William C; Guillermo Couto, C; Duval, Dawn L; Nielsen, Dahlia M; Modiano, Jaime F; Breen, Matthew

2011-11-01

Osteosarcoma (OS) is the most commonly diagnosed malignant bone tumor in humans and dogs, characterized in both species by extremely complex karyotypes exhibiting high frequencies of genomic imbalance. Evaluation of genomic signatures in human OS using array comparative genomic hybridization (aCGH) has assisted in uncovering genetic mechanisms that result in disease phenotype. Previous low-resolution (10-20 Mb) aCGH analysis of canine OS identified a wide range of recurrent DNA copy number aberrations, indicating extensive genomic instability. In this study, we profiled 123 canine OS tumors by 1 Mb-resolution aCGH to generate a dataset for direct comparison with current data for human OS, concluding that several high frequency aberrations in canine and human OS are orthologous. To ensure complete coverage of gene annotation, we identified the human refseq genes that map to these orthologous aberrant dog regions and found several candidate genes warranting evaluation for OS involvement. Specifically, subsequenct FISH and qRT-PCR analysis of RUNX2, TUSC3, and PTEN indicated that expression levels correlated with genomic copy number status, showcasing RUNX2 as an OS associated gene and TUSC3 as a possible tumor suppressor candidate. Together these data demonstrate the ability of genomic comparative oncology to identify genetic abberations which may be important for OS progression. Large scale screening of genomic imbalance in canine OS further validates the use of the dog as a suitable model for human cancers, supporting the idea that dysregulation discovered in canine cancers will provide an avenue for complementary study in human counterparts. Copyright © 2011 Wiley-Liss, Inc.

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)

2016-11-15

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.

Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

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Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

2008-01-01

Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

Genetisk udredning ved infantile spasmer

DEFF Research Database (Denmark)

Hansen, Lars Kjærsgaard; Ousager, Lilian Bomme; Møller, Rikke Steensbjerre

2012-01-01

Infantile spasms are a symptom of a severe epileptic encephalopathy. It is important to determine the aetiology for a child's disease. When a standard programme for evaluating the aetiology of the infantile spasms is unsuccessful genetic causes should be considered. We suggest array CGH as the fi......Infantile spasms are a symptom of a severe epileptic encephalopathy. It is important to determine the aetiology for a child's disease. When a standard programme for evaluating the aetiology of the infantile spasms is unsuccessful genetic causes should be considered. We suggest array CGH...

A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp.

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Boyang Cao

Full Text Available 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.

A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp.

Science.gov (United States)

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

2014-01-01

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. PMID:25469776

Copy number variations in Saudi family with intellectual disability and epilepsy.

Science.gov (United States)

Naseer, Muhammad I; Chaudhary, Adeel G; Rasool, Mahmood; Kalamegam, Gauthaman; Ashgan, Fai T; Assidi, Mourad; Ahmed, Farid; Ansari, Shakeel A; Zaidi, Syed Kashif; Jan, Mohammed M; Al-Qahtani, Mohammad H

2016-10-17

Epilepsy is genetically complex but common brain disorder of the world affecting millions of people with almost of all age groups. Novel Copy number variations (CNVs) are considered as important reason for the numerous neurodevelopmental disorders along with intellectual disability and epilepsy. DNA array based studies contribute to explain a more severe clinical presentation of the disease but interoperation of many detected CNVs are still challenging. In order to study novel CNVs with epilepsy related genes in Saudi family with six affected and two normal individuals with several forms of epileptic seizures, intellectual disability (ID), and minor dysmorphism, we performed the high density whole genome Agilent sure print G3 Hmn CGH 2x 400 K array-CGH chips analysis. Our results showed de novo deletions, duplications and deletion plus duplication on differential chromosomal regions in the affected individuals that were not shown in the normal fathe and normal kids by using Agilent CytoGenomics 3.0.6.6 softwear. Copy number gain were observed in the chromosome 1, 16 and 22 with LCE3C, HPR, GSTT2, GSTTP2, DDT and DDTL genes respectively whereas the deletions observed in the chromosomal regions 8p23-p21 (4303127-4337759) and the potential gene in this region is CSMD1 (OMIM: 612279). Moreover, the array CGH results deletions and duplication were also validated by using primer design of deleted regions utilizing the flanked SNPs using simple PCR and also by using quantitative real time PCR. We found some of the de novo deletions and duplication in our study in Saudi family with intellectual disability and epilepsy. Our results suggest that array-CGH should be used as a first line of genetic test for epilepsy except there is a strong indication for a monogenic syndrome. The advanced high through put array-CGH technique used in this study aim to collect the data base and to identify new mechanisms describing epileptic disorder, may help to improve the clinical

Surface modification of plasmonic nanostructured materials with thiolated oligonucleotides in 10 seconds using selective microwave heating

International Nuclear Information System (INIS)

Abel, B.; Aslan, K.

2012-01-01

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)

Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries.

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Murgha, Yusuf E; Rouillard, Jean-Marie; Gulari, Erdogan

2014-01-01

Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

18F-labelling of oligonucleotides using succinimido 4-[18F]fluorobenzoat

International Nuclear Information System (INIS)

Hedberg, Elisabeth; Laangstroem, Bengt

1998-01-01

A general method for the labelling of oligodeoxynucleotide and oligonucleoside phosphorothioates in the 5'-position with the positron-emitting radionuclide 18 F (t 1/2 = 110 min) is described. The label was incorporated by the reaction of succinimido 4 -[ 18 F]fluorobenzoate 4 with oligonucleotides (18- and 20-mers) modified in the 5'-position with a hexylamine linker. Oligodeoxynucleotides 5'-GCT,AAG,CGA,TGC,CTC,CGT-3' (MTCa) and 5'-GAA,CCT,CTG,AGA,GTT,CAT,CT-3' (CROa) were labelled in 20±3 % (MTCa) and 13±3 % (CROa) radiochemical yields (non-isolated, decay-corrected and based on 4). Oligonucleoside phosphorotioates MTCa (S-MTCa) and CROa (S-CROa) were labelled in 9 and 7% isolated radiochemical yield, respectively (decay-corrected and based on 4). Labelled oligonucleotides and phosphorothioate analogues were separated from their unlabelled counterparts using reversed-phase perfusion chromatography. The molecular mass of a labelled oligonucleotide CROa was determined by ESI-MS after a mixed 18 F/ 19 F fluorobenzoate labelling experiment and corresponded with the expected structure. (au)

NF2 tumor suppressor gene: a comprehensive and efficient detection of somatic mutations by denaturing HPLC and microarray-CGH.

Science.gov (United States)

Szijan, Irene; Rochefort, Daniel; Bruder, Carl; Surace, Ezequiel; Machiavelli, Gloria; Dalamon, Viviana; Cotignola, Javier; Ferreiro, Veronica; Campero, Alvaro; Basso, Armando; Dumanski, Jan P; Rouleau, Guy A

2003-01-01

The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.

Prenatal diagnosis of Wolf-Hirschhorn syndrome confirmed by comparative genomic hybridization array: report of two cases and review of the literature

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Sifakis Stavros

2012-02-01

Full Text Available Abstract Wolf-Hirschhorn syndrome (WHS is a well known genetic condition caused by a partial deletion of the short arm of chromosome 4. The great variability in the extent of the 4p deletion and the possible contribution of additional genetic rearrangements lead to a wide spectrum of clinical manifestations. The majority of the reports of prenatally diagnosed WHS cases are associated with large 4p deletions identified by conventional chromosome analysis; however, the widespread clinical use of novel molecular techniques such as array comparative genomic hybridization (a-CGH has increased the detection rate of submicroscopic chromosomal aberrations associated with WHS phenotype. We provide a report of two fetuses with WHS presenting with intrauterine growth restriction as an isolated finding or combined with oligohydramnios and abnormal Doppler waveform in umbilical artery and uterine arteries. Standard karyotyping demonstrated a deletion on chromosome 4 in both cases [del(4(p15.33 and del(4(p15.31, respectively] and further application of a-CGH confirmed the diagnosis and offered a precise characterization of the genetic defect. A detailed review of the currently available literature on the prenatal diagnostic approach of WHS in terms of fetal sonographic assessment and molecular cytogenetic investigation is also provided.

Preimplantation genetic screening for all 24 chromosomes by microarray comparative genomic hybridization significantly increases implantation rates and clinical pregnancy rates in patients undergoing in vitro fertilization with poor prognosis

Science.gov (United States)

Majumdar, Gaurav; Majumdar, Abha; Lall, Meena; Verma, Ishwar C.; Upadhyaya, Kailash C.

2016-01-01

CONTEXT: A majority of human embryos produced in vitro are aneuploid, especially in couples undergoing in vitro fertilization (IVF) with poor prognosis. Preimplantation genetic screening (PGS) for all 24 chromosomes has the potential to select the most euploid embryos for transfer in such cases. AIM: To study the efficacy of PGS for all 24 chromosomes by microarray comparative genomic hybridization (array CGH) in Indian couples undergoing IVF cycles with poor prognosis. SETTINGS AND DESIGN: A retrospective, case–control study was undertaken in an institution-based tertiary care IVF center to compare the clinical outcomes of twenty patients, who underwent 21 PGS cycles with poor prognosis, with 128 non-PGS patients in the control group, with the same inclusion criterion as for the PGS group. MATERIALS AND METHODS: Single cells were obtained by laser-assisted embryo biopsy from day 3 embryos and subsequently analyzed by array CGH for all 24 chromosomes. Once the array CGH results were available on the morning of day 5, only chromosomally normal embryos that had progressed to blastocyst stage were transferred. RESULTS: The implantation rate and clinical pregnancy rate (PR) per transfer were found to be significantly higher in the PGS group than in the control group (63.2% vs. 26.2%, P = 0.001 and 73.3% vs. 36.7%, P = 0.006, respectively), while the multiple PRs sharply declined from 31.9% to 9.1% in the PGS group. CONCLUSIONS: In this pilot study, we have shown that PGS by array CGH can improve the clinical outcome in patients undergoing IVF with poor prognosis. PMID:27382234

Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes

Energy Technology Data Exchange (ETDEWEB)

Hwang, Gyoyeon [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of); Lee, Hansol [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Jiyeon, E-mail: jylee@kist.re.kr [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of)

2015-11-13

The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.

Synthesis and Biophysical Investigations of Oligonucleotides Containing Galactose-Modified DNA, LNA and 2'-Amino-LNA Monomers

DEFF Research Database (Denmark)

Ries, Annika; Kumar, Rajesh; Lou, Chenguang

2016-01-01

Galactose-modified thymidine, LNA-T and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2'...

Comparison of the Agilent, ROMA/NimbleGen and Illumina platforms for classification of copy number alterations in human breast tumors

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Naume B

2008-08-01

Full Text Available Abstract Background Microarray Comparative Genomic Hybridization (array CGH provides a means to examine DNA copy number aberrations. Various platforms, brands and underlying technologies are available, facing the user with many choices regarding platform sensitivity and number, localization, and density distribution of probes. Results We evaluate three different platforms presenting different nature and arrangement of the probes: The Agilent Human Genome CGH Microarray 44 k, the ROMA/NimbleGen Representational Oligonucleotide Microarray 82 k, and the Illumina Human-1 Genotyping 109 k BeadChip, with Agilent being gene oriented, ROMA/NimbleGen being genome oriented, and Illumina being genotyping oriented. We investigated copy number changes in 20 human breast tumor samples representing different gene expression subclasses, using a suite of graphical and statistical methods designed to work across platforms. Despite substantial differences in the composition and spatial distribution of probes, the comparison revealed high overall concordance. Notably however, some short amplifications and deletions of potential biological importance were not detected by all platforms. Both correlation and cluster analysis indicate a somewhat higher similarity between ROMA/NimbleGen and Illumina than between Agilent and the other two platforms. The programs developed for the analysis are available from http://www.ifi.uio.no/bioinf/Projects/. Conclusion We conclude that platforms based on different technology principles reveal similar aberration patterns, although we observed some unique amplification or deletion peaks at various locations, only detected by one of the platforms. The correct platform choice for a particular study is dependent on whether the appointed research intention is gene, genome, or genotype oriented.

The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice

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Ingrid E C Verhaart

2014-01-01

Full Text Available Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides will be required. To investigate the dynamics and persistence of antisense 2′-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours–24 weeks. Oligonucleotide half-life was longer in heart (~65 days compared with that in skeletal muscle, liver, and kidney (~35 days. Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days. Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3–8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2′-O-methyl phosphorothioate oligos used for the treatment of DMD.

Small regions of overlapping deletions on 6q26 in human astrocytic tumours identified using chromosome 6 tile path array CGH

Science.gov (United States)

Ichimura, Koichi; Mungall, Andrew J; Fiegler, Heike; Pearson, Danita M.; Dunham, Ian; Carter, Nigel P; Collins, V. Peter

2009-01-01

Deletions of chromosome 6 are a common abnormality in diverse human malignancies including astrocytic tumours, suggesting the presence of tumour suppressor genes (TSG). In order to help identify candidate TSGs, we have constructed a chromosome 6 tile path microarray. The array contains 1780 clones (778 PACs and 1002 BACs) that cover 98.3% of the published chromosome 6 sequences. A total of 104 adult astrocytic tumours (10 diffuse astrocytomas, 30 anaplastic astrocytomas (AA), 64 glioblastomas (GB)) were analysed using this array. Single copy number change was successfully detected and the result was in general concordant with a microsatellite analysis. The pattern of copy number change was complex with multiple interstitial deletions/gains. However, a predominance of telomeric 6q deletions was seen. Two small common and overlapping regions of deletion at 6q26 were identified. One was 1002 kb in size and contained PACRG and QKI, while the second was 199 kb and harbours a single gene, ARID1B. The data show that the chromosome 6 tile path array is useful in mapping copy number changes with high resolution and accuracy. We confirmed the high frequency of chromosome 6 deletions in AA and GB, and identified two novel commonly deleted regions that may harbour TSGs. PMID:16205629

Streptavidin-coated gold nanoparticles: critical role of oligonucleotides on stability and fractal aggregation

Directory of Open Access Journals (Sweden)

Roberta D'Agata

2017-01-01

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.

FISH Oracle: a web server for flexible visualization of DNA copy number data in a genomic context.

Science.gov (United States)

Mader, Malte; Simon, Ronald; Steinbiss, Sascha; Kurtz, Stefan

2011-07-28

The rapidly growing amount of array CGH data requires improved visualization software supporting the process of identifying candidate cancer genes. Optimally, such software should work across multiple microarray platforms, should be able to cope with data from different sources and should be easy to operate. We have developed a web-based software FISH Oracle to visualize data from multiple array CGH experiments in a genomic context. Its fast visualization engine and advanced web and database technology supports highly interactive use. FISH Oracle comes with a convenient data import mechanism, powerful search options for genomic elements (e.g. gene names or karyobands), quick navigation and zooming into interesting regions, and mechanisms to export the visualization into different high quality formats. These features make the software especially suitable for the needs of life scientists. FISH Oracle offers a fast and easy to use visualization tool for array CGH and SNP array data. It allows for the identification of genomic regions representing minimal common changes based on data from one or more experiments. FISH Oracle will be instrumental to identify candidate onco and tumor suppressor genes based on the frequency and genomic position of DNA copy number changes. The FISH Oracle application and an installed demo web server are available at http://www.zbh.uni-hamburg.de/fishoracle.

GAB2 Amplification in Squamous Cell Lung Cancer of Non-Smokers.

Science.gov (United States)

Park, Yu Rang; Bae, Soo Hyeon; Ji, Wonjun; Seo, Eul Ju; Lee, Jae Cheol; Kim, Hyeong Ryul; Jang, Se Jin; Choi, Chang Min

2017-11-01

Lung squamous cell cancer (SCC) is typically found in smokers and has a very low incidence in non-smokers, indicating differences in the tumor biology of lung SCC in smokers and non-smokers. However, the specific mutations that drive tumor growth in non-smokers have not been identified. To identify mutations in lung SCC of non-smokers, we performed a genetic analysis using arrays comparative genomic hybridization (ArrayCGH). We analyzed 19 patients with lung SCC who underwent surgical treatment between April 2005 and April 2015. Clinical characteristics were reviewed, and DNA was extracted from fresh frozen lung cancer specimens. All of copy number alterations from ArrayCGH were validated using The Cancer Genome Atlas (TCGA) copy number variation (CNV) data of lung SCC. We examined the frequency of copy number changes according to the smoking status (non-smoker [n = 8] or smoker [n = 11]). We identified 16 significantly altered regions from ArrayCGH data, three gain and four loss regions overlapped with the TCGA lung squamous cell carcinoma (LUSC) patients. Within these overlapped significant regions, we detected 15 genes that have been reported in the Cancer Gene census. We also found that the proto-oncogene GAB2 (11q14.1) was significantly amplified in non-smokers patients and vice versa in both ArrayCGH and TCGA data. Immunohistochemical analyses showed that GAB2 protein was relatively upregulated in non-smoker than smoker tissues (37.5% vs. 9.0%, P = 0.007). GAB2 amplification may have an important role in the development of lung SCC in non-smokers. GAB2 may represent a potential biomarker for lung SCC in non-smokers. © 2017 The Korean Academy of Medical Sciences.

Calculation method of CGH for Binocular Eyepiece-Type Electro Holography

International Nuclear Information System (INIS)

Yang, Chanyoung; Yoneyama, Takuo; Sakamoto, Yuji; Okuyama, Fumio

2013-01-01

We had researched about eyepiece-type electro holography to display 3-D images of larger objects at wider angle. We had enlarged visual field considering depth of object with Fourier optical system using two lenses. In this paper, we extend our system for binocular. In the binocular system, we use two different holograms for each eye. The 3-D image for left eye should be observed like the real object observed using left eye and the same for right eye. So, we propose a method of calculation of computer-generated hologram (CGH) transforming the coordinate system of the model data to make two holograms for binocular eyepiece-type electro holography. The coordinate system of original model data is called the world coordinate system. The left and the right coordinate system are transformed from the world coordinate system. We also propose the method for correcting the installation error that occurs when placing the electronic and optical devices. The installation error is calculated and the model data is corrected using the distance between measured position and setup position of the reconstructed image Optical reconstruction experiments were carried out to verify the proposed method.

The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

Science.gov (United States)

2013-01-01

Background Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. Results Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. Conclusions Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative. PMID:23445545

DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY – HIGH RESOLUTION MASS SPECTROMETRY

Science.gov (United States)

Nikcevic, Irena; Wyrzykiewicz, Tadeusz K.; Limbach, Patrick A.

2010-01-01

Summary An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described. Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3′-terminal phosphate monoester and 3′-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N3 (2-cyanoethyl) adducts

Structural properties of oligonucleotide monolayers on gold surfaces probed by fluorescence investigations.

Science.gov (United States)

Rant, Ulrich; Arinaga, Kenji; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard; Tornow, Marc

2004-11-09

We present optical investigations on the conformation of oligonucleotide layers on Au surfaces. Our studies concentrate on the effect of varying surface coverage densities on the structural properties of layers of 12- and 24mer single-stranded DNA, tethered to the Au surface at one end while being labeled with a fluorescent marker at the opposing end. The distance-dependent energy transfer from the marker dye to the metal surface, which causes quenching of the observed fluorescence, is used to provide information on the orientation of the DNA strands relative to the surface. Variations in the oligonucleotide coverage density, as determined from electrochemical quantification, over 2 orders of magnitude are achieved by employing different preparation conditions. The observed enhancement in fluorescence intensity with increasing DNA coverage can be related to a model involving mutual steric interactions of oligonucleotides on the surface, as well as fluorescence quenching theory. Finally, the applicability of the presented concepts for investigations of heterogeneous monolayers is demonstrated by means of studying the coadsorption of mercaptohexanol onto DNA-modified Au surfaces.

An aCGH classifier derived from BRCA1-mutated breast cancer and benefit of high-dose platinum-based chemotherapy in HER2-negative breast cancer patients

NARCIS (Netherlands)

Vollebergh, M. A.; Lips, E. H.; Nederlof, P. M.; Wessels, L. F. A.; Schmidt, M. K.; van Beers, E. H.; Cornelissen, S.; Holtkamp, M.; Froklage, F. E.; de Vries, E. G. E.; Schrama, J. G.; Wesseling, J.; van de Vijver, M. J.; van Tinteren, H.; de Bruin, Michiel; Hauptmann, M.; Rodenhuis, S.; Linn, S. C.

2011-01-01

Breast cancer cells deficient for BRCA1 are hypersensitive to agents inducing DNA double-strand breaks (DSB), such as bifunctional alkylators and platinum agents. Earlier, we had developed a comparative genomic hybridisation (CGH) classifier based on BRCA1-mutated breast cancers. We hypothesised

Factor XI Antisense Oligonucleotide for Prevention of Venous Thrombosis

NARCIS (Netherlands)

Büller, Harry R.; Bethune, Claudette; Bhanot, Sanjay; Gailani, David; Monia, Brett P.; Raskob, Gary E.; Segers, Annelise; Verhamme, Peter; Weitz, Jeffrey I.; Weitz, Jeffrey; Prins, Martin; Beenen, Ludo; Otten, Hans-Martin; Roos, Yvo; Slagboom, Ton; Vandenbriele, Christophe; Vanassche, Thomas; Dani, Vidhi; Schulz, Dan; Shapiro, Cara; Kwoh, Katherine; Jung, Bill; Gawinek-Samelczak, Agata; Kaemmer, Christina; Angelov, S.; Stavrev, V.; Kinov, P.; Dessouki, E.; Abuzgaya, F.; Baurovskis, A.; Peredistijs, A.; Petronis, S.; Danilyak, V.; Driagin, V.; Kuropatkin, G.; Parfeev, S.; Safronov, A.; Ankin, M.; Korzh, M.; Olinichenko, G.; Polivoda, A.; Shevchenko, V.; Sulyma, V.

2015-01-01

Background 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

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon].

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Dmitrienko, E V; Pyshnaia, I A; Pyshnyĭ, D V

2010-01-01

The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

Design, validation and annotation of transcriptome-wide oligonucleotide probes for the oligochaete annelid Eisenia fetida.

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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

Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

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Hongguang Sun

2014-01-01

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.

Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

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Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

1994-09-01

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.

LNA-modified oligonucleotides mediate specific inhibition of microRNA function

DEFF Research Database (Denmark)

Ørom, Ulf Andersson; Kauppinen, Sakari; Lund, Anders H

2006-01-01

microRNAs are short, endogenous non-coding RNAs that act as post-transcriptional modulators of gene expression. Important functions for microRNAs have been found in the regulation of development, cellular proliferation and differentiation, while perturbed miRNA expression patterns have been...... observed in many human cancers. Here we present a method for specific inhibition of miRNA function through interaction with LNA-modified antisense oligonucleotides and report the specificity of this application. We show that LNA-modified oligonucleotides can inhibit exogenously introduced miRNAs with high...... specificity using a heterologous reporter assay, and furthermore demonstrate their ability to inhibit an endogenous miRNA in Drosophila melanogaster cells, leading to up-regulation of the cognate target protein. The method shows stoichiometric and reliable inhibition of the targeted miRNA and can thus...

Copy number variations in Saudi family with intellectual disability and epilepsy

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Muhammad I. Naseer

2016-10-01

Full Text Available Abstract Background Epilepsy is genetically complex but common brain disorder of the world affecting millions of people with almost of all age groups. Novel Copy number variations (CNVs are considered as important reason for the numerous neurodevelopmental disorders along with intellectual disability and epilepsy. DNA array based studies contribute to explain a more severe clinical presentation of the disease but interoperation of many detected CNVs are still challenging. Results In order to study novel CNVs with epilepsy related genes in Saudi family with six affected and two normal individuals with several forms of epileptic seizures, intellectual disability (ID, and minor dysmorphism, we performed the high density whole genome Agilent sure print G3 Hmn CGH 2x 400 K array-CGH chips analysis. Our results showed de novo deletions, duplications and deletion plus duplication on differential chromosomal regions in the affected individuals that were not shown in the normal fathe and normal kids by using Agilent CytoGenomics 3.0.6.6 softwear. Copy number gain were observed in the chromosome 1, 16 and 22 with LCE3C, HPR, GSTT2, GSTTP2, DDT and DDTL genes respectively whereas the deletions observed in the chromosomal regions 8p23-p21 (4303127–4337759 and the potential gene in this region is CSMD1 (OMIM: 612279. Moreover, the array CGH results deletions and duplication were also validated by using primer design of deleted regions utilizing the flanked SNPs using simple PCR and also by using quantitative real time PCR. Conclusions We found some of the de novo deletions and duplication in our study in Saudi family with intellectual disability and epilepsy. Our results suggest that array-CGH should be used as a first line of genetic test for epilepsy except there is a strong indication for a monogenic syndrome. The advanced high through put array-CGH technique used in this study aim to collect the data base and to identify new mechanisms describing

Reliability and applications of statistical methods based on oligonucleotide frequencies in bacterial and archaeal genomes

DEFF Research Database (Denmark)

Bohlin, J; Skjerve, E; Ussery, David

2008-01-01

with here are mainly used to examine similarities between archaeal and bacterial DNA from different genomes. These methods compare observed genomic frequencies of fixed-sized oligonucleotides with expected values, which can be determined by genomic nucleotide content, smaller oligonucleotide frequencies......, or be based on specific statistical distributions. Advantages with these statistical methods include measurements of phylogenetic relationship with relatively small pieces of DNA sampled from almost anywhere within genomes, detection of foreign/conserved DNA, and homology searches. Our aim was to explore...... the reliability and best suited applications for some popular methods, which include relative oligonucleotide frequencies (ROF), di- to hexanucleotide zero'th order Markov methods (ZOM) and 2.order Markov chain Method (MCM). Tests were performed on distant homology searches with large DNA sequences, detection...

Characterization of rat brain NCAM mRNA using DNA oligonucleotide probes

DEFF Research Database (Denmark)

Andersson, A M; Gaardsvoll, H; Giladi, E

1990-01-01

A number of different isoforms of the neural cell adhesion molecule (NCAM) have been identified. The difference between these is due to alternative splicing of a single NCAM gene. In rat brain NCAM mRNAs with sizes of 7.4, 6.7, 5.2, 4.3 and 2.9 kb have been reported. We have synthesized six DNA...... oligonucleotides, that hybridize to different exons in the NCAM gene. Furthermore we have constructed three oligonucleotides, that exclusively hybridize to mRNAs lacking certain exons, by letting them consist of sequences adjacent to both sides of the splice sites. By means of these probes we have characterized...... the five NCAM mRNAs in rat brain....

Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L.) gene expression oligonucleotide microarray.

Science.gov (United States)

Fernandez, Paula; Soria, Marcelo; Blesa, David; DiRienzo, Julio; Moschen, Sebastian; Rivarola, Maximo; Clavijo, Bernardo Jose; Gonzalez, Sergio; Peluffo, Lucila; Príncipi, Dario; Dosio, Guillermo; Aguirrezabal, Luis; García-García, Francisco; Conesa, Ana; Hopp, Esteban; Dopazo, Joaquín; Heinz, Ruth Amelia; Paniego, Norma

2012-01-01

Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (psunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.

Use of Array Comparative Genomic Hybridization for the Diagnosis of DiGeorge Syndrome in Saudi Arabian Population.

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Bahamat, Abeer A; Assidi, Mourad; Lary, Sahira A; Almughamsi, Muna M; Peer Zada, Abdul A; Chaudhary, Adeel; Abuzenadah, Adel; Abu-Elmagd, Muhammad; Al-Qahtani, Mohammed

2018-01-01

DiGeorge syndrome (DGS) is a genetic disorder known as a clinically variable syndrome with over 180 associated phenotypic features. It is caused by a common human deletion in the 22q11.2 chromosomal region and currently is affecting approximately 1 in 4,000 individuals. Despite the prevalence of inherited diseases mainly due to consanguineous marriages, the current diagnosis of DGS in Saudi Arabia is mainly based on conventional high-resolution chromosome banding (karyotyping) and FISH techniques. However, advanced genome-wide studies for detecting microdeletions or duplications across the whole genome are needed. The aim of this study is to implement and use aCGH technology in clinical diagnosis of the 22q11.2 deletion in Saudi Arabian DGS patients and to confirm its effectiveness compared to conventional FISH and chromosome banding techniques. Thirty suspected DGS patients were assessed for chromosome 22q11.2 deletion using high-resolution G-banding, FISH, and aCGH. The aCGH results were compared with those obtained by the other 2 cytogenetic techniques. G-banding detected the 22q11.2 deletion in only 1 patient in the cohort. Moreover, it detected additional chromosomal aberrations in 3 other patients. Using FISH, allowed for detection of the 22q11.2 deletion in 2 out of 30 patients. Interestingly, the use of aCGH technique showed deletions in the chromosome 22q11.2 region in 8 patients, indicating a 4-fold increase in diagnostic detection capacity compared to FISH. Our results show the effectiveness of aCGH to overcome the limitations of FISH and G-banding in terms of diagnostic yield and allow whole genome screening and detection of a larger number of deletions and/or duplications in Saudi Arabian DGS patients. Except for balanced translocations and inversions, our data demonstrate the suitability of aCGH in the diagnostics of submicroscopic deletion syndromes such as DGS and most chromosomal aberrations or complex abnormalities scattered throughout the human

Development of Multiexon Skipping Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy

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Yokota, Toshifumi; Wood, Matthew J. A.

2013-01-01

Duchenne muscular dystrophy (DMD) is an incurable, X-linked progressive muscle degenerative disorder that results from the absence of dystrophin protein and leads to premature death in affected individuals due to respiratory and/or cardiac failure typically by age of 30. Very recently the exciting prospect of an effective oligonucleotide therapy has emerged which restores dystrophin protein expression to affected tissues in DMD patients with highly promising data from a series of clinical trials. This therapeutic approach is highly mutation specific and thus is personalised. Therefore DMD has emerged as a model genetic disorder for understanding and overcoming of the challenges of developing personalised genetic medicines. One of the greatest weaknesses of the current oligonucleotide approach is that it is a mutation-specific therapy. To address this limitation, we have recently demonstrated that exons 45–55 skipping therapy has the potential to treat clusters of mutations that cause DMD, which could significantly reduce the number of compounds that would need to be developed in order to successfully treat all DMD patients. Here we discuss and review the latest preclinical work in this area as well as a variety of accompanying issues, including efficacy and potential toxicity of antisense oligonucleotides, prior to human clinical trials. PMID:23984357

Lipid-modified G4-decoy oligonucleotide anchored to nanoparticles

DEFF Research Database (Denmark)

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

2016-01-01

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 fa...

Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides

Science.gov (United States)

Basiri, Babak; Murph, Mandi M.; Bartlett, Michael G.

2017-08-01

Alkylamines are widely used as ion-pairing agents during LC-MS of oligonucleotides. In addition to a better chromatographic separation, they also assist with the desorption of oligonucleotide ions into the gas phase, cause charge state reduction, and decrease cation adduction. However, the choice of such ion-pairing agents has considerable influence on the MS signal intensity of oligonucleotides as they can also cause significant ion suppression. Interestingly, optimal ion-pairing agents should be selected on a case by case basis as their choice is strongly influenced by the sequence of the oligonucleotide under investigation. Despite imposing major practical difficulties to analytical method development, such a highly variable system that responds very strongly to the nuances of the electrospray composition provides an excellent opportunity for a fundamental study of the electrospray ionization process. Our investigations using this system quantitatively revealed the major factors that influenced the ESI ionization efficiency of oligonucleotides. Parameters such as boiling point, proton affinity, partition coefficient, water solubility, and Henry's law constants for the ion-pairing reagents and the hydrophobic thymine content of the oligonucleotides were found to be the most significant contributors. Identification of these parameters also allowed for the development of a statistical predictive algorithm that can assist with the choice of an optimum IP agent for each particular oligonucleotide sequence. We believe that research in the field of oligonucleotide bioanalysis will significantly benefit from this algorithm (included in Supplementary Material) as it advocates for the use of lesser-known but more suitable ion-pair alternatives to TEA for many oligonucleotide sequences.

Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

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Muyrers Joep PP

2003-01-01

Full Text Available Abstract Background The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined. Results Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα but only requires a phage annealing protein (RecT or Redβ. Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein. Conclusion Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids

DEFF Research Database (Denmark)

Nåbo, Lina J.; Madsen, Charlotte S.; Jensen, Knud J.

2015-01-01

Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid...... targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild...... conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our...

Oligonucleotide aptamers against tyrosine kinase receptors: Prospect for anticancer applications.

Science.gov (United States)

Camorani, Simona; Crescenzi, Elvira; Fedele, Monica; Cerchia, Laura

2018-04-01

Transmembrane receptor tyrosine kinases (RTKs) play crucial roles in cancer cell proliferation, survival, migration and differentiation. Area of intense research is searching for effective anticancer therapies targeting these receptors and, to date, several monoclonal antibodies and small-molecule tyrosine kinase inhibitors have entered the clinic. However, some of these drugs show limited efficacy and give rise to acquired resistance. Emerging highly selective compounds for anticancer therapy are oligonucleotide aptamers that interact with their targets by recognizing a specific three-dimensional structure. Because of their nucleic acid nature, the rational design of advanced strategies to manipulate aptamers for both diagnostic and therapeutic applications is greatly simplified over antibodies. In this manuscript, we will provide a comprehensive overview of oligonucleotide aptamers as next generation strategies to efficiently target RTKs in human cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

Science.gov (United States)

Alsop, Eric B; Raymond, Jason

2013-01-01

Oligonucleotide signatures, especially tetranucleotide signatures, have been used as method for homology binning by exploiting an organism's inherent biases towards the use of specific oligonucleotide words. Tetranucleotide signatures have been especially useful in environmental metagenomics samples as many of these samples contain organisms from poorly classified phyla which cannot be easily identified using traditional homology methods, including NCBI BLAST. This study examines oligonucleotide signatures across 1,424 completed genomes from across the tree of life, substantially expanding upon previous work. A comprehensive analysis of mononucleotide through nonanucleotide word lengths suggests that longer word lengths substantially improve the classification of DNA fragments across a range of sizes of relevance to high throughput sequencing. We find that, at present, heptanucleotide signatures represent an optimal balance between prediction accuracy and computational time for resolving taxonomy using both genomic and metagenomic fragments. We directly compare the ability of tetranucleotide and heptanucleotide world lengths (tetranucleotide signatures are the current standard for oligonucleotide word usage analyses) for taxonomic binning of metagenome reads. We present evidence that heptanucleotide word lengths consistently provide more taxonomic resolving power, particularly in distinguishing between closely related organisms that are often present in metagenomic samples. This implies that longer oligonucleotide word lengths should replace tetranucleotide signatures for most analyses. Finally, we show that the application of longer word lengths to metagenomic datasets leads to more accurate taxonomic binning of DNA scaffolds and have the potential to substantially improve taxonomic assignment and assembly of metagenomic data.

Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

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Eric B Alsop

Full Text Available Oligonucleotide signatures, especially tetranucleotide signatures, have been used as method for homology binning by exploiting an organism's inherent biases towards the use of specific oligonucleotide words. Tetranucleotide signatures have been especially useful in environmental metagenomics samples as many of these samples contain organisms from poorly classified phyla which cannot be easily identified using traditional homology methods, including NCBI BLAST. This study examines oligonucleotide signatures across 1,424 completed genomes from across the tree of life, substantially expanding upon previous work. A comprehensive analysis of mononucleotide through nonanucleotide word lengths suggests that longer word lengths substantially improve the classification of DNA fragments across a range of sizes of relevance to high throughput sequencing. We find that, at present, heptanucleotide signatures represent an optimal balance between prediction accuracy and computational time for resolving taxonomy using both genomic and metagenomic fragments. We directly compare the ability of tetranucleotide and heptanucleotide world lengths (tetranucleotide signatures are the current standard for oligonucleotide word usage analyses for taxonomic binning of metagenome reads. We present evidence that heptanucleotide word lengths consistently provide more taxonomic resolving power, particularly in distinguishing between closely related organisms that are often present in metagenomic samples. This implies that longer oligonucleotide word lengths should replace tetranucleotide signatures for most analyses. Finally, we show that the application of longer word lengths to metagenomic datasets leads to more accurate taxonomic binning of DNA scaffolds and have the potential to substantially improve taxonomic assignment and assembly of metagenomic data.

The database of chromosome imbalance regions and genes resided in lung cancer from Asian and Caucasian identified by array-comparative genomic hybridization

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Lo Fang-Yi

2012-06-01

Full Text Available Abstract Background Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis. Methods Array-comparative genomic hybridization (array-CGH was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR, chromogenic in situ hybridization (CISH, reverse transcriptase-qPCR (RT-qPCR, and immunohistochemistry (IHC in more patients. Results We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1 functioning in Rho activity control, FRAT2 (10q24.1 involved in Wnt signaling, PAFAH1B1 (17p13.3 functioning in motility control, and ZNF322A (6p22.1 involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (PP=0.06. In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of

The database of chromosome imbalance regions and genes resided in lung cancer from Asian and Caucasian identified by array-comparative genomic hybridization

International Nuclear Information System (INIS)

Lo, Fang-Yi; Nandi, Suvobroto; Salgia, Ravi; Wang, Yi-Ching; Chang, Jer-Wei; Chang, I-Shou; Chen, Yann-Jang; Hsu, Han-Shui; Huang, Shiu-Feng Kathy; Tsai, Fang-Yu; Jiang, Shih Sheng; Kanteti, Rajani

2012-01-01

Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis. Array-comparative genomic hybridization (array-CGH) was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR), chromogenic in situ hybridization (CISH), reverse transcriptase-qPCR (RT-qPCR), and immunohistochemistry (IHC) in more patients. We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1) functioning in Rho activity control, FRAT2 (10q24.1) involved in Wnt signaling, PAFAH1B1 (17p13.3) functioning in motility control, and ZNF322A (6p22.1) involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (P<0.001~P=0.06). In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of PAFAH1B1 protein overexpression was 68

Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate nanocapsules in cells

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Tomcin S

2014-11-01

Full Text Available Stephanie Tomcin,1 Grit Baier,1 Katharina Landfester,1 Volker Mailänder1,21Max Planck Institute for Polymer Research, 2University Medical Center of the Johannes Gutenberg University, III Medical Clinic, Mainz, GermanyAbstract: For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly(n-butylcyanoacrylate (PBCA nanocapsules as a shell and separately an oligonucleotide (20 mer as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.Keywords: drug delivery, mitochondria, miniemulsion, colocalization

A novel deletion in the thyrotropin Beta-subunit gene identified by array comparative genomic hybridization analysis causes central congenital hypothyroidism in a boy originating from Turkey.

Science.gov (United States)

Hermanns, Pia; Couch, Robert; Leonard, Norma; Klotz, Cherise; Pohlenz, Joachim

2014-01-01

Isolated central congenital hypothyroidism (ICCH) is rare but important. Most ICCH patients are diagnosed later, which results in severe growth failure and intellectual disability. We describe a boy with ICCH due to a large homozygous TSHβ gene deletion. A 51-day-old male Turkish infant, whose parents were first cousins, was admitted for evaluation of prolonged jaundice. His clinical appearance was compatible with hypothyroidism. Venous thyrotropin (TSH) was undetectably low, with a subsequent low free T4 and a low free T3, suggestive of central hypothyroidism. Using different PCR protocols, we could not amplify both coding exons of the boy's TSHβ gene, which suggested a deletion. An array comparative genomic hybridization (aCGH) using specific probes around the TSHβ gene locus showed him to be homozygous for a 6-kb deletion spanning all exons and parts of the 5' untranslated region of the gene. Infants who are clinically suspected of having hypothyroidism should be evaluated thoroughly, even if their TSH-based screening result is normal. In cases with ICCH and undetectably low TSH serum concentrations, a TSHβ gene deletion should be considered; aCGH should be performed when gene deletions are suspected. In such cases, PCR-based sequencing techniques give negative results.

Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.

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Mannion James W

2002-10-01

Full Text Available Abstract Background Rat oligonucleotide microarrays were used to detect changes in gene expression in the dorsal root ganglion (DRG 3 days following sciatic nerve transection (axotomy. Two comparisons were made using two sets of triplicate microarrays, naïve versus naïve and naïve versus axotomy. Results Microarray variability was assessed using the naïve versus naïve comparison. These results support use of a P 1.5-fold expression change and P 1.5-fold and P in situ hybridization verified the expression of 24 transcripts. These data showed an 83% concordance rate with the arrays; most mismatches represent genes with low expression levels reflecting limits of array sensitivity. A significant correlation was found between actual mRNA differences and relative changes between microarrays (r2 = 0.8567. Temporal patterns of individual genes regulation varied. Conclusions We identify parameters for microarray analysis which reduce error while identifying many putatively regulated genes. Functional classification of these genes suggest reorganization of cell structural components, activation of genes expressed by immune and inflammatory cells and down-regulation of genes involved in neurotransmission.

Live birth from a patient with a three-way balanced translocation t(5 ...

African Journals Online (AJOL)

Objectives: Array comparative genomic hybridisation (array-CGH) was used to screen embryos for chromosome imbalances. Methods: Embryo biopsy, preimplantation genetic diagnosis using a 24sure+ kit to detect translocations in embryos. Results: Of 10 embryos tested, 2 were found to have an unbalanced translocation, ...

An oral oligonucleotide delivery system based on a thiolated polymer: Development and in vitro evaluation.

Science.gov (United States)

Martien, Ronny; Hoyer, Herbert; Perera, Glen; Schnürch, Andreas Bernkop

2011-08-01

The purpose of this study was to develop and evaluate an oral oligonucleotide delivery system based on a thiolated polymer/reduced glutathione (GSH) system providing a protective effect toward nucleases and permeation enhancement. A polycarbophil-cysteine conjugate (PCP-Cys) was synthesized. Enzymatic degradation of a model oligonucleotide by DNase I and within freshly collected intestinal fluid was investigated in the absence and presence of PCP-Cys. Permeation studies with PCP-Cys/GSH versus control were performed in vitro on Caco-2 cell monolayers and ex vivo on rat intestinal mucosa. PCP-Cys displayed 223 ± 13.8 μmol thiol groups per gram polymer. After 4h, 61% of the free oligonucleotides were degraded by DNase I and 80% within intestinal fluid. In contrast, less than 41% (DNase I) and 60% (intestinal fluid) were degraded in the presence of 0.02% (m/v) PCP-Cys. Permeation studies revealed an 8-fold (Caco-2) and 10-fold (intestinal mucosa) increase in apparent permeability compared to buffer control. Hence, this PCP-Cys/GSH system might be a promising tool for the oral administration of oligonucleotides as it allows a significant protection toward degrading enzymes and facilitates their transport across intestinal membranes. Copyright © 2011 Elsevier B.V. All rights reserved.

Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics

Directory of Open Access Journals (Sweden)

Moizza Mansoor

2008-01-01

Full Text Available Antisense oligonucleotides (As-ODNs are single stranded, synthetically prepared strands of deoxynucleotide sequences, usually 18–21 nucleotides in length, complementary to the mRNA sequence of the target gene. As-ODNs are able to selectively bind cognate mRNA sequences by sequence-specific hybridization. This results in cleavage or disablement of the mRNA and, thus, inhibits the expression of the target gene. The specificity of the As approach is based on the probability that, in the human genome, any sequence longer than a minimal number of nucleotides (nt, 13 for RNA and 17 for DNA, normally occurs only once. The potential applications of As-ODNs are numerous because mRNA is ubiquitous and is more accessible to manipulation than DNA. With the publication of the human genome sequence, it has become theoretically possible to inhibit mRNA of almost any gene by As-ODNs, in order to get a better understanding of gene function, investigate its role in disease pathology and to study novel therapeutic targets for the diseases caused by dysregulated gene expression. The conceptual simplicity, the availability of gene sequence information from the human genome, the inexpensive availability of synthetic oligonucleotides and the possibility of rational drug design makes As-ODNs powerful tools for target identification, validation and therapeutic intervention. In this review we discuss the latest developments in antisense oligonucleotide design, delivery, pharmacokinetics and potential side effects, as well as its uses in target identification and validation, and finally focus on the current developments of antisense oligonucleotides in therapeutic intervention in various diseases.

Sequence-Dependent Mechanism of DNA Oligonucleotide Dehybridization Resolved through Infrared Spectroscopy.

Science.gov (United States)

Sanstead, Paul J; Stevenson, Paul; Tokmakoff, Andrei

2016-09-14

Despite its important role in biology and nanotechnology, many questions remain regarding the molecular mechanism and dynamics by which oligonucleotides recognize and hybridize to their complementary sequence. The thermodynamics and kinetics of DNA oligonucleotide hybridization and dehybridization are often assumed to involve an all-or-nothing two-state dissociation pathway, but deviations from this behavior can be considerable even for short sequences. We introduce a new strategy to characterize the base-pair-specific thermal dissociation mechanism of DNA oligonucleotides through steady-state and time-resolved infrared spectroscopy. Experiments are interpreted with a lattice model to provide a structure-specific interpretation. This method is applied to a model set of self-complementary 10-base-pair sequences in which the placement of GC base pairs is varied in an otherwise AT strand. Through a combination of Fourier transform infrared and two-dimensional infrared spectroscopy, experiments reveal varying degrees of deviation from simple two-state behavior. As the temperature is increased, duplexes dissociate through a path in which the terminal bases fray, without any significant contribution from loop configurations. Transient temperature jump experiments reveal time scales of 70-100 ns for fraying and 10-30 μs for complete dissociation near the melting temperature. Whether or not frayed states are metastable intermediates or short-lived configurations during the full dissociation of the duplex is dictated by the nucleobase sequence.

A High-Throughput Computational Framework for Identifying Significant Copy Number Aberrations from Array Comparative Genomic Hybridisation Data

Directory of Open Access Journals (Sweden)

Ian Roberts

2012-01-01

Full Text Available Reliable identification of copy number aberrations (CNA from comparative genomic hybridization data would be improved by the availability of a generalised method for processing large datasets. To this end, we developed swatCGH, a data analysis framework and region detection heuristic for computational grids. swatCGH analyses sequentially displaced (sliding windows of neighbouring probes and applies adaptive thresholds of varying stringency to identify the 10% of each chromosome that contains the most frequently occurring CNAs. We used the method to analyse a published dataset, comparing data preprocessed using four different DNA segmentation algorithms, and two methods for prioritising the detected CNAs. The consolidated list of the most commonly detected aberrations confirmed the value of swatCGH as a simplified high-throughput method for identifying biologically significant CNA regions of interest.

De novo microdeletions of chromosome 6q14.1-q14.3 and 6q12.1-q14.1 in two patients with intellectual disability - further delineation of the 6q14 microdeletion syndrome and review of the literature

DEFF Research Database (Denmark)

Becker, Kerstin; Di Donato, Nataliya; Holder-Espinasse, Muriel

2012-01-01

with broad nasal tip, anteverted nares, long philtrum, and thin upper lip. In this study we describe two patients with overlapping 6q14 deletions presenting with developmental delay and characteristic dysmorphism. Molecular karyotyping using array CGH analysis revealed a de novo 8.9 Mb deletion at 6q14.1-q14.......3 and a de novo 11.3 Mb deletion at 6q12.1-6q14.1, respectively. We provide a review of the clinical features of twelve other patients with 6q14 deletions detected by array CGH analysis. By assessing all reported data we could not identify a single common region of deletion. Possible candidate genes in 6q14...

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: alessandro.paciaroni@fisica.unipg.it [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)

2015-07-07

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.

Polynucleotides. XXXII. Further studies on the synthesis of oligonucleotides containing 8,2'-S-cycloadenosine.

Science.gov (United States)

Ikehara, M; Tezuka, T

1975-01-01

A dinucleoside monophosphate, 8,2'-anhydro-8-mercapto-9-beta-D-arabinofuranosyladenine phosphoryl-(3'-5')-inosine (AspI) was synthesized by the condensation of protected 8-mercapto-adenosine 2',3'-cyclic phosphate and 2',3'-isopropylideneinosine with diphenylphosphorochloridate. 8-Mercaptoadenosine 2',3'-cyclic phosphate was polymerized by using tetraphenyl pyrophosphate as the condensing reagent. As oligonucleotides, thus obtained, contained some uncyclized 8-mercaptoadenosine residues and were cleaved at these sites with 0.3N KOH. As 5'-phosphate was synthesized and polymerized with DCC to give oligonucleotides with chain lengths 2 to 9. PMID:170595

Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques

Energy Technology Data Exchange (ETDEWEB)

Awsiuk, K., E-mail: kamil.awsiuk@uj.edu.pl [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Rysz, J. [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Petrou, P. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece); Budkowski, A. [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Bernasik, A. [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, Kraków 30-059 (Poland); Kakabakos, S. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece); Marzec, M.M. [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, Kraków 30-059 (Poland); Raptis, I. [Institute for Advanced Materials, Physicochemical Processes, Nanotechnology and Microsystems, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece)

2014-01-30

To immobilize effectively oligonucleotide probes on SiO{sub 2} modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin–biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin–b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin–b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m{sup 2}) and second (1.31(±0.22) mg/m{sup 2}) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m{sup 2}) and fourth (0.41(±0.11) mg/m{sup 2}) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.

Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques

International Nuclear Information System (INIS)

Awsiuk, K.; Rysz, J.; Petrou, P.; Budkowski, A.; Bernasik, A.; Kakabakos, S.; Marzec, M.M.; Raptis, I.

2014-01-01

To immobilize effectively oligonucleotide probes on SiO 2 modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin–biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin–b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin–b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m 2 ) and second (1.31(±0.22) mg/m 2 ) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m 2 ) and fourth (0.41(±0.11) mg/m 2 ) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.

Tandem Oligonucleotide Probe Annealing and Elongation To Discriminate Viral Sequence

DEFF Research Database (Denmark)

Taskova, Maria; Uhd, Jesper; Miotke, Laura

2017-01-01

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...

Genome dynamics of short oligonucleotides: the example of bacterial DNA uptake enhancing sequences.

Directory of Open Access Journals (Sweden)

Mohammed Bakkali

Full Text Available Among the many bacteria naturally competent for transformation by DNA uptake-a phenomenon with significant clinical and financial implications- Pasteurellaceae and Neisseriaceae species preferentially take up DNA containing specific short sequences. The genomic overrepresentation of these DNA uptake enhancing sequences (DUES causes preferential uptake of conspecific DNA, but the function(s behind this overrepresentation and its evolution are still a matter for discovery. Here I analyze DUES genome dynamics and evolution and test the validity of the results to other selectively constrained oligonucleotides. I use statistical methods and computer simulations to examine DUESs accumulation in Haemophilus influenzae and Neisseria gonorrhoeae genomes. I analyze DUESs sequence and nucleotide frequencies, as well as those of all their mismatched forms, and prove the dependence of DUESs genomic overrepresentation on their preferential uptake by quantifying and correlating both characteristics. I then argue that mutation, uptake bias, and weak selection against DUESs in less constrained parts of the genome combined are sufficient enough to cause DUESs accumulation in susceptible parts of the genome with no need for other DUES function. The distribution of overrepresentation values across sequences with different mismatch loads compared to the DUES suggests a gradual yet not linear molecular drive of DNA sequences depending on their similarity to the DUES. Other genomically overrepresented sequences, both pro- and eukaryotic, show similar distribution of frequencies suggesting that the molecular drive reported above applies to other frequent oligonucleotides. Rare oligonucleotides, however, seem to be gradually drawn to genomic underrepresentation, thus, suggesting a molecular drag. To my knowledge this work provides the first clear evidence of the gradual evolution of selectively constrained oligonucleotides, including repeated, palindromic and protein

Detection of recurrent transmission of 17q12 microdeletion by array comparative genomic hybridization in a fetus with prenatally diagnosed hydronephrosis, hydroureter, and multicystic kidney, and variable clinical spectrum in the family.

Science.gov (United States)

Chen, Chih-Ping; Chang, Shuenn-Dyh; Wang, Tzu-Hao; Wang, Liang-Kai; Tsai, Jeng-Daw; Liu, Yu-Peng; Chern, Schu-Rern; Wu, Peih-Shan; Su, Jun-Wei; Chen, Yu-Ting; Wang, Wayseen

2013-12-01

This study was aimed at detection of recurrent transmission of the 17q12 microdeletion in a fetus with congenital anomalies of the kidney and urinary tract. A 35-year-old woman was referred to the hospital at 20 weeks' gestation because of hydronephrosis in the fetus. The mother was normal and healthy. Her second child was a girl who had bilateral dysplastic kidneys that required hemodialysis, and died at the age of 5 years. During this pregnancy, the woman underwent amniocentesis at 18 weeks' gestation because of advanced maternal age. Cytogenetic analysis revealed a karyotype of 46,XY. Prenatal ultrasound showed left hydronephrosis with a tortuous ureter, right hydronephrosis, and increased echogenicity of the kidneys. Fetal magnetic resonance imaging showed right dilated renal calyces, left hydronephrosis, hydroureter, and multicystic kidney. The pregnancy was subsequently terminated. Array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization were applied for genetic analysis using umbilical cord, maternal blood, and cultured amniocytes. aCGH analysis on umbilical cord detected a 1.75-Mb deletion at 17q12 including haploinsufficiency of LHX1 and HNF1B. aCGH analysis on maternal blood detected a 1.54-Mb deletion at 17q12 including haploinsufficiency of LHX1 and HNF1B. Metaphase fluorescence in situ hybridization analysis on cultured amniocytes and maternal blood lymphocytes using 17q12-specific bacterial artificial chromosome probe showed 17q12 microdeletion in the fetus and the mother. Prenatal diagnosis of recurrent renal and urinary tract abnormalities in the fetus should include a differential diagnosis of familial 17q12 microdeletion. Copyright © 2013. Published by Elsevier B.V.

“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: mlototskyy@uwc.ac.za [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)

2015-10-05

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.

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

International Nuclear Information System (INIS)

Lototskyy, M.; Tolj, I.; Davids, M.W.; Bujlo, P.; Smith, F.; Pollet, B.G.

2015-01-01

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 2 -type MH material (H 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 2 charge pressure; (ii) shorter charging times in the refuelling mode and smoother peaks of H 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

Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

Science.gov (United States)

Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

2006-03-01

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

Single-Labeled Oligonucleotides Showing Fluorescence Changes upon Hybridization with Target Nucleic Acids

Directory of Open Access Journals (Sweden)

Gil Tae Hwang

2018-01-01

Full Text Available Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection. Interestingly, DNA mutations can be detected using fluorescently labeled oligonucleotide probes with only one fluorophore. This review summarizes recent research on single-labeled oligonucleotide probes that exhibit fluorescence changes after encountering target nucleic acids, such as guanine-quenching probes, cyanine-containing probes, probes containing a fluorophore-labeled base, and microenvironment-sensitive probes.

Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L. gene expression oligonucleotide microarray.

Directory of Open Access Journals (Sweden)

Paula Fernandez

Full Text Available Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de. The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons. The resulting Sunflower Unigen Resource (SUR version 1.0 was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01 allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.

Correlating In Vitro Splice Switching Activity With Systemic In Vivo Delivery Using Novel ZEN-modified Oligonucleotides

Directory of Open Access Journals (Sweden)

Suzan M Hammond

2014-01-01

Full Text Available Splice switching oligonucleotides (SSOs induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, “ZEN™” to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2′-O-methyl (2′OMe oligonucleotide, increasing melting temperature and potency over unmodified 2′OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2′OMe phosphorothioate (PS oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2′OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized.

Cloth-based hybridization array system for expanded identification of the animal species origin of derived materials in feeds.

Science.gov (United States)

Murphy, Johanna; Armour, Jennifer; Blais, Burton W

2007-12-01

A cloth-based hybridization array system (CHAS) previously developed for the detection of animal species for which prohibited materials have been specified (cattle, sheep, goat, elk, and deer) has been expanded to include the detection of animal species for which there are no prohibitions (pig and horse) in Canadian and American animal feeds. Animal species were identified by amplification of mitochondrial DNA sequences by PCR and subsequent hybridization of the amplicons with an array of species-specific oligonucleotide capture probes immobilized on a polyester cloth support, followed by an immunoenzymatic assay of the bound PCR products. The CHAS permitted sensitive and specific detection of meat meals from different animal species blended in a grain-based feed and should provide a useful adjunct to microscopic examination for the identification of prohibited materials in animal feeds.

Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

Science.gov (United States)

Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

2016-01-01

Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

Characterization of peptide-oligonucleotide heteroconjugates by mass spectrometry.

OpenAIRE

Jensen, O N; Kulkarni, S; Aldrich, J V; Barofsky, D F

1996-01-01

Two peptide-oligothymidylic acids, prepared by joining an 11 residue synthetic peptide containing one internal carboxyl group (Asp side chain) to amino-linker-5'pdT6 and amino-linker-5'pdT10 oligonucleotides, were analyzed by matrix-assisted laser desorption/ionization (MALDI) on a linear time-of-flight mass spectrometer and by electrospray ionization (ESI) on a triple-quadrupole system. These synthetic compounds model peptide-nucleic acid heteroconjugates encountered in antisense research an...

Direct Mutagenesis of Thousands of Genomic Targets using Microarray-derived Oligonucleotides

DEFF Research Database (Denmark)

Bonde, Mads; Kosuri, Sriram; Genee, Hans Jasper

2015-01-01

Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by ...

Rapid Induction of Protective Immunity Against Biothreat Agents Using CPG-Based Oligonucleotides

National Research Council Canada - National Science Library

Klinman, Dennis

2003-01-01

This research project examines the ability of synthetic oligonucleotides (ODN) containing immunostimulatory "CpG motifs' to trigger the innate immune system, thereby improving the host's ability to survive infection by biowarfare agents...

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

DEFF Research Database (Denmark)

Straarup, Ellen Marie; Fisker, Niels; Hedtjärn, Maj

2010-01-01

-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non...... using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates....

Technetium-99m labeled antisense oligonucleotide-noninvasive tumor imaging in mice

International Nuclear Information System (INIS)

Qin, G.M.; Zhang, Y.X.; An, R.; Gao, Z.R.; Cao, W.; Cao, G.X.; Hnatowich, D.J.

2002-01-01

Single-stranded RNA and DNA oligonucleotides may be useful as radiopharmaceuticals for antisense and other in vivo applications if convenient methods for stably attaching radionuclides such as 99m Tc can be developed. The c-myc oncogene works in cooperation with other oncogenes in a variety of malignant tumors. The concentration of c-myc messenger RNA increases rapidly 30 to 50 fold during DNA synthesis, thus making it a suitable target for following the progression of malignancy by noninvasive imaging with radiolabeled antisense oligonucleotide probes. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. 2 Oligonucleotide Labeling: A fresh 50mg/ml solution of sodium tartrate was prepared in sterile 0.5 M ammonium The ability of the labeled DNA to hybridize to its complement was analyzed by Sep-Pak column chromatography before and after the addition of the complementary DNA. 3 Biodistribution and Tumor Imaging Studies: A colony of KM mice (15-20g) were inoculated with 1x10 6 Ehrlich carcinoma tumor cells in the right thigh, and the tumors were allowed to grow for 6-7 days to a size of 1.0-1.5 cm in diameter. Biodistribution studies were performed in 32 KM mice after 50 μCi per mouse of 99m Tc-labeled oncogene probes were injected intravenously. A total of 8 mice were injected intravenously in the tail vein with 1-2 mCi of 99m Tc-labeled sense or antisense probes, immobilized with ketamine hydrochloride and imaged periodically from 0.5hr to 24hr with a gamma camera. Results: Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. The highest accumulation of label was in the liver first, with the kidney and small bowel next. The injected activity localized in the lesion

Analytical Devices Based on Direct Synthesis of DNA on Paper.

Science.gov (United States)

Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

2016-01-05

This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

Novel approaches to study low-energy electron-induced damage to DNA oligonucleotides

International Nuclear Information System (INIS)

Rackwitz, Jenny; Bald, Ilko; Ranković, Miloš Lj; Milosavljević, Aleksandar R

2015-01-01

The novel approach of DNA origami structures as templates for precise quantification of various well- defined oligonucleotides provides the opportunity to determine the sensitivity of complex DNA sequences towards low-energy electrons. (paper)

Synthesis of modified oligonucleotides for repair and replication studies of single and double radio-induced DNA lesions

International Nuclear Information System (INIS)

Muller, E.

2002-01-01

Several oxidative processes induce the formation of DNA lesions. In order to evaluate the biological and structural significance of such damage, several DNA lesions were inserted into synthetic oligonucleotides at defined sites. The research work aimed at describing the preparation of oligonucleotides t hat contained DNA damage and the evaluation of the biological properties of the lesions. A first part described the incorporation of radiation-induced lesions, namely (5'S,6S)-5',6-cyclo-5,6-dihydro-2'-deoxyuridine and (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-desoxyuridine into oligonucleotides. The modified DNA fragments were characterised by several spectroscopic and biochemical analyses including ESI MS, MALDI-TOF MS, CLHP and enzymatic digestions. During in vitro DNA synthesis by Taq DNA polymerase and Klenow exo fragment, the pyrimidine cyclo-nucleosides were found to block the progression of the enzymes. Then, repair studies by ADN N glycosylases, operating in the base excision repair pathway, have shown that the anhydro-nucleoside lesions were not recognised nor excised by Fpg, endo III, endo VIII, yNtg1 yNtg2 and yOgg1. Interestingly, the Latococcus lactis Fpg protein recognises (formation of a non covalent complex) but do not excise the damage. The incorporation into oligonucleotides of the (5R*) and (5S*) diastereoisomers of 1-[2-deoxy-β-D-erythro-pentofuranosyl]-5-hydroxy-hydantoin, generated by several oxidative processes was then described. In vitro DNA replication assays using modified oligonucleotides matrix showed a lethal potential of the latter base damage. Repair studies by ADN N-glycosylases showed that the damage was substrate for Fpg, endo III, endo VIII, Ntg1, Ntg2 and Fpg-L1. The rates of excision as inferred from the determination of the Michaelis kinetics constants were found to be affected by the presence of the damage. MALDI-TOF MS was used in order to gain insights into mechanistic aspects of oligonucleotides cleavage by the

Automated synthesis of an {sup 18}F-labelled pyridine-based alkylating agent for high yield oligonucleotide conjugation

Energy Technology Data Exchange (ETDEWEB)

Guggenberg, Elisabeth von; Sader, Jayden A.; Wilson, John S.; Shahhosseini, Soraya; Koslowsky, Ingrid; Wuest, Frank [Edmonton PET Centre, Division of Oncologic Imaging, Department of Oncology, Cross Cancer Institute, 11560 University Ave, Edmonton, AB, T6G 1Z2 (Canada); Mercer, John R. [Edmonton PET Centre, Division of Oncologic Imaging, Department of Oncology, Cross Cancer Institute, 11560 University Ave, Edmonton, AB, T6G 1Z2 (Canada)], E-mail: johnmerc@cancerboard.ab.ca

2009-09-15

Alkylating agents have been shown to be very promising for the radiolabelling of oligonucleotides with fluorine-18. In this report we describe the fully automated synthesis of 2-bromo-N-[3-(2-[{sup 18}F]fluoropyridin-3-yloxy)propyl]acetamide ([{sup 18}F]FPyBrA) utilizing a modular synthesis unit. Reaction conditions for the coupling of this pyridine-based alkylating agent at the 5' end of a fully phosphorothioated random 20-mer DNA sequence were optimized to achieve very high radiochemical yields (>90%) and a maximum specific activity of 5-6 GBq/{mu}moL. The potential for rapid purification by solid phase extraction without need of chromatographic isolation of the radiolabelled oligonucleotide presents an overall benefit for the application of oligonucleotides in preclinical studies and potential clinical applications.

Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

DEFF Research Database (Denmark)

Astakhova, Irina; Kumar, Santhosh T.; Wengel, Jesper

2011-01-01

efficiency of the resulting perylene-2'-amino-alpha-L-LNA monomer (T*) into synthetic oligonucleotides was significantly improved by replacement of the typically used 1H-tetrazole activator with pyridine hydrochloride. Generally, oligonucleotides containing monomer T* showed high binding affinity towards...... incorporations of monomers T* was quenched (quantum yield Phi(F) = 0.21) relative to duplexes of this probe with complementary DNA and RNA (Phi(F) = 0.42 and 0.35, respectively). On the contrary, a strong fluorescence quenching upon target binding was demonstrated by two short oligonucleotides of analogues...... sequences containing monomers T* at 5'- and 3'-terminal positions. We explain the hybridization-induced light-up effect observed for double-labeled probe by a reduction of fluorescence quenching due to precise positioning of the fluorophores within the double-stranded complexes. Furthermore, we propose...

Spectroscopic (UV/VIS, Raman and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

Directory of Open Access Journals (Sweden)

Seyedeh Maryam Banihashemian

Full Text Available Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100, is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT. As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

DEFF Research Database (Denmark)

Bestas, Burcu; Moreno, Pedro M D; Blomberg, K Emelie M

2014-01-01

, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we...

Diamond cubic phase of monoolein and water as an amphiphilic matrix for electrophoresis of oligonucleotides.

Science.gov (United States)

Carlsson, Nils; Winge, Ann-Sofie; Engström, Sven; Akerman, Björn

2005-10-06

We used a cubic liquid crystal formed by the nonionic monoglyceride monoolein and water as a porous matrix for the electrophoresis of oligonucleotides. The diamond cubic phase is thermodynamically stable when in contact with a water-rich phase, which we exploit to run the electrophoresis in the useful submarine mode. Oligonucleotides are separated according to size and secondary structure by migration through the space-filling aqueous nanometer pores of the regular liquid crystal, but the comparatively slow migration means the cubic phase will not be a replacement for the conventional DNA gels. However, our demonstration that the cubic phase can be used in submarine electrophoresis opens up the possibility for a new matrix for electrophoresis of amphiphilic molecules. From this perspective, the results on the oligonucleotides show that water-soluble particles of nanometer size, typical for the hydrophilic parts of membrane-bound proteins, may be a useful separation motif. A charged contamination in the commercial sample of monoolein, most likely oleic acid that arises from its hydrolysis, restricts useful buffer conditions to a pH below 5.6.

Application of four dyes in gene expression analyses by microarrays

Directory of Open Access Journals (Sweden)

van Schooten Frederik J

2005-07-01

Full Text Available Abstract Background DNA microarrays are widely used in gene expression analyses. To increase throughput and minimize costs without reducing gene expression data obtained, we investigated whether four mRNA samples can be analyzed simultaneously by applying four different fluorescent dyes. Results Following tests for cross-talk of fluorescence signals, Alexa 488, Alexa 594, Cyanine 3 and Cyanine 5 were selected for hybridizations. For self-hybridizations, a single RNA sample was labelled with all dyes and hybridized on commercial cDNA arrays or on in-house spotted oligonucleotide arrays. Correlation coefficients for all combinations of dyes were above 0.9 on the cDNA array. On the oligonucleotide array they were above 0.8, except combinations with Alexa 488, which were approximately 0.5. Standard deviation of expression differences for replicate spots were similar on the cDNA array for all dye combinations, but on the oligonucleotide array combinations with Alexa 488 showed a higher variation. Conclusion In conclusion, the four dyes can be used simultaneously for gene expression experiments on the tested cDNA array, but only three dyes can be used on the tested oligonucleotide array. This was confirmed by hybridizations of control with test samples, as all combinations returned similar numbers of differentially expressed genes with comparable effects on gene expression.

DNA-Catalytically Active Gold Nanoparticle Conjugates-Based Colorimetric Multidimensional Sensor Array for Protein Discrimination.

Science.gov (United States)

Wei, Xiangcong; Chen, Zhengbo; Tan, Lulu; Lou, Tianhong; Zhao, Yan

2017-01-03

A series of single-strand oligonucleotides functionalized catalytically active gold nanoparticle (AuNPs) as nonspecific receptors have been designed to build a protein sensing array. We take advantage of the correlation between the catalytic activity and the exposed surface area of AuNPs, i.e., DNA-proteins interactions mask the surface area of AuNPs, leading to poor catalytic performance of AuNPs. As the number of DNA-bound proteins increases, the surfaces of AuNPs become more masked; thus, the time of 4- nitrophenol/NaBH 4 reaction for color change (yellow → colorless) of the solution increases. Taking advantage of three nonspecific SH-labeled DNA sequences (A15, C15, and T15) as array sensing elements and the color-change time (CCT) of the solution as signal readout, colorimetric response patterns can be obtained on the array and identified via linear discriminant analysis (LDA). Eleven proteins have been completely distinguished with 100% accuracy with the naked eye at the 30 nM level. Remarkably, two similar proteins (bovine serum albumin and human serum albumin), two different proteins (bovine serum albumin and concanavalin) at the same concentration, and the mixtures of the two proteins with different molar ratios have been discriminated with 100%. The practicability of this sensor array is further validated by high accuracy (100%) identification of 11 proteins in human serum samples.

Unsupervised Analysis of Array Comparative Genomic Hybridization Data from Early-Onset Colorectal Cancer Reveals Equivalence with Molecular Classification and Phenotypes

Directory of Open Access Journals (Sweden)

María Arriba

2017-01-01

Full Text Available AIM: To investigate whether chromosomal instability (CIN is associated with tumor phenotypes and/or with global genomic status based on MSI (microsatellite instability and CIMP (CpG island methylator phenotype in early-onset colorectal cancer (EOCRC. METHODS: Taking as a starting point our previous work in which tumors from 60 EOCRC cases (≤45 years at the time of diagnosis were analyzed by array comparative genomic hybridization (aCGH, in the present study we performed an unsupervised hierarchical clustering analysis of those aCGH data in order to unveil possible associations between the CIN profile and the clinical features of the tumors. In addition, we evaluated the MSI and the CIMP statuses of the samples with the aim of investigating a possible relationship between copy number alterations (CNAs and the MSI/CIMP condition in EOCRC. RESULTS: Based on the similarity of the CNAs detected, the unsupervised analysis stratified samples into two main clusters (A, B and four secondary clusters (A1, A2, B3, B4. The different subgroups showed a certain correspondence with the molecular classification of colorectal cancer (CRC, which enabled us to outline an algorithm to categorize tumors according to their CIMP status. Interestingly, each subcluster showed some distinctive clinicopathological features. But more interestingly, the CIN of each subcluster mainly affected particular chromosomes, allowing us to define chromosomal regions more specifically affected depending on the CIMP/MSI status of the samples. CONCLUSIONS: Our findings may provide a basis for a new form of classifying EOCRC according to the genomic status of the tumors.

Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis.

Science.gov (United States)

Harsch, A; Marzilli, L A; Bunt, R C; Stubbe, J; Vouros, P

2000-05-01

Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.

A facile one-step fluorescence method for the quantitation of low-content single base deamination impurity in synthetic oligonucleotides.

Science.gov (United States)

Su, Xiaoye; Liang, Ruiting; Stolee, Jessica A

2018-06-05

Oligonucleotides are being researched and developed as potential drug candidates for the treatment of a broad spectrum of diseases. The characterization of antisense oligonucleotide (ASO) impurities caused by base mutations (e.g. deamination) which are closely related to the target ASO is a significant analytical challenge. Herein, we describe a novel one-step method, utilizing a strategy that combines fluorescence-ON detection with competitive hybridization, to achieve single base mutation quantitation in extensively modified synthetic ASOs. Given that this method is highly specific and sensitive (LoQ = 4 nM), we envision that it will find utility for screening other impurities as well as sequencing modified oligonucleotides. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array.

Science.gov (United States)

Aydin, Muhsin; Carter-Conger, Jacqueline; Gao, Ning; Gilmore, David F; Ricke, Steven C; Ahn, Soohyoun

2018-04-01

Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

Effective intracellular delivery of oligonucleotides in order to make sense of antisense

NARCIS (Netherlands)

Shi, FX; Hoekstra, D

2004-01-01

For more than two decades, antisense oligonucleotides (ODNs) have been used to modulate gene expression for the purpose of applications in cell biology and for development of novel sophisticated medical therapeutics. Conceptually, the antisense approach represents an elegant strategy, involving the

Specificity tests of an oligonucleotide probe against food-outbreak salmonella for biosensor detection

Science.gov (United States)

Chen, I.-H.; Horikawa, S.; Xi, J.; Wikle, H. C.; Barbaree, J. M.; Chin, B. A.

2017-05-01

Phage based magneto-elastic (ME) biosensors have been shown to be able to rapidly detect Salmonella in various food systems to serve food pathogen monitoring purposes. In this ME biosensor platform, the free-standing strip-shaped magneto-elastic sensor is the transducer and the phage probe that recognizes Salmonella in food serves as the bio-recognition element. According to Sorokulova et al. at 2005, a developed oligonucleotide probe E2 was reported to have high specificity to Salmonella enterica Typhimurium. In the report, the specificity tests were focused in most of Enterobacterace groups outside of Salmonella family. Here, to understand the specificity of phage E2 to different Salmonella enterica serotypes within Salmonella Family, we further tested the specificity of the phage probe to thirty-two Salmonella serotypes that were present in the major foodborne outbreaks during the past ten years (according to Centers for Disease Control and Prevention). The tests were conducted through an Enzyme linked Immunosorbent Assay (ELISA) format. This assay can mimic probe immobilized conditions on the magnetoelastic biosensor platform and also enable to study the binding specificity of oligonucleotide probes toward different Salmonella while avoiding phage/ sensor lot variations. Test results confirmed that this oligonucleotide probe E2 was high specific to Salmonella Typhimurium cells but showed cross reactivity to Salmonella Tennessee and four other serotypes among the thirty-two tested Salmonella serotypes.

Ultrasensitive Detection of Ebola Virus Oligonucleotide Based on Upconversion Nanoprobe/Nanoporous Membrane System.

Science.gov (United States)

Tsang, Ming-Kiu; Ye, WeiWei; Wang, Guojing; Li, Jingming; Yang, Mo; Hao, Jianhua

2016-01-26

Ebola outbreaks are currently of great concern, and therefore, development of effective diagnosis methods is urgently needed. The key for lethal virus detection is high sensitivity, since early-stage detection of virus may increase the probability of survival. Here, we propose a luminescence scheme of assay consisting of BaGdF5:Yb/Er upconversion nanoparticles (UCNPs) conjugated with oligonucleotide probe and gold nanoparticles (AuNPs) linked with target Ebola virus oligonucleotide. As a proof of concept, a homogeneous assay was fabricated and tested, yielding a detection limit at picomolar level. The luminescence resonance energy transfer is ascribed to the spectral overlapping of upconversion luminescence and the absorption characteristics of AuNPs. Moreover, we anchored the UCNPs and AuNPs on a nanoporous alumina (NAAO) membrane to form a heterogeneous assay. Importantly, the detection limit was greatly improved, exhibiting a remarkable value at the femtomolar level. The enhancement is attributed to the increased light-matter interaction throughout the nanopore walls of the NAAO membrane. The specificity test suggested that the nanoprobes were specific to Ebola virus oligonucleotides. The strategy combining UCNPs, AuNPs, and NAAO membrane provides new insight into low-cost, rapid, and ultrasensitive detection of different diseases. Furthermore, we explored the feasibility of clinical application by using inactivated Ebola virus samples. The detection results showed great potential of our heterogeneous design for practical application.

Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

Science.gov (United States)

Shahmuradyan, Anna; Krull, Ulrich J

2016-03-15

Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

The use of oligonucleotide probes for meningococcal serotype characterization

Directory of Open Access Journals (Sweden)

SACCHI Claudio Tavares

1998-01-01

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.

Hetero-oligonucleotide Nanoscale Tiles Capable of Two-Dimensional Lattice Formation as Testbeds for a Rapid, Affordable Purification Methodology

Science.gov (United States)

2013-01-01

devices; however, DNA is not the only polymer that can take advantage of the specicity of the Watson – Crick base-pair to achieve these goals. Central to...14. ABSTRACT 16. SECURITY CLASSIFICATION OF: New nanoscale hetero-oligonucleotide tiles are assembled from DNA , RNA and morpholino oligos and...purified using size exclusion filtration. Homo-oligonucleotide tiles assembled from RP-cartridge processed DNA oligos are purified by nondenaturing gel

[Analysis of genetics mechanism for the phenotypic diversity in a patient carrying a rare ring chromosome 9].

Science.gov (United States)

Qin, Shengfang; Wang, Xueyan; Li, Yunxing; Wei, Ping; Chen, Chun; Zeng, Lan

2016-02-01

To explore the genetics mechanism for the phenotypic variability in a patient carrying a rare ring chromosome 9. The karyotype of the patient was analyzed with cytogenetics method. Presence of sex chromosome was confirmed with fluorescence in situ hybridization. The SRY gene was subjected to PCR amplification and direct sequencing. Potential deletion and duplication were detected with array-based comparative genomic hybridization (array-CGH). The karyotype of the patient has comprised 6 types of cell lines containing a ring chromosome 9. The SRY gene sequence was normal. By array-CGH, the patient has carried a hemizygous deletion at 9p24.3-p23 (174 201-9 721 761) encompassing 30 genes from Online Mendelian Inheritance in Man. The phenotypic variability of the 9p deletion syndrome in conjunct with ring chromosome 9 may be attributable to multiple factors including loss of chromosomal material, insufficient dosage of genes, instability of ring chromosome, and pattern of inheritance.

Thermoplastic polymers surfaces for Dip-Pen Nanolithography of oligonucleotides

International Nuclear Information System (INIS)

Suriano, Raffaella; Biella, Serena; Cesura, Federico; Levi, Marinella; Turri, Stefano

2013-01-01

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.

Classification of Dukes' B and C colorectal cancers using expression arrays

DEFF Research Database (Denmark)

Frederiksen, C.M.; Knudsen, Steen; Laurberg, S.

2003-01-01

Purpose. Colorectal cancer is one of the most common malignancies. Substaging of the cancer is of importance not only to prognosis but also to treatment. Classification of substages based on DNA microarray technology is currently the most promising approach. We therefore investigated if gene...... expression microarrays could be used to classify colorectal tumors. Methods. We used the Affymetrix oligonucleotide arrays to analyze the expression of more than 5,000 genes in samples from the sigmoid and upper rectum of the left colon. Five samples were from normal mucosa and five samples from each...... expression of one of the most common malignancies, colorectal cancer, now seems to be within reach. The data indicates that it is possible at least to classify Dukes' B and C colorectal tumors with microarrays....

Drop drying on surfaces determines chemical reactivity - the specific case of immobilization of oligonucleotides on microarrays

Science.gov (United States)

2013-01-01

Background Drop drying is a key factor in a wide range of technical applications, including spotted microarrays. The applied nL liquid volume provides specific reaction conditions for the immobilization of probe molecules to a chemically modified surface. Results We investigated the influence of nL and μL liquid drop volumes on the process of probe immobilization and compare the results obtained to the situation in liquid solution. In our data, we observe a strong relationship between drop drying effects on immobilization and surface chemistry. In this work, we present results on the immobilization of dye labeled 20mer oligonucleotides with and without an activating 5′-aminoheptyl linker onto a 2D epoxysilane and a 3D NHS activated hydrogel surface. Conclusions Our experiments identified two basic processes determining immobilization. First, the rate of drop drying that depends on the drop volume and the ambient relative humidity. Oligonucleotides in a dried spot react unspecifically with the surface and long reaction times are needed. 3D hydrogel surfaces allow for immobilization in a liquid environment under diffusive conditions. Here, oligonucleotide immobilization is much faster and a specific reaction with the reactive linker group is observed. Second, the effect of increasing probe concentration as a result of drop drying. On a 3D hydrogel, the increasing concentration of probe molecules in nL spotting volumes accelerates immobilization dramatically. In case of μL volumes, immobilization depends on whether the drop is allowed to dry completely. At non-drying conditions, very limited immobilization is observed due to the low oligonucleotide concentration used in microarray spotting solutions. The results of our study provide a general guideline for microarray assay development. They allow for the initial definition and further optimization of reaction conditions for the immobilization of oligonucleotides and other probe molecule classes to different

Encephalopathy and bilateral cataract in a boy with an interstitial deletion of Xp22 comprising the CDKL5 and NHS genes.

Science.gov (United States)

Van Esch, Hilde; Jansen, Anna; Bauters, Marijke; Froyen, Guy; Fryns, Jean-Pierre

2007-02-15

We describe a male patient with a deletion at Xp22, detected by high resolution X-array CGH. The clinical phenotype present in this infant boy, consists of severe encephalopathy, congenital cataracts and tetralogy of Fallot and can be attributed to the deletion of the genes within the interval. Among these deleted genes are the gene for Nance-Horan syndrome and the cyclin-dependent kinase-like 5 gene (CDKL5), responsible for the early seizure variant of Rett syndrome. This is the first description of a male patient with a deletion of these genes, showing the involvement of CDKL5 in severe epileptic encephalopathy in males. Moreover it illustrates the added value of high resolution array-CGH in molecular diagnosis of mental retardation-multiple congenital anomaly cases. (c) 2007 Wiley-Liss, Inc.

Differentiation of the seven major lyssavirus species by oligonucleotide microarray.

Science.gov (United States)

Xi, Jin; Guo, Huancheng; Feng, Ye; Xu, Yunbin; Shao, Mingfu; Su, Nan; Wan, Jiayu; Li, Jiping; Tu, Changchun

2012-03-01

An oligonucleotide microarray, LyssaChip, has been developed and verified as a highly specific diagnostic tool for differentiation of the 7 major lyssavirus species. As with conventional typing microarray methods, the LyssaChip relies on sequence differences in the 371-nucleotide region coding for the nucleoprotein. This region was amplified using nested reverse transcription-PCR primers that bind to the 7 major lyssaviruses. The LyssaChip includes 57 pairs of species typing and corresponding control oligonucleotide probes (oligoprobes) immobilized on glass slides, and it can analyze 12 samples on a single slide within 8 h. Analysis of 111 clinical brain specimens (65 from animals with suspected rabies submitted to the laboratory and 46 of butchered dog brain tissues collected from restaurants) showed that the chip method was 100% sensitive and highly consistent with the "gold standard," a fluorescent antibody test (FAT). The chip method could detect rabies virus in highly decayed brain tissues, whereas the FAT did not, and therefore the chip test may be more applicable to highly decayed brain tissues than the FAT. LyssaChip may provide a convenient and inexpensive alternative for diagnosis and differentiation of rabies and rabies-related diseases.

Array-CGH detection of three cryptic submicroscopic imbalances in ...

Indian Academy of Sciences (India)

2The Second Clinical Medical College (Shenzhen People's Hospital), Jinan University, Guangdong Province. 518020, People's Republic of China ... Here, we describe a phenotypically normal prenatal case who was found to carry an apparently balanced .... Precise definitions of CCRs and their real complexity can only be ...

PCR amplification on microarrays of gel immobilized oligonucleotides

Science.gov (United States)

Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

2003-11-04

The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

DEFF Research Database (Denmark)

Chen, Fuqiang; Pruett-Miller, Shondra M; Huang, Yuping

2011-01-01

Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point...

Discovery of novel variants in genotyping arrays improves genotype retention and reduces ascertainment bias

Directory of Open Access Journals (Sweden)

Didion John P

2012-01-01

Full Text Available Abstract Background High-density genotyping arrays that measure hybridization of genomic DNA fragments to allele-specific oligonucleotide probes are widely used to genotype single nucleotide polymorphisms (SNPs in genetic studies, including human genome-wide association studies. Hybridization intensities are converted to genotype calls by clustering algorithms that assign each sample to a genotype class at each SNP. Data for SNP probes that do not conform to the expected pattern of clustering are often discarded, contributing to ascertainment bias and resulting in lost information - as much as 50% in a recent genome-wide association study in dogs. Results We identified atypical patterns of hybridization intensities that were highly reproducible and demonstrated that these patterns represent genetic variants that were not accounted for in the design of the array platform. We characterized variable intensity oligonucleotide (VINO probes that display such patterns and are found in all hybridization-based genotyping platforms, including those developed for human, dog, cattle, and mouse. When recognized and properly interpreted, VINOs recovered a substantial fraction of discarded probes and counteracted SNP ascertainment bias. We developed software (MouseDivGeno that identifies VINOs and improves the accuracy of genotype calling. MouseDivGeno produced highly concordant genotype calls when compared with other methods but it uniquely identified more than 786000 VINOs in 351 mouse samples. We used whole-genome sequence from 14 mouse strains to confirm the presence of novel variants explaining 28000 VINOs in those strains. We also identified VINOs in human HapMap 3 samples, many of which were specific to an African population. Incorporating VINOs in phylogenetic analyses substantially improved the accuracy of a Mus species tree and local haplotype assignment in laboratory mouse strains. Conclusion The problems of ascertainment bias and missing

Combined M-FISH and CGH analysis allows comprehensive description of genetic alterations in neuroblastoma cell lines.

Science.gov (United States)

Van Roy, N; Van Limbergen, H; Vandesompele, J; Van Gele, M; Poppe, B; Salwen, H; Laureys, G; Manoel, N; De Paepe, A; Speleman, F

2001-10-01

Cancer cell lines are essential gene discovery tools and have often served as models in genetic and functional studies of particular tumor types. One of the future challenges is comparison and interpretation of gene expression data with the available knowledge on the genomic abnormalities in these cell lines. In this context, accurate description of these genomic abnormalities is required. Here, we show that a combination of M-FISH with banding analysis, standard FISH, and CGH allowed a detailed description of the genetic alterations in 16 neuroblastoma cell lines. In total, 14 cryptic chromosome rearrangements were detected, including a balanced t(2;4)(p24.3;q34.3) translocation in cell line NBL-S, with the 2p24 breakpoint located at about 40 kb from MYCN. The chromosomal origin of 22 marker chromosomes and 41 cytogenetically undefined translocated segments was determined. Chromosome arm 2 short arm translocations were observed in six cell lines (38%) with and five (31%) without MYCN amplification, leading to partial chromosome arm 2p gain in all but one cell line and loss of material in the various partner chromosomes, including 1p and 11q. These 2p gains were often masked in the GGH profiles due to MYCN amplification. The commonly overrepresented region was chromosome segment 2pter-2p22, which contains the MYCN gene, and five out of eleven 2p breakpoints clustered to the interface of chromosome bands 2p16 and 2p21. In neuroblastoma cell line SJNB-12, with double minutes (dmins) but no MYCN amplification, the dmins were shown to be derived from 16q22-q23 sequences. The ATBF1 gene, an AT-binding transcription factor involved in normal neurogenesis and located at 16q22.2, was shown to be present in the amplicon. This is the first report describing the possible implication of ATBF1 in neuroblastoma cells. We conclude that a combined approach of M-FISH, cytogenetics, and CGH allowed a more complete and accurate description of the genetic alterations occurring in the

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: serena.biella@polimi.it [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)

2013-05-15

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.

Pure partial monosomy 3p (3p25.3 → pter: Prenatal diagnosis and array comparative genomic hybridization characterization

Directory of Open Access Journals (Sweden)

Chih-Ping Chen

2012-09-01

Conclusion: In this case, aCGH has characterized a 3p deleted region with haploinsufficiency of the neurodevelopmental genes associated with cognitive deficit and mental retardation but without involvement of the congenital heart disease susceptibility locus, and QF-PCR has determined a paternal origin of the deletion. aCGH and QF-PCR help to delineate the genomic imbalance in prenatally detected de novo chromosome aberration, and the information acquired is useful for genetic counseling.

Oral tongue cancer gene expression profiling: Identification of novel potential prognosticators by oligonucleotide microarray analysis

International Nuclear Information System (INIS)

Estilo, Cherry L; Boyle, Jay O; Kraus, Dennis H; Patel, Snehal; Shaha, Ashok R; Wong, Richard J; Huryn, Joseph M; Shah, Jatin P; Singh, Bhuvanesh; O-charoenrat, Pornchai; Talbot, Simon; Socci, Nicholas D; Carlson, Diane L; Ghossein, Ronald; Williams, Tijaana; Yonekawa, Yoshihiro; Ramanathan, Yegnanarayana

2009-01-01

The present study is aimed at identifying potential candidate genes as prognostic markers in human oral tongue squamous cell carcinoma (SCC) by large scale gene expression profiling. The gene expression profile of patients (n=37) with oral tongue SCC were analyzed using Affymetrix HG-U95Av2 high-density oligonucleotide arrays. Patients (n=20) from which there were available tumor and matched normal mucosa were grouped into stage (early vs. late) and nodal disease (node positive vs. node negative) subgroups and genes differentially expressed in tumor vs. normal and between the subgroups were identified. Three genes, GLUT3, HSAL2, and PACE4, were selected for their potential biological significance in a larger cohort of 49 patients via quantitative real-time RT-PCR. Hierarchical clustering analyses failed to show significant segregation of patients. In patients (n=20) with available tumor and matched normal mucosa, 77 genes were found to be differentially expressed (P< 0.05) in the tongue tumor samples compared to their matched normal controls. Among the 45 over-expressed genes, MMP-1 encoding interstitial collagenase showed the highest level of increase (average: 34.18 folds). Using the criterion of two-fold or greater as overexpression, 30.6%, 24.5% and 26.5% of patients showed high levels of GLUT3, HSAL2 and PACE4, respectively. Univariate analyses demonstrated that GLUT3 over-expression correlated with depth of invasion (P<0.0001), tumor size (P=0.024), pathological stage (P=0.009) and recurrence (P=0.038). HSAL2 was positively associated with depth of invasion (P=0.015) and advanced T stage (P=0.047). In survival studies, only GLUT3 showed a prognostic value with disease-free (P=0.049), relapse-free (P=0.002) and overall survival (P=0.003). PACE4 mRNA expression failed to show correlation with any of the relevant parameters. The characterization of genes identified to be significant predictors of prognosis by oligonucleotide microarray and further validation by

Nucleoside-O-Methyl-(H)-Phosphinates: Novel Monomers for the Synthesis of Methylphosphonate Oligonucleotides Using H-Phosphonate Chemistry.

Science.gov (United States)

Kostov, Ondřej; Páv, Ondřej; Rosenberg, Ivan

2017-09-18

This unit comprises the straightforward synthesis of protected 2'-deoxyribonucleoside-O-methyl-(H)-phosphinates in both 3'- and 5'-series. These compounds represent a new class of monomers compatible with the solid-phase synthesis of oligonucleotides using H-phosphonate chemistry and are suitable for the preparation of both 3'- and 5'-O-methylphosphonate oligonucleotides. The synthesis of 4-toluenesulfonyloxymethyl-(H)-phosphinic acid as a new reagent for the preparation of O-methyl-(H)-phosphinic acid derivatives is described. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A new achiral reagent for the incorporation of multiple amino groups into oligonucleotides

DEFF Research Database (Denmark)

Behrens, Carsten; Petersen, Kenneth H.; Egholm, Michael

1995-01-01

The synthesis of a new functionalized achiral linker reagent (10) for the incorporation of multiple primary amino groups into oligonucleotides is described. The linker reagent is compatible with conventional DNA-synthesis following the phosphoramidite methodology, and the linker can be incorporated...

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

NARCIS (Netherlands)

Windpassinger, Christian; Piard, Juliette; Bonnard, Carine; Alfadhel, Majid; Lim, Shuhui; Bisteau, Xavier; Blouin, Stéphane; Ali, Nur'Ain B; Ng, Alvin Yu Jin; Lu, Hao; Tohari, Sumanty; Talib, S Zakiah A; van Hul, Noémi; Caldez, Matias J; Van Maldergem, Lionel; Yigit, Gökhan; Kayserili, Hülya; Youssef, Sameh A; Coppola, Vincenzo; de Bruin, Alain; Tessarollo, Lino; Choi, Hyungwon; Rupp, Verena; Roetzer, Katharina; Roschger, Paul; Klaushofer, Klaus; Altmüller, Janine; Roy, Sudipto; Venkatesh, Byrappa; Ganger, Rudolf; Grill, Franz; Ben Chehida, Farid; Wollnik, Bernd; Altunoglu, Umut; Al Kaissi, Ali; Reversade, Bruno; Kaldis, Philipp

2017-01-01

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic

Preimplantation genetic diagnosis

Directory of Open Access Journals (Sweden)

Karin Writzl

2013-02-01

Conclusions: Over the last two decades, PGD has been shown to be a reliable and safe genetic test for couples who are at risk of a specific inher - ited disorder. For PGS, the results from several ongoing randomized controlled trials performed at different cell biopsy stage, using array-CGH and SNP array will provide the data needed to evaluate the clinical efficacy.

Chemically modified oligonucleotides with efficient RNase H response

DEFF Research Database (Denmark)

Vester, Birte; Boel, Anne Marie; Lobedanz, Sune

2008-01-01

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....

Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

CSIR Research Space (South Africa)

Becker, JVW

2010-01-01

Full Text Available ] for hybridization to an Operon oligonucleotide array, interrogating 7797 70-mer oligonucleotides which repre- sent 4585 unique genes. RNA was extracted from several untreated, unsynchronized P. falciparum 3D7 cultures and cDNA synthesized for construction of a... background- subtracted intensity of a spot exceeded the average back- ground intensity of all spots in that channel [31]. cDNA synthesis and array hybridization A reference design was employed for array hybridisation, utilising the URR pool described...

Dose-Dependent Lowering of Mutant Huntingtin Using Antisense Oligonucleotides in Huntington Disease Patients.

Science.gov (United States)

van Roon-Mom, Willeke M C; Roos, Raymund A C; de Bot, Susanne T

2018-04-01

On December 11 of 2017, Ionis Pharmaceuticals published a press release announcing dose-dependent reductions of mutant huntingtin protein in their HTTRx Phase 1/2a study in Huntington disease (HD) patients. The results from this Ionis trial have gained much attention from the patient community and the oligonucleotide therapeutics field, since it is the first trial targeting the cause of HD, namely the mutant huntingtin protein, using antisense oligonucleotides (ASOs). The press release also states that the primary endpoints of the study (safety and tolerability) were met, but does not contain data. This news follows the approval of another therapeutic ASO nusinersen (trade name Spinraza) for a neurological disease, spinal muscular atrophy, by the U.S. Food and Drug Administration and European Medicines Agency, in 2016 and 2017, respectively. Combined, this offers hope for the development of the HTTRx therapy for HD patients.

CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression

DEFF Research Database (Denmark)

Zaghloul, Eman M; Gissberg, Olof; Moreno, Pedro M D

2017-01-01

Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion....... Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT...

CARAT: A novel method for allelic detection of DNA copy number changes using high density oligonucleotide arrays

Directory of Open Access Journals (Sweden)

Ishikawa Shumpei

2006-02-01

Full Text Available Abstract Background DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell. Results We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100, 000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods. Conclusion Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.

Partial trisomy 8 mosaicism not detected by cultured amniotic-fluid cells

Directory of Open Access Journals (Sweden)

Meng-Che Tsai

2014-12-01

Conclusion: Conventional karyotyping through amniocentesis has limitations particularly in detecting rare trisomy mosaicism if trisomic cells show growth disadvantage. Array-CGH using uncultured cells may be of help in providing more information on genetic dosage variations in such cases.

Fulltext PDF

Indian Academy of Sciences (India)

healthy children and three early childhood deaths (two sons and a daughter) with an ... Array CGH in proband's parents did not show any deletion in this region. .... omphalocele, hypospadia and high blood pressure associated to a deletion at ...

Synthetic Nucleic Acid Analogues in Gene Therapy: An Update for Peptide–Oligonucleotide Conjugates

DEFF Research Database (Denmark)

Taskova, Maria; Mantsiou, Anna; Astakhova, Kira

2017-01-01

The main objective of this work is to provide an update on synthetic nucleic acid analogues and nanoassemblies as tools in gene therapy. In particular, the synthesis and properties of peptide–oligonucleotide conjugates (POCs), which have high potential in research and as therapeutics, are described...

Directing polyallylamine adsorption on microlens array patterned silicon for microarray fabrication.

Science.gov (United States)

Saini, Gaurav; Gates, Richard; Asplund, Matthew C; Blair, Steve; Attavar, Sachin; Linford, Matthew R

2009-06-21

The selective adsorption of reagents is often essential for bioarray and lab-on-a-chip type devices. As the starting point for a bioarray, alkyl monolayer terminated silicon shards were photopatterned in a few nanoseconds with thousands of wells (spots) using an optical element, a microlens array. Polyallylamine (PAAm), a primary amine containing polymer, adsorbed with little selectivity to the spots, i.e., silicon oxide, over the hydrophobic background. However, at appropriate concentrations, addition of a cationic surfactant to the PAAm deposition solution, cetyltrimethylammonium chloride, prevented the nonspecific adsorption of PAAm onto the hydrophobic monolayer, while directing it effectively to the active spots on the device. A nonionic surfactant was less effective in preventing the nonspecific adsorption of PAAm onto the hydrophobic monolayer. The localized reactions/interactions of adsorbed PAAm with four species that are useful for bioconjugate chemistry: glutaric anhydride, phenylenediisothiocyanate, biotin NHS ester, and an oligonucleotide (DNA) were shown in the spots of an array. The reactivity of PAAm was further demonstrated with an isocyanate. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) played an important role in confirming selective surface reactivity and adsorption. X-ray photoelectron spectroscopy (XPS), spectroscopic ellipsometry, and wetting confirmed PAAm reactivity on planar substrates.

Molecular imaging of atherosclerotic plaques with technetium-99m-labelled antisense oligonucleotides

International Nuclear Information System (INIS)

Qin Guangming; Zhang Yongxue; Cao Wei; An Rui; Gao Zairong; Xu Wendai; Zhang Kaijun; Li Guiling; Li Shuren

2005-01-01

The purpose of this study was to visualise experimental atherosclerotic lesions using radiolabelled antisense oligonucleotides (ASONs). Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for approximately 60 days. In vivo and ex vivo imaging was performed in atherosclerotic rabbits and normal control rabbits after i.v. injection of 92.5±18.5 MBq 99m Tc-labelled ASON or 99m Tc-labelled sense oligonucleotides. Immediately after the in vivo imaging, the animals were sacrificed and ex vivo imaging of the aortic specimens was performed. Biodistribution of radiolabelled c-mycASON was evaluated in vivo in atherosclerotic rabbits. Planar imaging revealed accumulation of 99m Tc-labelled c-mycASON in atherosclerotic lesions along the artery wall. Ex vivo imaging further demonstrated that the area of activity accumulation matched the area of atherosclerotic lesions. In contrast, no atherosclerotic lesions were found in the vessel wall and no positive imaging results were obtained in animals of the control group. This molecular imaging approach has potential for non-invasive imaging of atherosclerotic plaques at an early stage. (orig.)

Integrative Genomics Reveals Mechanisms of Copy Number Alterations Responsible for Transcriptional Deregulation in Colorectal Cancer

Science.gov (United States)

Camps, Jordi; Nguyen, Quang Tri; Padilla-Nash, Hesed M.; Knutsen, Turid; McNeil, Nicole E.; Wangsa, Danny; Hummon, Amanda B.; Grade, Marian; Ried, Thomas; Difilippantonio, Michael J.

2016-01-01

To evaluate the mechanisms and consequences of chromosomal aberrations in colorectal cancer (CRC), we used a combination of spectral karyotyping, array comparative genomic hybridization (aCGH), and array-based global gene expression profiling on 31 primary carcinomas and 15 established cell lines. Importantly, aCGH showed that the genomic profiles of primary tumors are recapitulated in the cell lines. We revealed a preponderance of chromosome breakpoints at sites of copy number variants (CNVs) in the CRC cell lines, a novel mechanism of DNA breakage in cancer. The integration of gene expression and aCGH led to the identification of 157 genes localized within high-level copy number changes whose transcriptional deregulation was significantly affected across all of the samples, thereby suggesting that these genes play a functional role in CRC. Genomic amplification at 8q24 was the most recurrent event and led to the overexpression of MYC and FAM84B. Copy number dependent gene expression resulted in deregulation of known cancer genes such as APC, FGFR2, and ERBB2. The identification of only 36 genes whose localization near a breakpoint could account for their observed deregulated expression demonstrates that the major mechanism for transcriptional deregulation in CRC is genomic copy number changes resulting from chromosomal aberrations. PMID:19691111

Comparative genomic hybridization.

Science.gov (United States)

Pinkel, Daniel; Albertson, Donna G

2005-01-01

Altering DNA copy number is one of the many ways that gene expression and function may be modified. Some variations are found among normal individuals ( 14, 35, 103 ), others occur in the course of normal processes in some species ( 33 ), and still others participate in causing various disease states. For example, many defects in human development are due to gains and losses of chromosomes and chromosomal segments that occur prior to or shortly after fertilization, whereas DNA dosage alterations that occur in somatic cells are frequent contributors to cancer. Detecting these aberrations, and interpreting them within the context of broader knowledge, facilitates identification of critical genes and pathways involved in biological processes and diseases, and provides clinically relevant information. Over the past several years array comparative genomic hybridization (array CGH) has demonstrated its value for analyzing DNA copy number variations. In this review we discuss the state of the art of array CGH and its applications in medical genetics and cancer, emphasizing general concepts rather than specific results.

A novel setup for the determination of absolute cross sections for low-energy electron induced strand breaks in oligonucleotides - The effect of the radiosensitizer 5-fluorouracil

International Nuclear Information System (INIS)

Rackwitz, J.; Rankovic, M.L.; Milosavljevic, A.R.; Bald, I.

2017-01-01

Low-energy electrons (LEEs) play an important role in DNA radiation damage. Here we present a method to quantify LEE induced strand breakage in well-defined oligonucleotide single strands in terms of absolute cross sections. An LEE irradiation setup covering electron energies <500 eV is constructed and optimized to irradiate DNA origami triangles carrying well-defined oligonucleotide target strands. Measurements are presented for 10.0 and 5.5 eV for different oligonucleotide targets. The determination of absolute strand break cross sections is performed by atomic force microscopy analysis. An accurate fluence determination ensures small margins of error of the determined absolute single strand break cross sections σ SSB . In this way, the influence of sequence modification with the radiosensitive 5-Fluorouracil ( 5F U) is studied using an absolute and relative data analysis. We demonstrate an increase in the strand break yields of 5F U containing oligonucleotides by a factor of 1.5 to 1.6 compared with non-modified oligonucleotide sequences when irradiated with 10 eV electrons. (authors)

Optical properties and electronic transitions of DNA oligonucleotides as a function of composition and stacking sequence.

Science.gov (United States)

Schimelman, Jacob B; Dryden, Daniel M; Poudel, Lokendra; Krawiec, Katherine E; Ma, Yingfang; Podgornik, Rudolf; Parsegian, V Adrian; Denoyer, Linda K; Ching, Wai-Yim; Steinmetz, Nicole F; French, Roger H

2015-02-14

The role of base pair composition and stacking sequence in the optical properties and electronic transitions of DNA is of fundamental interest. We present and compare the optical properties of DNA oligonucleotides (AT)10, (AT)5(GC)5, and (AT-GC)5 using both ab initio methods and UV-vis molar absorbance measurements. Our data indicate a strong dependence of both the position and intensity of UV absorbance features on oligonucleotide composition and stacking sequence. The partial densities of states for each oligonucleotide indicate that the valence band edge arises from a feature associated with the PO4(3-) complex anion, and the conduction band edge arises from anti-bonding states in DNA base pairs. The results show a strong correspondence between the ab initio and experimentally determined optical properties. These results highlight the benefit of full spectral analysis of DNA, as opposed to reductive methods that consider only the 260 nm absorbance (A260) or simple purity ratios, such as A260/A230 or A260/A280, and suggest that the slope of the absorption edge onset may provide a useful metric for the degree of base pair stacking in DNA. These insights may prove useful for applications in biology, bioelectronics, and mesoscale self-assembly.

Chemosensitization of Human Renal Cell Cancer Using Antisense Oligonucleotides Targeting the Antiapoptotic Gene Clusterin

Directory of Open Access Journals (Sweden)

Tobias Zellweger

2001-01-01

Full Text Available BACKGROUND: Renal cell cancer (RCC is a chemoresistant disease with no active chemotherapeutic agent achieving objective response rates higher than 15%. Clusterin is a cell survival gene that increases in human renal tubular epithelial cells after various states of injury and disease. Downregulation of clusterin, using antisense oligonucleotides (ASO, has recently been shown to increase chemosensitivity in several prostate cancer models. The objectives in this study were to evaluate clusterin expression levels in human RCC and normal kidney tissue, and to test whether clusterin ASO could also enhance chemosensitivity in human RCC Caki-2 cells both in vitro and in vivo. METHODS: Immunohistochemical staining was used to characterize clusterin expression in 67 RCC and normal kidney tissues obtained from radical nephrectomy specimens. Northern blot analysis was used to assess changes in clusterin mRNA expression after ASO and paclitaxel treatment. The effects of combined clusterin ASO and paclitaxel treatment on Caki-2 cell growth was examined using an MTT assay. Athymic mice bearing Caki-2 tumors were treated with clusterin ASO alone, clusterin ASO plus paclitaxel, and mismatch control oligonucleotides plus paclitaxel, over a period of 28 days with measurement of tumor volumes once weekly over 8 weeks. RESULTS: Immunohistochemistry of normal and malignant kidney tissue sections of 67 patients demonstrated positive clusterin staining for almost all RCC (98% and an overexpression, compared to normal tissue, in a majority of RCC (69%. Clusterin ASO, but not mismatch control oligonucleotides, decreased clusterin mRNA expression in Caki-2 cells in a dosedependent and sequence-specific manner. Pretreatment of Caki-2 cells with clusterin ASO significantly enhanced chemosensitivity to paclitaxel in vitro. Characteristic apoptotic DNA laddering was observed after combined treatment with ASO plus paclitaxel, but not with either agent alone. In vivo

A new microduplication syndrome encompassing the region of the Miller-Dieker (17p13 deletion) syndrome

DEFF Research Database (Denmark)

Roos, L; Jønch, A E; Kjaergaard, S

2009-01-01

BACKGROUND: The use of array comparative genome hybridisation (CGH) analyses for investigation of children with mental retardation has led to the identification of a growing number of new microdeletion and microduplication syndromes, some of which have become clinically well characterised and som...

The Utility of Chromosomal Microarray Analysis in Developmental and Behavioral Pediatrics

Science.gov (United States)

Beaudet, Arthur L.

2013-01-01

Chromosomal microarray analysis (CMA) has emerged as a powerful new tool to identify genomic abnormalities associated with a wide range of developmental disabilities including congenital malformations, cognitive impairment, and behavioral abnormalities. CMA includes array comparative genomic hybridization (CGH) and single nucleotide polymorphism…

Left ventricular hypertrabeculation/noncompaction with epilepsy, other heart defects, minor facial anomalies and new copy number variants

Directory of Open Access Journals (Sweden)

Nagel Bert

2012-07-01

Full Text Available Abstract Background Left ventricular hypertrabeculation/noncompaction (LVHT is a cardiac abnormality of unknown etiology which has been described in children as well as in adults with and without chromosomal aberrations. LVHT has been reported in association with various cardiac and extracardiac abnormalities like epilepsy and facial dysmorphism. Case presentation A unique combination of LVHT, atrial septal defect, pulmonary valve stenosis, aortic stenosis, epilepsy and minor facial anomalies is presented in a 5.5 years old girl. Microarray-based genomic hybridization (array-CGH detected six previously not described copy number variants (CNVs inherited from a clinically unaffected father and minimally affected mother, thus, most likely, not clinically significant but rare benign variants. Conclusions Despite this complex phenotype de novo microdeletions or microduplications were not detected by array CGH. Further investigations, such as whole exome sequencing, could reveal point mutations and small indels as the possible cause.

Obtaining a genetic diagnosis in a child with disability: impact on parental quality of life.

Science.gov (United States)

Lingen, M; Albers, L; Borchers, M; Haass, S; Gärtner, J; Schröder, S; Goldbeck, L; von Kries, R; Brockmann, K; Zirn, B

2016-02-01

Recent progress in genetic testing has facilitated obtaining an etiologic diagnosis in children with developmental delay/intellectual disability (DD/ID) or multiple congenital anomalies (MCA) or both. Little is known about the benefits of diagnostic elucidation for affected families. We studied the impact of a genetic diagnosis on parental quality of life (QoL) using a validated semiquantitative questionnaire in families with a disabled child investigated by array-based comparative genomic hybridization (aCGH). We received completed questionnaires from 95 mothers and 76 fathers of 99 families. We used multivariate analysis for adjustment of potential confounders. Taken all 99 families together, maternal QoL score (percentile rank scale 51.05) was significantly lower than fathers' QoL (61.83, p = 0.01). Maternal QoL score was 20.17 [95% CI (5.49; 34.82)] percentile rank scales higher in mothers of children with diagnostic (n = 34) aCGH as opposed to mothers of children with inconclusive (n = 65) aCGH (Hedges' g = 0.71). Comparison of these QoL scores with retrospectively recalled QoL before aCGH revealed an increase of maternal QoL after diagnostic clarification. Our results indicate a benefit for maternal QoL if a genetic test, here aCGH, succeeds to clarify the etiologic diagnosis in a disabled child. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

Science.gov (United States)

Panpradist, Nuttada; Beck, Ingrid A.; Chung, Michael H.; Kiarie, James N.; Frenkel, Lisa M.; Lutz, Barry R.

2016-01-01

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories. PMID:26751207

Dissecting the hybridization of oligonucleotides to structured complementary sequences.

Science.gov (United States)

Peracchi, Alessio

2016-06-01

When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and >20 complementary oligonucleotides ('antisenses'). Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process. Copyright © 2016 Elsevier B.V. All rights reserved.

The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays.

Science.gov (United States)

Wang, Lih-Chiann; Kuo, Ya-Ting; Chueh, Ling-Ling; Huang, Dean; Lin, Jiunn-Horng

2017-05-01

Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n=33) and 41.1% (n=23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control. Copyright © 2017 Elsevier B.V. All rights reserved.

N-Branched acyclic nucleoside phosphonates as monomers for the synthesis of modified oligonucleotides

Czech Academy of Sciences Publication Activity Database

Hocková, Dana; Rosenbergová, Šárka; Ménová, Petra; Páv, Ondřej; Pohl, Radek; Novák, Pavel; Rosenberg, Ivan

2015-01-01

Roč. 13, č. 15 (2015), s. 4449-4458 ISSN 1477-0520 R&D Projects: GA ČR GAP207/11/0108; GA ČR GA13-26526S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * oligonucleotides * solid phase synthesis Subject RIV: CC - Organic Chemistry Impact factor: 3.559, year: 2015

Evaluation of fluorine-18-labeled alkylating agents as potential synthons for the labeling of oligonucleotides

NARCIS (Netherlands)

de Vries, EFJ; Vroegh, J; Elsinga, PH; Vaalburg, W

Six fluorine-18-labeled alkylating agents were selected as potentially suitable synthons for the labeling of antisense oligonucleotides. The selected synthons were evaluated in a model reaction with the monomer adenosine 5'-O-thiomonophosphate. Of these synthons,

Ultra high-resolution gene centric genomic structural analysis of a non-syndromic congenital heart defect, Tetralogy of Fallot.

Directory of Open Access Journals (Sweden)

Douglas C Bittel

Full Text Available Tetralogy of Fallot (TOF is one of the most common severe congenital heart malformations. Great progress has been made in identifying key genes that regulate heart development, yet approximately 70% of TOF cases are sporadic and nonsyndromic with no known genetic cause. We created an ultra high-resolution gene centric comparative genomic hybridization (gcCGH microarray based on 591 genes with a validated association with cardiovascular development or function. We used our gcCGH array to analyze the genomic structure of 34 infants with sporadic TOF without a deletion on chromosome 22q11.2 (n male = 20; n female = 14; age range of 2 to 10 months. Using our custom-made gcCGH microarray platform, we identified a total of 613 copy number variations (CNVs ranging in size from 78 base pairs to 19.5 Mb. We identified 16 subjects with 33 CNVs that contained 13 different genes which are known to be directly associated with heart development. Additionally, there were 79 genes from the broader list of genes that were partially or completely contained in a CNV. All 34 individuals examined had at least one CNV involving these 79 genes. Furthermore, we had available whole genome exon arrays from right ventricular tissue in 13 of our subjects. We analyzed these for correlations between copy number and gene expression level. Surprisingly, we could detect only one clear association between CNVs and expression (GSTT1 for any of the 591 focal genes on the gcCGH array. The expression levels of GSTT1 were correlated with copy number in all cases examined (r = 0.95, p = 0.001. We identified a large number of small CNVs in genes with varying associations with heart development. Our results illustrate the complexity of human genome structural variation and underscore the need for multifactorial assessment of potential genetic/genomic factors that contribute to congenital heart defects.

Combination of vascular endothelial growth factor antisense oligonucleotide therapy and radiotherapy increases the curative effects against maxillofacial VX2 tumors in rabbits

Energy Technology Data Exchange (ETDEWEB)

Zheng Linfeng, E-mail: zhenglinfeng04@yahoo.com.cn [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Li Yujie, E-mail: yujieli01@yahoo.com.cn [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Wang Han, E-mail: bingowh@hotmail.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Zhao Jinglong, E-mail: jinglongz@yahoo.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Wang Xifu, E-mail: wangxiechen001@163.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Hu Yunsheng, E-mail: springmorninghu@163.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Zhang Guixiang, E-mail: guixiangzhang@sina.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China)

2011-05-15

Purpose: To study the effects of combination of vascular endothelial growth factor (VEGF) antisense oligonucleotide therapy and radiotherapy on maxillofacial VX2 tumors in rabbits. Methods: We used 24 New Zealand white rabbits as a model to induce maxillofacial VX2 tumor. The rabbits were randomly divided into the following 4 groups: radiotherapy group (group A), treated with 16 Gy of radiotherapy; VEGF antisense oligonucleotide treatment group (group B), treated with an injection of 150 {mu}g of VEGF antisense oligonucleotide into the local tumor; VEGF antisense oligonucleotide combined with radiotherapy group (group C), treated with an injection of 150 {mu}g of VEGF antisense oligonucleotide into the local tumor immediately after 16 Gy of radiotherapy; and control group (group D), treated with an injection of 300 {mu}l 5% aqueous glucose solution into the local tumor. On days 3 and 14 after treatment, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed to calculate maximal enhancement ratio (MER), slope of enhancement (SLE), and tumor volume change. Rabbits were killed on day 14 to obtain samples for pathological examination and immunohistochemical staining for VEGF. Results: In group C, tumor volume was significantly reduced on day 14 after treatment, and the difference was statistically different as compared to that before treatment, on day 3 after treatment and other groups (P < 0.01). Values of both MER and SLE after treatment were significantly lower than the values before treatment (P < 0.05). Pathological specimen revealed tumor cell edema, bleeding, necrosis, vascular wall thickening and occlusion, and decreased VEGF expression. The immunohistochemical score (IHS) of group C was significantly different from groups A and D respectively (P < 0.05). Conclusion: Injecting the tumor with VEGF antisense oligonucleotide immediately after radiotherapy can enhance the curative effect on rabbit maxillofacial VX2 tumor, and DCE-MRI can serve

Oligonucleotides with 1,4-dioxane-based nucleotide monomers

DEFF Research Database (Denmark)

Madsen, Andreas S; Wengel, Jesper

2012-01-01

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...

A statistical method for predicting splice variants between two groups of samples using GeneChip® expression array data

Directory of Open Access Journals (Sweden)

Olson James M

2006-04-01

Full Text Available Abstract Background Alternative splicing of pre-messenger RNA results in RNA variants with combinations of selected exons. It is one of the essential biological functions and regulatory components in higher eukaryotic cells. Some of these variants are detectable with the Affymetrix GeneChip® that uses multiple oligonucleotide probes (i.e. probe set, since the target sequences for the multiple probes are adjacent within each gene. Hybridization intensity from a probe correlates with abundance of the corresponding transcript. Although the multiple-probe feature in the current GeneChip® was designed to assess expression values of individual genes, it also measures transcriptional abundance for a sub-region of a gene sequence. This additional capacity motivated us to develop a method to predict alternative splicing, taking advance of extensive repositories of GeneChip® gene expression array data. Results We developed a two-step approach to predict alternative splicing from GeneChip® data. First, we clustered the probes from a probe set into pseudo-exons based on similarity of probe intensities and physical adjacency. A pseudo-exon is defined as a sequence in the gene within which multiple probes have comparable probe intensity values. Second, for each pseudo-exon, we assessed the statistical significance of the difference in probe intensity between two groups of samples. Differentially expressed pseudo-exons are predicted to be alternatively spliced. We applied our method to empirical data generated from GeneChip® Hu6800 arrays, which include 7129 probe sets and twenty probes per probe set. The dataset consists of sixty-nine medulloblastoma (27 metastatic and 42 non-metastatic samples and four cerebellum samples as normal controls. We predicted that 577 genes would be alternatively spliced when we compared normal cerebellum samples to medulloblastomas, and predicted that thirteen genes would be alternatively spliced when we compared metastatic

Generalized pustular psoriasis in infant with heterozygous mutation in the IL36RN gene successfully treated with infliximab

DEFF Research Database (Denmark)

Glerup, Mia; Herlin, Troels; Veirum, Jens Erik

, but to our knowledge heterozygous IL36RN mutation related to severe generalized pustular psoriasis in early childhood has not been described. Case presentation: First child of non-consanguineous caucasian (Danish) parents prenatally diagnosed with tetralogy of Fallot. Array CGH revealed normal karyotype...

Analysis of genomic imbalances and gene expression changes in transformed follicular lymphoma (FL)

DEFF Research Database (Denmark)

Obel, G.; Farinha, P.; Lam, W.

2005-01-01

American patients with transformed FL. Methods: High-resolution BAC-array comparative genomic hybridisation (CGH) was used to detect genomic imbalances. Gene expression profiling was performed using cDNA microarrays (Affymetrix). Results: Of 9 biopsy pairs identified so far, analysis results of the first 4...

Genetic profiling differentiates second primary tumors from metastases in adult metachronous soft tissue sarcoma

DEFF Research Database (Denmark)

Fernebro, Josefin; Carneiro, Ana; Rydholm, Anders

2008-01-01

Purpose. Patients with soft tissue sarcomas (STS) are at increased risk of second primary malignancies, including a second STS, but distinction between metastases and a second primary STS is difficult. Patients and Methods. Array-based comparative genomic hybridization (aCGH) was applied to 30 mu...

Moving towards personalised therapy in head and neck squamous cell carcinoma through analysis of next generation sequencing data

DEFF Research Database (Denmark)

Giefing, M; Wierzbicka, M; Szyfter, K

2016-01-01

of the discovery and functional impact of recurrent genetic lesions that are likely to influence the management of this disease in the near future. This manuscript integrates genetic data from publicly available array comparative genome hybridization (aCGH) and next-generation sequencing genetics databases...

Ultrasensitive electrochemical biosensor based on the oligonucleotide self-assembled monolayer-mediated immunosensing interface

Energy Technology Data Exchange (ETDEWEB)

Liu, Dengyou; Luo, Qimei [Science College of Hunan Agricultural University, Changsha 410128 (China); Deng, Fawen [The Fourth Hospital of Chansha, Changsha 410006 (China); Li, Zhen [Science College of Hunan Agricultural University, Changsha 410128 (China); Li, Benxiang, E-mail: 172170960@qq.com [Science College of Hunan Agricultural University, Changsha 410128 (China); Shen, Zhifa, E-mail: shenzhifa@wmu.edu.cn [Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035 (China)

2017-06-08

Highly sensitive and selective quantitation of a variety of proteins over a wide concentration range is highly desirable for increased accuracy of biomarker detection or for multidisease diagnostics. In the present contribution, using human immunoglobulin G (HIgG) as the model target protein, an electrochemical ultrasensitive immunosensing platform was developed based on the oligonucleotide self-assembled monolayer-mediated (OSAM) sensing interface. For this immunosensor, the “signal-on” signaling mechanism and enzymatic signal amplification effect were integrated into one sensing architecture. Moreover, the thiolated flexible single-stranded DNAs immobilized onto gold electrode surface not only performs the wobbling motion to facilitate the electron transfer between the electrode surface and biosensing layer but also fundamentally prohibiting the direct interaction of proteins with gold substrate. Thus, the electrochemical signal could be efficiently enhanced and the unspecific adsorption or cross-reaction might be eliminated. As a result, utilizing the newly-proposed immunosensor, the HIgG can be detected down to 0.5 ng/mL, and the high detection specificity is offered. The successful design of OSAM and the highly desirable detection capability of new immunosensor are expected to provide a perspective for fabricating new robust immunosensing platform and for promising potential of oligonucleotide probe in biological research and biomedical diagnosis. - Highlights: • An electrochemical ultrasensitive immunosensing platform was developed based on the oligonucleotide self-assembled monolayer (OASM). • OASM severs as a flexible monolayer to promote electron transfer and prohibits the direct interaction of proteins with gold substrate. • The electrochemical signal is efficiently enhanced and the unspecific adsorption or cross-reaction is eliminated. • Target protein can be detected down to 0.5 ng/mL, and the high detection specificity can be obtained. �

Synthesis and Excellent Duplex Stability of Oligonucleotides Containing 2'-Amino-LNA Functionalized with Galactose Units

DEFF Research Database (Denmark)

Kumar, Rajesh; Ries, Annika; Wengel, Jesper

2017-01-01

A convenient method for the preparation of oligonucleotides containing internally-attached galactose and triantennary galactose units has been developed based on click chemistry between 2'-N-alkyne 2'-amino-LNA nucleosides and azido-functionalized galactosyl building blocks. The synthesized...

Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides

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Palta Priit

2010-04-01

Full Text Available Abstract Background The hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities. Results We demonstrate that adding specific DNA helper oligonucleotides (chaperones to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule. Conclusions The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.

Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

OpenAIRE

Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

1994-01-01

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 prelim...

Short Oligonucleotides Aligned in Stretched Humid Matrix: Secondary DNA Structure in Poly(vinyl alcohol) Environment

KAUST Repository

Hanczyc, Piotr; Å kerman, Bjö rn; Nordé n, Bengt

2012-01-01

) spectroscopy. Oligonucleotides of lengths varying between 10 (3.4 nm) and 60 bases (20.4 nm) were investigated with respect to structural properties in the gel-like polymer environment. The DNA conformation as a function of relative humidity reveals a strong

Colloidal silica films for high-capacity DNA arrays

Science.gov (United States)

Glazer, Marc Irving

The human genome project has greatly expanded the amount of genetic information available to researchers, but before this vast new source of data can be fully utilized, techniques for rapid, large-scale analysis of DNA and RNA must continue to develop. DNA arrays have emerged as a powerful new technology for analyzing genomic samples in a highly parallel format. The detection sensitivity of these arrays is dependent on the quantity and density of immobilized probe molecules. We have investigated substrates with a porous, "three-dimensional" surface layer as a means of increasing the surface area available for the synthesis of oligonucleotide probes, thereby increasing the number of available probes and the amount of detectable bound target. Porous colloidal silica films were created by two techniques. In the first approach, films were deposited by spin-coating silica colloid suspensions onto flat glass substrates, with the pores being formed by the natural voids between the solid particles (typically 23nm pores, 35% porosity). In the second approach, latex particles were co-deposited with the silica and then pyrolyzed, creating films with larger pores (36 nm), higher porosity (65%), and higher surface area. For 0.3 mum films, enhancements of eight to ten-fold and 12- to 14-fold were achieved with the pure silica films and the films "templated" with polymer latex, respectively. In gene expression assays for up to 7,000 genes using complex biological samples, the high-capacity films provided enhanced signals and performed equivalently or better than planar glass on all other functional measures, confirming that colloidal silica films are a promising platform for high-capacity DNA arrays. We have also investigated the kinetics of hybridization on planar glass and high-capacity substrates. Adsorption on planar arrays is similar to ideal Langmuir-type adsorption, although with an "overshoot" at high solution concentration. Hybridization on high-capacity films is

Clinical validation of integrated nucleic acid and protein detection on an electrochemical biosensor array for urinary tract infection diagnosis.

Directory of Open Access Journals (Sweden)

Ruchika Mohan

Full Text Available BACKGROUND: Urinary tract infection (UTI is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management. METHODOLOGY/PRINCIPAL FINDINGS: The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both. CONCLUSION/SIGNIFICANCE: We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for

Différentes structures des marqueurs chromosomiques

African Journals Online (AJOL)

abp

hétérochromatine et d'euchromatine des chromosomes accrocentriques. En 1992, la technique de la CGH puis sa dérivée la "CGHarray µ array comparative genomic Hybridization", techniques basées sur la comparaison entre l'ADN d'un patient avec ...

Nonparametric testing for DNA copy number induced differential mRNA gene expression

NARCIS (Netherlands)

van Wieringen, W.N.; van de Wiel, M.A.

2009-01-01

The central dogma of molecular biology relates DNA with mRNA. Array CGH measures DNA copy number and gene expression microarrays measure the amount of mRNA. Methods that integrate data from these two platforms may uncover meaningful biological relationships that further our understanding of cancer.

Butyrate induces profound changes in gene expression related to multiple signal pathways in bovine kidney epithelial cells

OpenAIRE

Li, Robert W; Li, CongJun

2006-01-01

Abstract Background Global gene expression profiles of bovine kidney epithelial cells regulated by sodium butyrate were investigated with high-density oligonucleotide microarrays. The bovine microarray with 86,191 distinct 60mer oligonucleotides, each with 4 replicates, was designed and produced with Maskless Array Synthesizer technology. These oligonucleotides represent approximately 45,383 unique cattle sequences. Results 450 genes significantly regulated by butyrate with a median False Dis...

Design and analysis of effects of triplet repeat oligonucleotides in cell models for myotonic dystrophy

NARCIS (Netherlands)

Gonzalez-Barriga, A.; Mulders, S.A.M.; Giessen, J. van der; Hooijer, J.D.; Bijl, S.; Kessel, I.D.G. van; Beers, J. van; Deutekom, J.C. van; Fransen, J.A.M.; Wieringa, B.; Wansink, D.G.

2013-01-01

Myotonic dystrophy type 1 (DM1) is caused by DM protein kinase (DMPK) transcripts containing an expanded (CUG)n repeat. Antisense oligonucleotide (AON)-mediated suppression of these mutant RNAs is considered a promising therapeutic strategy for this severe disorder. Earlier, we identified a

Synthesis of modified oligonucleotides that contain purine and pyrimidine radio-induced base lesions

International Nuclear Information System (INIS)

Romieu, Anthony

1999-01-01

Different factors as oxidizing or carcinogenesis agents, UV and ionizing radiations,.... can generate a wide, spectrum of DNA base damages. In order to study the biochemical and structural features of these DNA damages, it is important to prepare short DNA fragments (20 to 50 bases long) bearing a single or several modifications at specific sites in their sequences. The chemical synthesis is a powerful tool to prepare such modified DNA fragments. This work focusses on the chemical incorporation of several modified nucleosides formed in DNA by ionizing radiations or by photo-sensitization. The first part of this study describes the preparation of a phosphoramidite synthon of 5-hydroxy-2'-deoxyuridine and its subsequent incorporation into synthetic oligonucleotides ranging from 14 to 33 bases long. In a second part (chapters Ill and IV), the synthesis and incorporation of original radiation-induced tandem lesions: the carbon-bridged cyclo-nucleosides are presented. Both (5'R)- and (5'S) diastereomers of 5',8-cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyguanosine have been individually inserted into various different oligonucleotides (3 to 22 bases long) by using the standard phosphoramidite chemistry. The chemical incorporation of a pyrimidine analogue: (5'S, 6S)-5',6-cyclo-5,6-dihydro-thymidine has been also achieved. The loss of aromaticity of this modified nucleoside and its poor reactivity required the development of a synthetic strategy entirely different from that used for the preparation and subsequent incorporation of the phosphoramidite synthons of 5',8-cyclo-purine-2'-deoxyribo-nucleosides. The third part of this study deals with the synthesis of a phosphoramidite synthon of 4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine. The two (4R)- and (4S)- diastereomers of this oxidized purine have been separated and individually inserted in several synthetic DNA fragments. No epimerization of C-4 position was observed during the solid-phase synthesis and during the

Matrix-comparative genomic hybridization from multicenter formalin-fixed paraffin-embedded colorectal cancer tissue blocks

Directory of Open Access Journals (Sweden)

Köhne Claus-Henning

2007-04-01

Full Text Available Abstract Background The identification of genomic signatures of colorectal cancer for risk stratification requires the study of large series of cancer patients with an extensive clinical follow-up. Multicentric clinical studies represent an ideal source of well documented archived material for this type of analyses. Methods To verify if this material is technically suitable to perform matrix-CGH, we performed a pilot study using macrodissected 29 formalin-fixed, paraffin-embedded tissue samples collected within the framework of the EORTC-GI/PETACC-2 trial for colorectal cancer. The scientific aim was to identify prognostic genomic signatures differentiating locally restricted (UICC stages II-III from systemically advanced (UICC stage IV colorectal tumours. Results The majority of archived tissue samples collected in the different centers was suitable to perform matrix-CGH. 5/7 advanced tumours displayed 13q-gain and 18q-loss. In locally restricted tumours, only 6/12 tumours showed a gain on 13q and 7/12 tumours showed a loss on 18q. Interphase-FISH and high-resolution array-mapping of the gain on 13q confirmed the validity of the array-data and narrowed the chromosomal interval containing potential oncogenes. Conclusion Archival, paraffin-embedded tissue samples collected in multicentric clinical trials are suitable for matrix-CGH analyses and allow the identification of prognostic signatures and aberrations harbouring potential new oncogenes.

Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease

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Erik van der Wal

2017-06-01

Full Text Available The most common variant causing Pompe disease is c.-32-13T>G (IVS1 in the acid α-glucosidase (GAA gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult phenotype. We hypothesized that cis-acting splicing motifs may exist that could be blocked using antisense oligonucleotides (AONs to promote exon inclusion. To test this, a screen was performed in patient-derived primary fibroblasts using a tiling array of U7 small nuclear RNA (snRNA-based AONs. This resulted in the identification of a splicing regulatory element in GAA intron 1. We designed phosphorodiamidate morpholino oligomer-based AONs to this element, and these promoted exon 2 inclusion and enhanced GAA enzyme activity to levels above the disease threshold. These results indicate that the common IVS1 GAA splicing variant in Pompe disease is subject to negative regulation, and inhibition of a splicing regulatory element using AONs is able to restore canonical GAA splicing and endogenous GAA enzyme activity.

Genetical stability and osteogenic ability of mesenchimal stem cells on demineralized bone matrices

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A. Pozzuoli

2015-03-01

Full Text Available Aim Tissue engineering is a rapidly expanding field with regard to the use of biomaterials and stem cells in the orthopedic surgery. Many experimental studies have been done to understand the best characteristics of cells, materials and laboratory methods for safe clinical applications. The aim of this study was to compare the ability of 2 different human demineralized bone matrices (DBMs, the one enriched and the other not enriched with hyaluronic acid, to stimulate in vitro the proliferation and the osteogenic differentiation of human adipose-derived stem cells (ADSCs seeded onto an osteoconductive scaffold. Materials and Methods ADSCs were isolated, by enzymatic digestion, from abdominal adipose tissue of 5 patients undergoing cosmetic lipoaspiration surgery. ADSCs were then seeded onto a 3D scaffold in the presence of the two different osteoinductive matrices of human demineralized bone and evaluated for proliferation and osteogenic differentiation. The safety of the methods was verified using array-Comparative Genomic Hybridization (array-CGH. Results ADSCs were able to differentiate in osteogenic sense. Both DBMs showed the ability to induce osteogenic differentiation of the cells. Conclusion array-CGH showed no changes at genome level, thus confirming the safety of materials and methods.

Reliable Assessment and Quantification of the Fluorescence-Labeled Antisense Oligonucleotides In Vivo

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Maria Chiara Munisso

2014-01-01

Full Text Available The availability of fluorescent dyes and the advances in the optical systems for in vivo imaging have stimulated an increasing interest in developing new methodologies to study and quantify the biodistribution of labeled agents. However, despite these great achievements, we are facing significant challenges in determining if the observed fluorescence does correspond to the quantity of the dye in the tissues. In fact, although the far-red and near-infrared lights can propagate through several centimetres of tissue, they diffuse within a few millimetres as consequence of the elastic scattering of photons. In addition, when dye-labeled oligonucleotides form stable complex with cationic carriers, a large change in the fluorescence intensity of the dye is observed. Therefore, the measured fluorescence intensity is altered by the tissue heterogeneity and by the fluctuation of dye intensity. Hence, in this study a quantification strategy for fluorescence-labeled oligonucleotides was developed to solve these disadvantageous effects. Our results proved that upon efficient homogenization and dilution with chaotropic agents, such as guanidinium thiocyanate, it is possible to achieve a complete fluorescence intensity recovery. Furthermore, we demonstrated that this method has the advantage of good sensitivity and reproducibility, as well as easy handling of the tissue samples.

Template-Independent Enzymatic Oligonucleotide Synthesis (TiEOS): Its History, Prospects, and Challenges.

Science.gov (United States)

Jensen, Michael A; Davis, Ronald W

2018-03-27

There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is comprised of additions > n+1 and deletions. By tapping into the physical and biochemical properties of TdT, we strive to further elucidate its mercurial behavior and offer ways to better optimize TiEOS for production-grade oligonucleotide synthesis.

Functionalization of silicon oxide using supercritical fluid deposition of 3,4-epoxybutyltrimethoxysilane for the immobilization of amino-modified oligonucleotide

Energy Technology Data Exchange (ETDEWEB)

Rull, Jordi [Université Grenoble Alpes, Grenoble F38000 (France); CEA, LETI, MINATEC Campus, Grenoble Cedex 9 F38054 (France); CEA, iRTSV, LCBM, Grenoble 38054 (France); CNRS, UMR 5249, Grenoble (France); Nonglaton, Guillaume, E-mail: guillaume.nonglaton@cea.fr [Université Grenoble Alpes, Grenoble F38000 (France); CEA, LETI, MINATEC Campus, Grenoble Cedex 9 F38054 (France); Costa, Guillaume; Fontelaye, Caroline [Université Grenoble Alpes, Grenoble F38000 (France); CEA, LETI, MINATEC Campus, Grenoble Cedex 9 F38054 (France); Marchi-Delapierre, Caroline; Ménage, Stéphane [Université Grenoble Alpes, Grenoble F38000 (France); CEA, iRTSV, LCBM, Grenoble 38054 (France); CNRS, UMR 5249, Grenoble (France); Marchand, Gilles [Université Grenoble Alpes, Grenoble F38000 (France); CEA, LETI, MINATEC Campus, Grenoble Cedex 9 F38054 (France)

2015-11-01

Graphical abstract: - Highlights: • First example of grafting of 3,4-epoxybutyltrimethoxysilane (EBTMOS) onto silicon oxide by supercritical fluid deposition. • Extraordinary efficiency of the supercritical fluid deposition for the grafting of the EBTMOS compared with the conventional solution or vapor phase methodologies. • Demonstration of the efficiency of this functionalization process for the immobilization of amino-modified oligonucleotides. - Abstract: The functionalization of silicon oxide based substrates using silanes is generally performed through liquid phase methodologies. These processes involve a huge quantity of potentially toxic solvents and present some important disadvantages for the functionalization of microdevices or porous materials, for example the low diffusion. To overcome this drawback, solvent-free methodologies like molecular vapor deposition (MVD) or supercritical fluid deposition (SFD) have been developed. In this paper, the deposition process of 3,4-epoxybutyltrimethoxysilane (EBTMOS) on silicon oxide using supercritical carbon dioxide (scCO{sub 2}) as a solvent is studied for the first time. The oxirane ring of epoxy silanes readily reacts with amine group and is of particular interest for the grafting of amino-modified oligonucleotides or antibodies for diagnostic application. Then the ability of this specific EBTMOS layer to react with amine functions has been evaluated using the immobilization of amino-modified oligonucleotide probes. The presence of the probes is revealed by fluorescence using hybridization with a fluorescent target oligonucleotide. The performances of SFD of EBTMOS have been optimized and then compared with the dip coating and molecular vapor deposition methods, evidencing a better grafting efficiency and homogeneity, a lower reaction time in addition to the eco-friendly properties of the supercritical carbon dioxide. The epoxysilane layers have been characterized by surface enhanced ellipsometric

Prenatal diagnosis of microdeletion 16p13.11 combination with partial monosomy of 2q37.1-qter and partial trisomy of 7p15.3-pter in a fetus with bilateral ventriculomegaly, agenesis of corpus callosum, and polydactyly

Directory of Open Access Journals (Sweden)

Pi-Lin Sung

2012-06-01

Conclusion: Array CGH is a useful tool not only to discover the genomic imbalance at the breakpoints as well as to detect unexpectedly complex rearrangements in other chromosomes. Our case also provided evidence that genomic aberration at chromosome 16p13.11 involves in the formation of polydactyly.

3q27.3 microdeletional syndrome

DEFF Research Database (Denmark)

Thevenon, Julien; Callier, Patrick; Poquet, Hélène

2014-01-01

BACKGROUND: Since the advent of array-CGH, numerous new microdeletional syndromes have been delineated while others remain to be described. Although 3q29 subtelomeric deletion is a well-described syndrome, there is no report on 3q interstitial deletions. METHODS: We report for the first time seve...

TIA-1 RRM23 binding and recognition of target oligonucleotides.

Science.gov (United States)

Waris, Saboora; García-Mauriño, Sofía M; Sivakumaran, Andrew; Beckham, Simone A; Loughlin, Fionna E; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C J; Wilce, Jacqueline A

2017-05-05

TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Application of hierarchical oligonucleotide primer extension (HOPE) to assess relative abundances of ammonia- and nitrite-oxidizing bacteria

KAUST Repository

Scarascia, Giantommaso; Cheng, Hong; Harb, Moustapha; Hong, Pei-Ying

2017-01-01

for ensuring the efficiency of nitrification in water treatment systems. Hierarchical oligonucleotide primer extension (HOPE), previously developed to rapidly quantify relative abundances of specific microbial groups of interest, was applied in this study

Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals

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Punit P Seth

2012-01-01

Full Text Available We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs modified with α-L-locked nucleic acid (LNA and related modifications targeting phosphatase and tensin homologue (PTEN messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3′- and 5′-flanks with R-5′-Me-α-L-LNA but not R-6′-Me- or 3′-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5′-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5′-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.

Development and production of an oligonucleotide MuscleChip: use for validation of ambiguous ESTs

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Lanfranchi Gerolamo

2002-10-01

Full Text Available Abstract Background We describe the development, validation, and use of a highly redundant 120,000 oligonucleotide microarray (MuscleChip containing 4,601 probe sets representing 1,150 known genes expressed in muscle and 2,075 EST clusters from a non-normalized subtracted muscle EST sequencing project (28,074 EST sequences. This set included 369 novel EST clusters showing no match to previously characterized proteins in any database. Each probe set was designed to contain 20–32 25 mer oligonucleotides (10–16 paired perfect match and mismatch probe pairs per gene, with each probe evaluated for hybridization kinetics (Tm and similarity to other sequences. The 120,000 oligonucleotides were synthesized by photolithography and light-activated chemistry on each microarray. Results Hybridization of human muscle cRNAs to this MuscleChip (33 samples showed a correlation of 0.6 between the number of ESTs sequenced in each cluster and hybridization intensity. Out of 369 novel EST clusters not showing any similarity to previously characterized proteins, we focused on 250 EST clusters that were represented by robust probe sets on the MuscleChip fulfilling all stringent rules. 102 (41% were found to be consistently "present" by analysis of hybridization to human muscle RNA, of which 40 ESTs (39% could be genome anchored to potential transcription units in the human genome sequence. 19 ESTs of the 40 ESTs were furthermore computer-predicted as exons by one or more than three gene identification algorithms. Conclusion Our analysis found 40 transcriptionally validated, genome-anchored novel EST clusters to be expressed in human muscle. As most of these ESTs were low copy clusters (duplex and triplex in the original 28,000 EST project, the identification of these as significantly expressed is a robust validation of the transcript units that permits subsequent focus on the novel proteins encoded by these genes.

Membrane-Assisted Growth of DNA Origami Nanostructure Arrays

Science.gov (United States)

2015-01-01

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors—a three-layered rectangular block and a Y-shaped DNA structure—to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes. PMID:25734977

Membrane-assisted growth of DNA origami nanostructure arrays.

Science.gov (United States)

Kocabey, Samet; Kempter, Susanne; List, Jonathan; Xing, Yongzheng; Bae, Wooli; Schiffels, Daniel; Shih, William M; Simmel, Friedrich C; Liedl, Tim

2015-01-01

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors--a three-layered rectangular block and a Y-shaped DNA structure--to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes.

Global genomic analysis of intraductal papillary mucinous neoplasms of the pancreas reveals significant molecular differences compared to ductal adenocarcinoma.

Science.gov (United States)

Fritz, Stefan; Fernandez-del Castillo, Carlos; Mino-Kenudson, Mari; Crippa, Stefano; Deshpande, Vikram; Lauwers, Gregory Y; Warshaw, Andrew L; Thayer, Sarah P; Iafrate, A John

2009-03-01

To determine whether intraductal papillary mucinous neoplasms of the pancreas (IPMNs) have a different genetic background compared with ductal adenocarcinoma (PDAC). The biologic and clinical behavior of IPMNs and IPMN-associated adenocarcinomas is different from PDAC in having a less aggressive tumor growth and significantly improved survival. Up to date, the molecular mechanisms underlying the clinical behavior of IPMNs are incompletely understood. 128 cystic pancreatic lesions were prospectively identified during the course of 2 years. From the corresponding surgical specimens, 57 IPMNs were separated and subdivided by histologic criteria into those with low-grade dysplasia, moderate dysplasia, high-grade dysplasia, and invasive cancer. Twenty specimens were suitable for DNA isolation and subsequent performance of array CGH. While none of the IPMNs with low-grade dysplasia displayed detectable chromosomal aberrations, IPMNs with moderate and high-grade dysplasia showed frequent copy number alterations. Commonly lost regions were located on chromosome 5q, 6q, 10q, 11q, 13q, 18q, and 22q. The incidence of loss of chromosome 5q, 6q, and 11q was significantly higher in IPMNs with high-grade dysplasia or invasion compared with PDAC. Ten of 13 IPMNs with moderate dysplasia or malignancy had loss of part or all of chromosome 6q, with a minimal deleted region between linear positions 78.0 and 130.0. This study is the first to use array CGH to characterize IPMNs. Recurrent cytogenetic alterations were identified and were different than those described in PDAC. Array CGH may help distinguish between these 2 entities and give insight into the differences in their biology and prognosis.

Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

Science.gov (United States)

Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

2012-06-01

Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

Antiproliferation effects of an androgen receptor triple-helix forming oligonucleotide on prostate cancer cells

International Nuclear Information System (INIS)

Zhang Yong; Chen Weizhen; Xie Yao; Gao Jinhui

2005-01-01

Objective: To provide experimental basis for antigene radiation therapy through exploring the effects of antigene strategy on androgen receptor (AR) expression and proliferation of prostate cancer cells. Methods: The triple-helix forming oligonucleotide (TFO) targeting 2447-2461nt of AR cDNA was designed and transfected LNCaP prostate cancer cells with liposome. 24-72 h after transfection, the cellular proliferation was detected by 3 H-thymidine (TdR) incorporation test, the expression of AR gene was examined by reverse transcription-polymerase chain reaction (RT-PCR) and expression of AR protein was performed by radioligand binding assay. The results of TFO were compared with antisense oligonucleotide (ASON). Results: At all time points, the AR expression levels in TFO group were markedly lower than that of ASON group (P<0.05). The inhibitory rate of TFO for cellular proliferation was significantly higher than that of ASON (P<0.05). Conclusion: The TFO was a potent inhibitor for AR expression and cell proliferation of LNCaP cells , and could be used in antigene radiotherapy. (authors)

Canine urothelial carcinoma: genomically aberrant and comparatively relevant.

Science.gov (United States)

Shapiro, S G; Raghunath, S; Williams, C; Motsinger-Reif, A A; Cullen, J M; Liu, T; Albertson, D; Ruvolo, M; Bergstrom Lucas, A; Jin, J; Knapp, D W; Schiffman, J D; Breen, M

2015-06-01

Urothelial carcinoma (UC), also referred to as transitional cell carcinoma (TCC), is the most common bladder malignancy in both human and canine populations. In human UC, numerous studies have demonstrated the prevalence of chromosomal imbalances. Although the histopathology of the disease is similar in both species, studies evaluating the genomic profile of canine UC are lacking, limiting the discovery of key comparative molecular markers associated with driving UC pathogenesis. In the present study, we evaluated 31 primary canine UC biopsies by oligonucleotide array comparative genomic hybridization (oaCGH). Results highlighted the presence of three highly recurrent numerical aberrations: gain of dog chromosome (CFA) 13 and 36 and loss of CFA 19. Regional gains of CFA 13 and 36 were present in 97 % and 84 % of cases, respectively, and losses on CFA 19 were present in 77 % of cases. Fluorescence in situ hybridization (FISH), using targeted bacterial artificial chromosome (BAC) clones and custom Agilent SureFISH probes, was performed to detect and quantify these regions in paraffin-embedded biopsy sections and urine-derived urothelial cells. The data indicate that these three aberrations are potentially diagnostic of UC. Comparison of our canine oaCGH data with that of 285 human cases identified a series of shared copy number aberrations. Using an informatics approach to interrogate the frequency of copy number aberrations across both species, we identified those that had the highest joint probability of association with UC. The most significant joint region contained the gene PABPC1, which should be considered further for its role in UC progression. In addition, cross-species filtering of genome-wide copy number data highlighted several genes as high-profile candidates for further analysis, including CDKN2A, S100A8/9, and LRP1B. We propose that these common aberrations are indicative of an evolutionarily conserved mechanism of pathogenesis and harbor genes

Distribution of Sulfate-Reducing Bacteria, O2, and H2s in Photosynthetic Biofilms Determined by Oligonucleotide Probes and Microelectrodes Rid A-1977-2009

DEFF Research Database (Denmark)

RAMSING, NB; KUHL, M.; JØRGENSEN, BB

1993-01-01

The vertical distribution of sulfate-reducing bacteria (SRB) in photosynthetic biofilms from the trickling filter of a sewage treatment plant was investigated with oligonucleotide probes binding to 16S rRNA. To demonstrate the effect of daylight and photosynthesis and thereby of increased oxygen....... Fluorescent-dye-conjugated oligonucleotides were used as ''phylogenetic'' probes to identify single cells in the slices. Oligonucleotide sequences were selected which were complementary to short sequence elements (16 to 20 nucleotides) within the 16S rRNA of sulfate-reducing bacteria. The probes were labeled...... with fluorescein or rhodamine derivatives for subsequent visualization by epifluorescence microscopy. Five probes were synthesized for eukaryotes, eubacteria, SRB (including most species of the delta group of purple bacteria), Desulfobacter spp., and a nonhybridizing control. The SRB were unevenly distributed...

A 1.37-Mb 12p11.22-p11.21 deletion coincident with a 367-kb 22q11.2 duplication detected by array comparative genomic hybridization in an adolescent girl with autism and difficulty in self-care of menstruation.

Science.gov (United States)

Chen, Chih-Ping; Lin, Shuan-Pei; Chern, Schu-Rern; Wu, Peih-Shan; Su, Jun-Wei; Lee, Chen-Chi; Wang, Wayseen

2014-03-01

To present an array comparative genomic hybridization (aCGH) characterization of a 12p11.22-p11.21 microdeletion and 22q11.2 microduplication in an adolescent girl with autism, mental retardation, facial dysmorphism, microcephaly, behavior problems, and an apparently balanced reciprocal translocation of t(8;12)(q24.3;p11.2). A 13-year-old girl was referred to the hospital because of autism, mental retardation, and difficulty in the self-care of her menstruation. Cytogenetic analysis revealed an apparently balanced reciprocal translocation and a karyotype of 46,XX,t(8;12) (q24.3;p11.2)dn. The girl manifested microcephaly, hypertelorism, flat facial profile, prominent forehead, thick scalp hair, upslanting palpebral fissures, broad nasal bridge, bulbous nose, right simian crease, bilateral clinodactyly of the fifth fingers, bilateral pes cavus, learning difficulties, mental retardation, emotional instability, cognitive impairment, behavior problems, jumping-like gaits, and autistic spectrum disorder. aCGH was performed to evaluate genomic imbalance in this patient. aCGH analysis revealed a 1.37-Mb 12p11.22-p11.21 microdeletion or arr [hg 19] 12p11.22-p11.21 (30,645,008-32,014,774)×1 and a 367-kb 22q11.21 microduplication or arr [hg 19] 22q11.21 (18,657,470-19,024,306)×3. The 1.37-Mb 12p11.22-p11.21 microdeletion encompassed 26 genes including IPO8, CAPRIN2, and DDX11, and the 367-kb 22q11.21 microduplication encompassed 20 genes including USP18, DGCR6, PRODH, and DGCR2. An apparently balanced translocation may be in fact affected by concurrent deletion and duplication in two different chromosomal regions. Our presentation provides information on diagnostic phenotype of 12p11.22-p11.21 microdeletion and 22q11.2 microduplication. Copyright © 2014. Published by Elsevier B.V.

Duplication of 7q36.3 encompassing the Sonic Hedgehog (SHH) gene is associated with congenital muscular hypertrophy

DEFF Research Database (Denmark)

Kristensen, Lone Krøldrup; Kjaergaard, S; Kirchhoff, Marianne

2012-01-01

with muscular hypertrophy and mildly retarded psychomotor development. Array-CGH identified a small duplication of 7q36.3 including the Sonic Hedgehog (SHH) gene in both the aborted foetus and the live born male sib. Neither of the parents carried the 7q36.3 duplication. The consequences of overexpression...

Detection of genomic deletions in rice using oligonucleotide microarrays

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Bordeos Alicia

2009-03-01

Full Text Available Abstract Background The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL. However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions. Results We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip® allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to ~500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations http://irfgc.irri.org/cgi-bin/gbrowse/IR64_deletion_mutants/. Conclusion We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a

Array capabilities and future arrays

International Nuclear Information System (INIS)

Radford, D.

1993-01-01

Early results from the new third-generation instruments GAMMASPHERE and EUROGAM are confirming the expectation that such arrays will have a revolutionary effect on the field of high-spin nuclear structure. When completed, GAMMASHPERE will have a resolving power am order of magnitude greater that of the best second-generation arrays. When combined with other instruments such as particle-detector arrays and fragment mass analysers, the capabilites of the arrays for the study of more exotic nuclei will be further enhanced. In order to better understand the limitations of these instruments, and to design improved future detector systems, it is important to have some intelligible and reliable calculation for the relative resolving power of different instrument designs. The derivation of such a figure of merit will be briefly presented, and the relative sensitivities of arrays currently proposed or under construction presented. The design of TRIGAM, a new third-generation array proposed for Chalk River, will also be discussed. It is instructive to consider how far arrays of Compton-suppressed Ge detectors could be taken. For example, it will be shown that an idealised open-quote perfectclose quotes third-generation array of 1000 detectors has a sensitivity an order of magnitude higher again than that of GAMMASPHERE. Less conventional options for new arrays will also be explored

A genome-wide map of aberrantly expressed chromosomal islands in colorectal cancer

Directory of Open Access Journals (Sweden)

Castanos-Velez Esmeralda

2006-09-01

Full Text Available Abstract Background Cancer development is accompanied by genetic phenomena like deletion and amplification of chromosome parts or alterations of chromatin structure. It is expected that these mechanisms have a strong effect on regional gene expression. Results We investigated genome-wide gene expression in colorectal carcinoma (CRC and normal epithelial tissues from 25 patients using oligonucleotide arrays. This allowed us to identify 81 distinct chromosomal islands with aberrant gene expression. Of these, 38 islands show a gain in expression and 43 a loss of expression. In total, 7.892 genes (25.3% of all human genes are located in aberrantly expressed islands. Many chromosomal regions that are linked to hereditary colorectal cancer show deregulated expression. Also, many known tumor genes localize to chromosomal islands of misregulated expression in CRC. Conclusion An extensive comparison with published CGH data suggests that chromosomal regions known for frequent deletions in colon cancer tend to show reduced expression. In contrast, regions that are often amplified in colorectal tumors exhibit heterogeneous expression patterns: even show a decrease of mRNA expression. Because for several islands of deregulated expression chromosomal aberrations have never been observed, we speculate that additional mechanisms (like abnormal states of regional chromatin also have a substantial impact on the formation of co-expression islands in colorectal carcinoma.

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: apeluso@unisa.it [Dipartimento di Chimica e Biologia, Università di Salerno, Via Giovanni Paolo II, I-84084 Fisciano, SA (Italy)

2014-08-31

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.

Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic

DEFF Research Database (Denmark)

Lou, Chenguang; Martos-Maldonado, Manuel C.; Madsen, Charlotte Stahl

2016-01-01

Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain ...

Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

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Xavier Gérard

2015-01-01

Full Text Available Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15% creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2’-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA-approved adeno-associated virus (AAV-vectors, thus hampering gene augmentation therapy.

Label-Free Electrochemical Detection of the Specific Oligonucleotide Sequence of Dengue Virus Type 1 on Pencil Graphite Electrodes

Science.gov (United States)

Souza, Elaine; Nascimento, Gustavo; Santana, Nataly; Ferreira, Danielly; Lima, Manoel; Natividade, Edna; Martins, Danyelly; Lima-Filho, José

2011-01-01

A biosensor that relies on the adsorption immobilization of the 18-mer single-stranded nucleic acid related to dengue virus gene 1 on activated pencil graphite was developed. Hybridization between the probe and its complementary oligonucleotides (the target) was investigated by monitoring guanine oxidation by differential pulse voltammetry (DPV). The pencil graphite electrode was made of ordinary pencil lead (type 4B). The polished surface of the working electrode was activated by applying a potential of 1.8 V for 5 min. Afterward, the dengue oligonucleotides probe was immobilized on the activated electrode by applying 0.5 V to the electrode in 0.5 M acetate buffer (pH 5.0) for 5 min. The hybridization process was carried out by incubating at the annealing temperature of the oligonucleotides. A time of five minutes and concentration of 1 μM were found to be the optimal conditions for probe immobilization. The electrochemical detection of annealing between the DNA probe (TS-1P) immobilized on the modified electrode, and the target (TS-1T) was achieved. The target could be quantified in a range from 1 to 40 nM with good linearity and a detection limit of 0.92 nM. The specificity of the electrochemical biosensor was tested using non-complementary sequences of dengue virus 2 and 3. PMID:22163916

Complementarily addressed modification and cleavage of a single-stranded fragment of DNA with the aid of alkylating derivatives of oligonucleotides

International Nuclear Information System (INIS)

Brosalina, E.B.; Vlasov, V.V.; Kutyavin, I.V.; Mamaev, S.V.; Pletnev, A.G.; Podyminogin, M.A.

1986-01-01

The chemical modification of a 303-nucleotide single-stranded fragment of DNA by alkylating oligonucleotide derivatives bearing 4-[N-methyl-N-(2-chloroethyl)amino]benzyl groups in the 5'-terminal phosphate of the 3'-terminal ribose residue has been investigated. It has been shown that under the conditions of the formation of a complex with the DNA fragment both types of derivatives specifically alkylate nucleotides of the DNA fragments that are located directly adjacent to the sections complementary to the oligonucleotides bearing the reactive groups. Alkylation takes place with a high efficiency, and the DNA fragment can be cleaved specifically at the position of the alkylated nucleotides

Pd0-Catalyzed Methyl Transfer on Nucleosides and Oligonucleotides, Envisaged as a PET Tracer

Directory of Open Access Journals (Sweden)

Eric Fouquet

2013-11-01

Full Text Available The methyl transfer reaction from activated monomethyltin, via a modified Stille coupling reaction, was studied under “ligandless” conditions on fully deprotected 5'-modified nucleosides and one dinucleotide. The reaction was optimized to proceed in a few minutes and quantitative yield, even under dilute conditions, thus affording a rapid and efficient new method for oligonucleotide labelling with carbon-11.

Insertional translocation leading to a 4q13 duplication including the EPHA5 gene in two siblings with attention-deficit hyperactivity disorder.

Science.gov (United States)

Matoso, Eunice; Melo, Joana B; Ferreira, Susana I; Jardim, Ana; Castelo, Teresa M; Weise, Anja; Carreira, Isabel M

2013-08-01

An insertional translocation (IT) can result in pure segmental aneusomy for the inserted genomic segment allowing to define a more accurate clinical phenotype. Here, we report on two siblings sharing an unbalanced IT inherited from the mother with a history of learning difficulty. An 8-year-old girl with developmental delay, speech disability, and attention-deficit hyperactivity disorder (ADHD), showed by GTG banding analysis a subtle interstitial alteration in 21q21. Oligonucleotide array comparative genomic hybridization (array-CGH) analysis showed a 4q13.1-q13.3 duplication spanning 8.6 Mb. Fluorescence in situ hybridization (FISH) with bacterial artificial chromosome (BAC) clones confirmed the rearrangement, a der(21)ins(21;4)(q21;q13.1q13.3). The duplication described involves 50 RefSeq genes including the EPHA5 gene that encodes for the EphA5 receptor involved in embryonic development of the brain and also in synaptic remodeling and plasticity thought to underlie learning and memory. The same rearrangement was observed in a younger brother with behavioral problems and also exhibiting ADHD. ADHD is among the most heritable of neuropsychiatric disorders. There are few reports of patients with duplications involving the proximal region of 4q and a mild phenotype. To the best of our knowledge this is the first report of a duplication restricted to band 4q13. This abnormality could be easily missed in children who have nonspecific cognitive impairment. The presence of this behavioral disorder in the two siblings reinforces the hypothesis that the region involved could include genes involved in ADHD. Copyright © 2013 Wiley Periodicals, Inc.

Evidence for increased SOX3 dosage as a risk factor for X-linked hypopituitarism and neural tube defects

NARCIS (Netherlands)

Bauters, M.; Frints, S.G.; Esch, H. van; Spruijt, L.; Baldewijns, M.M.; Die-Smulders, C.E.M. de; Fryns, J.P.; Marynen, P.; Froyen, G.

2014-01-01

Genomic duplications of varying lengths at Xq26-q27 involving SOX3 have been described in families with X-linked hypopituitarism. Using array-CGH we detected a 1.1 Mb microduplication at Xq27 in a large family with three males suffering from X-linked hypopituitarism. The duplication was mapped from

The value of blastocyst culture on preimplantation genetic diagnosis%囊胚培养在植入前遗传学诊断中的价值

Institute of Scientific and Technical Information of China (English)

偶健; 王玮; 马燕琳; 周知; 丁洁; 王馥新; 段程颖; 李林江; 郑爱燕

2015-01-01

Objective To estimate the value of blastocyst culture for preimplantation genetic diagnosis (PGD).Methods Day 3 embryos were biopsied and analyzed with fluorescence in situ hybridization (FISH) technique.Embryos with normal FISH results were cultured into blastocysts,and the ones with better morphology scores were transferred.Fourteen embryos with abnormal FISH results were cultured into blastocysts.Part of the cells taken from the blastocysts were amplified by whole genomic amplification (WGA) and assessed by array-based comparative genomic hybridization (array-CGH) analysis.Results Six blastocysts with normal FISH results were transferred in 5 cycles.Four healthy babies of 3 cycles were delivered.Another one was a singleton pregnancy but with embryo growth arrest,whose villus karyotype was normal.Fourteen embryos with abnormal FISH results were cultured into blastocysts and analyzed by array-CGH.Six blastocysts were normal by array-CGH.Conclusion FISH combined with blastocyst culture may further ensure the accuracy of PGD result.Detection at the blastocyst stage can avoid false positive results and mosaic interferences on Day 3 stage and are therefore more authentic.%目的 探讨囊胚培养在植入前遗传学诊断(preimplantation genetic diagnosis,PGD)中的应用价值.方法 受精后第3天(Day 3)行胚胎活检,进行荧光原位杂交(fluorescent in situ hybridization,FISH).对于诊断为正常的胚胎,培养到囊胚阶段后选择形态评分优良的囊胚进行移植;对于诊断为异常的胚胎,有14个培养到囊胚阶段,各取其一部分细胞用于全基因组扩增(whole genomic amplification,WGA),将扩增后的DNA用微阵列比较基因组杂交(array-based comparative genomic hybridization,arrayCGH)进行再次检测.结果 FISH诊断为正常的6个囊胚进行了5个周期的胚胎移植,3个周期成功生育了4个健康婴儿,1个周期单胎妊娠见胚囊后流产,绒毛染色体检测为正常核型.FISH诊断为异常的14个囊胚用array

Lineage analysis of early and advanced tubular adenocarcinomas of the stomach: continuous or discontinuous?

International Nuclear Information System (INIS)

Nakayama, Takahisa; Ling, Zhi-Qiang; Mukaisho, Ken-ichi; Hattori, Takanori; Sugihara, Hiroyuki

2010-01-01

Eradication of early gastric carcinoma (GC) is thought to contribute to reduction in the mortality of GC, given that most of the early GCs progress to the advanced GCs. However, early GC is alternatively considered a dormant variant of GC, and it infrequently progresses to advanced GC. The aim of this study was to clarify the extent of overlap of genetic lineages between early and advanced tubular adenocarcinomas (TUBs) of the stomach. Immunohistochemical staining for p53 was performed using 28 surgically resected stomachs with 13 intramucosal and 15 invasive TUBs. By chromosome- and array-based comparative genomic hybridization (CGH), genomic copy number constitution was compared between the mucosal and invasive parts of the invasive TUBs and between the mucosal parts of the invasive and intramucosal TUBs, using 25 and 22 TUBs, respectively. TP53 mutation in exons 5-8 was examined in 20 TUBs. Chromosomal CGH revealed that 4q+ and 11q+ were more common in advanced and early TUBs, respectively, whereas copy number changes in 8q and 17p showed no significant differences between early and advanced TUBs. However, array CGH revealed that, of the 13 intramucosal TUBs examined, loss of MYC (MYC-) and gain of TP53 (TP53+) was detected in 9 TUBs and MYC+ and/or TP53- was detected in 3 TUBs. Of the mucosal samples of 9 invasive TUBs, 7 showed MYC-/TP53+ and none showed MYC+ and/or TP53-. Of the 9 samples from the invasive parts, 1 (from submucosal cancers) showed MYC-/TP53+ and 6 (1 from submucosal and 5 from advanced cancers) showed MYC+ and/or TP53-. The latter 6 tumours commonly showed a mutant pattern (diffuse or null) in p53 immunohistochemistry, and 4 of the 6 tumours assessable for TP53 sequence analysis revealed mutations. The overall array CGH pattern indicated that, between the mucosal and invasive parts, genetic lineage was found discontinuous in 5 advanced cancers and continuous in 3 submucosal cancers. Genetic lineages often differed between early and advanced

Gene expression profile and genomic alterations in colonic tumours induced by 1,2-dimethylhydrazine (DMH) in rats

International Nuclear Information System (INIS)

Femia, Angelo Pietro; Luceri, Cristina; Toti, Simona; Giannini, Augusto; Dolara, Piero; Caderni, Giovanna

2010-01-01

Azoxymethane (AOM) or 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats shares many phenotypical similarities with human sporadic colon cancer and is a reliable model for identifying chemopreventive agents. Genetic mutations relevant to human colon cancer have been described in this model, but comprehensive gene expression and genomic analysis have not been reported so far. Therefore, we applied genome-wide technologies to study variations in gene expression and genomic alterations in DMH-induced colon cancer in F344 rats. For gene expression analysis, 9 tumours (TUM) and their paired normal mucosa (NM) were hybridized on 4 × 44K Whole rat arrays (Agilent) and selected genes were validated by semi-quantitative RT-PCR. Functional analysis on microarray data was performed by GenMAPP/MappFinder analysis. Array-comparative genomic hybridization (a-CGH) was performed on 10 paired TUM-NM samples hybridized on Rat genome arrays 2 × 105K (Agilent) and the results were analyzed by CGH Analytics (Agilent). Microarray gene expression analysis showed that Defcr4, Igfbp5, Mmp7, Nos2, S100A8 and S100A9 were among the most up-regulated genes in tumours (Fold Change (FC) compared with NM: 183, 48, 39, 38, 36 and 32, respectively), while Slc26a3, Mptx, Retlna and Muc2 were strongly down-regulated (FC: -500; -376, -167, -79, respectively). Functional analysis showed that pathways controlling cell cycle, protein synthesis, matrix metalloproteinases, TNFα/NFkB, and inflammatory responses were up-regulated in tumours, while Krebs cycle, the electron transport chain, and fatty acid beta oxidation were down-regulated. a-CGH analysis showed that four TUM out of ten had one or two chromosomal aberrations. Importantly, one sample showed a deletion on chromosome 18 including Apc. The results showed complex gene expression alterations in adenocarcinomas encompassing many altered pathways. While a-CGH analysis showed a low degree of genomic imbalance, it is interesting to

Oligonucleotide fishing for STAT6: cross-talk between IL-4 and chemokines

DEFF Research Database (Denmark)

Eriksen, K W; Nielsen, M; Kaltoft, K

2001-01-01

Signal transducer and activator of transcription 6 (STAT6) is essential for the biological activities of interleukin-4 (IL-4) and the development of allergic responses in mice. Here we report on a sensitive and specific assay for STAT6 activation in response to IL-4. We took advantage of double-s...... activation, whereas other chemokines and cytokines do not. In conclusion, our data show that oligonucleotide fishing is a supplementary tool for studying cytokine cross-talk at a genomic level....

Importance of length and sequence order on magnesium binding to surface-bound oligonucleotides studied by second harmonic generation and atomic force microscopy.

Science.gov (United States)

Holland, Joseph G; Geiger, Franz M

2012-06-07

The binding of magnesium ions to surface-bound single-stranded oligonucleotides was studied under aqueous conditions using second harmonic generation (SHG) and atomic force microscopy (AFM). The effect of strand length on the number of Mg(II) ions bound and their free binding energy was examined for 5-, 10-, 15-, and 20-mers of adenine and guanine at pH 7, 298 K, and 10 mM NaCl. The binding free energies for adenine and guanine sequences were calculated to be -32.1(4) and -35.6(2) kJ/mol, respectively, and invariant with strand length. Furthermore, the ion density for adenine oligonucleotides did not change as strand length increased, with an average value of 2(1) ions/strand. In sharp contrast, guanine oligonucleotides displayed a linear relationship between strand length and ion density, suggesting that cooperativity is important. This data gives predictive capabilities for mixed strands of various lengths, which we exploit for 20-mers of adenines and guanines. In addition, the role sequence order plays in strands of hetero-oligonucleotides was examined for 5'-A(10)G(10)-3', 5'-(AG)(10)-3', and 5'-G(10)A(10)-3' (here the -3' end is chemically modified to bind to the surface). Although the free energy of binding is the same for these three strands (averaged to be -33.3(4) kJ/mol), the total ion density increases when several guanine residues are close to the 3' end (and thus close to the solid support substrate). To further understand these results, we analyzed the height profiles of the functionalized surfaces with tapping-mode atomic force microscopy (AFM). When comparing the average surface height profiles of the oligonucleotide surfaces pre- and post- Mg(II) binding, a positive correlation was found between ion density and the subsequent height decrease following Mg(II) binding, which we attribute to reductions in Coulomb repulsion and strand collapse once a critical number of Mg(II) ions are bound to the strand.

Inhibition of cyclic AMP response element-directed transcription by decoy oligonucleotides enhances tumor-specific radiosensitivity

Energy Technology Data Exchange (ETDEWEB)

Park, Serk In, E-mail: serkin@korea.edu [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); The BK21 Plus Program for Biomedical Sciences, Korea University College of Medicine, Seoul (Korea, Republic of); Department of Medicine and Center for Bone Biology, Vanderbilt University School of Medicine, Nashville, TN (United States); Park, Sung-Jun [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); Laboratory of Obesity and Aging Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD (United States); Lee, Junghan; Kim, Hye Eun; Park, Su Jin; Sohn, Jeong-Won [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); Park, Yun Gyu, E-mail: parkyg@korea.ac.kr [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of)

2016-01-15

The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while there was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach. - Highlights: • γ-Irradiation induced CREB phosphorylation and CRE-directed transcription in tumor. • γ-Irradiation-induced transcriptional activation of CREB was via p38 MAPK pathway. • CRE blockade increased radiosensitivity of tumor cells but not of normal cells. • CRE decoy oligonucleotides or p38 MAPK inhibitors can be used as radiosensitizers.

Inhibition of cyclic AMP response element-directed transcription by decoy oligonucleotides enhances tumor-specific radiosensitivity

International Nuclear Information System (INIS)

Park, Serk In; Park, Sung-Jun; Lee, Junghan; Kim, Hye Eun; Park, Su Jin; Sohn, Jeong-Won; Park, Yun Gyu

2016-01-01

The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while there was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach. - Highlights: • γ-Irradiation induced CREB phosphorylation and CRE-directed transcription in tumor. • γ-Irradiation-induced transcriptional activation of CREB was via p38 MAPK pathway. • CRE blockade increased radiosensitivity of tumor cells but not of normal cells. • CRE decoy oligonucleotides or p38 MAPK inhibitors can be used as radiosensitizers.

Labelling of nucleosides and oligonucleotides by solvatochromic 4-aminophthalimide fluorophore for studying DNA–protein interactions

Czech Academy of Sciences Publication Activity Database

Riedl, Jan; Pohl, Radek; Ernsting, N. P.; Orság, Petr; Fojta, Miroslav; Hocek, Michal

2012-01-01

Roč. 3, č. 9 (2012), s. 2797-2806 ISSN 2041-6520 R&D Projects: GA ČR GA203/09/0317; GA ČR GBP206/12/G151 Institutional support: RVO:61388963 ; RVO:68081707 Keywords : DNA * oligonucleotides * polymerase * phthalimide * nucleotides * fluorescent labeling Subject RIV: CC - Organic Chemistry Impact factor: 8.314, year: 2012

Short Oligonucleotides Aligned in Stretched Humid Matrix: Secondary DNA Structure in Poly(vinyl alcohol) Environment

KAUST Repository

Hanczyc, Piotr

2012-04-24

We report that short, synthetic, double- as well as single-stranded DNA can be aligned in stretched humid poly(vinyl alcohol) (PVA) matrix, and the secondary structure (nucleobase orientation) can be characterized with linear dichroism (LD) spectroscopy. Oligonucleotides of lengths varying between 10 (3.4 nm) and 60 bases (20.4 nm) were investigated with respect to structural properties in the gel-like polymer environment. The DNA conformation as a function of relative humidity reveals a strong dependence of helical structure of DNA on PVA hydration level, results of relevance for nanotechnical studies of DNA-based supramolecular systems. Also, the PVA gel could provide possibilities to test models for nucleic acid interactions and distribution in cell contexts, including structural stability of genetic material in the cell and PVA-packaging for gene delivery. A method by which duplex oligonucleotides, with sequences designed to provide specific binding sites, become amenable to polarized-light spectroscopy opens up new possibilities for studying structure in DNA complexes with small adduct molecules as well as proteins. © 2012 American Chemical Society.

Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment.

Science.gov (United States)

Kozlowska, Anna Karolina; Florczak, Anna; Smialek, Maciej; Dondajewska, Ewelina; Mackiewicz, Andrzej; Kortylewski, Marcin; Dams-Kozlowska, Hanna

2017-09-01

Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG-STAT3siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG-STAT3siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel

Identification of novel candidate target genes in amplicons of Glioblastoma multiforme tumors detected by expression and CGH microarray profiling

Directory of Open Access Journals (Sweden)

Hernández-Moneo Jose-Luis

2006-09-01

Full Text Available Abstract Background Conventional cytogenetic and comparative genomic hybridization (CGH studies in brain malignancies have shown that glioblastoma multiforme (GBM is characterized by complex structural and numerical alterations. However, the limited resolution of these techniques has precluded the precise identification of detailed specific gene copy number alterations. Results We performed a genome-wide survey of gene copy number changes in 20 primary GBMs by CGH on cDNA microarrays. A novel amplicon at 4p15, and previously uncharacterized amplicons at 13q32-34 and 1q32 were detected and are analyzed here. These amplicons contained amplified genes not previously reported. Other amplified regions containg well-known oncogenes in GBMs were also detected at 7p12 (EGFR, 7q21 (CDK6, 4q12 (PDGFRA, and 12q13-15 (MDM2 and CDK4. In order to identify the putative target genes of the amplifications, and to determine the changes in gene expression levels associated with copy number change events, we carried out parallel gene expression profiling analyses using the same cDNA microarrays. We detected overexpression of the novel amplified genes SLA/LP and STIM2 (4p15, and TNFSF13B and COL4A2 (13q32-34. Some of the candidate target genes of amplification (EGFR, CDK6, MDM2, CDK4, and TNFSF13B were tested in an independent set of 111 primary GBMs by using FISH and immunohistological assays. The novel candidate 13q-amplification target TNFSF13B was amplified in 8% of the tumors, and showed protein expression in 20% of the GBMs. Conclusion This high-resolution analysis allowed us to propose novel candidate target genes such as STIM2 at 4p15, and TNFSF13B or COL4A2 at 13q32-34 that could potentially contribute to the pathogenesis of these tumors and which would require futher investigations. We showed that overexpression of the amplified genes could be attributable to gene dosage and speculate that deregulation of those genes could be important in the development

Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

DEFF Research Database (Denmark)

Moreno, Pedro M D; Geny, Sylvain; Pabon, Y Vladimir

2013-01-01

In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasi...

Introduction on Using the FastPCR Software and the Related Java Web Tools for PCR and Oligonucleotide Assembly and Analysis.

Science.gov (United States)

Kalendar, Ruslan; Tselykh, Timofey V; Khassenov, Bekbolat; Ramanculov, Erlan M

2017-01-01

This chapter introduces the FastPCR software as an integrated tool environment for PCR primer and probe design, which predicts properties of oligonucleotides based on experimental studies of the PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations. These include the standard PCR as well as the multiplex, long-distance, inverse, real-time, group-specific, unique, overlap extension PCR for multi-fragments assembling cloning and loop-mediated isothermal amplification (LAMP). It also contains a built-in program to design oligonucleotide sets both for long sequence assembly by ligase chain reaction and for design of amplicons that tile across a region(s) of interest. The software calculates the melting temperature for the standard and degenerate oligonucleotides including locked nucleic acid (LNA) and other modifications. It also provides analyses for a set of primers with the prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity as well as a primer dilution and resuspension calculator. The program consists of various bioinformatical tools for analysis of sequences with the GC or AT skew, CG% and GA% content, and the purine-pyrimidine skew. It also analyzes the linguistic sequence complexity and performs generation of random DNA sequence as well as restriction endonucleases analysis. The program allows to find or create restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It performs efficient and complete detection of various repeat types with visual display. The FastPCR software allows the sequence file batch processing that is essential for automation. The program is available for download at http://primerdigital.com/fastpcr.html , and its online version is located at http://primerdigital.com/tools/pcr.html .

Development of specific oligonucleotide probes for the identification and in situ detection of hydrocarbon-degrading Alcanivorax strains.

Science.gov (United States)

Syutsubo, K; Kishira, H; Harayama, S

2001-06-01

The genus Alcanivorax comprises diverse hydrocarbon-degrading marine bacteria. Novel 16S rRNA-targeted oligonucleotide DNA probes (ALV735 and ALV735-b) were developed to quantify two subgroups of the Alcanivorax/Fundibacter group by fluorescence in situ hybridization (FISH), and the conditions for the single-mismatch discrimination of the probes were optimized. The specificity of the probes was improved further using a singly mismatched oligonucleotide as a competitor. The growth of Alcanivorax cells in crude oil-contaminated sea water under the biostimulation condition was investigated by FISH with the probe ALV735, which targeted the main cluster of the Alcanivorax/Fundibacter group. The size of the Alcanivorax population increased with increasing incubation time and accounted for 91% of the 4',6-diamidino-2-phenylindole (DAPI) count after incubation for 2 weeks. The probes developed in this study are useful for detecting Alcanivorax populations in petroleum hydrocarbon-degrading microbial consortia.

Assessment of copy number variations in 120 patients with Poland syndrome.

Science.gov (United States)

Vaccari, Carlotta Maria; Tassano, Elisa; Torre, Michele; Gimelli, Stefania; Divizia, Maria Teresa; Romanini, Maria Victoria; Bossi, Simone; Musante, Ilaria; Valle, Maura; Senes, Filippo; Catena, Nunzio; Bedeschi, Maria Francesca; Baban, Anwar; Calevo, Maria Grazia; Acquaviva, Massimo; Lerone, Margherita; Ravazzolo, Roberto; Puliti, Aldamaria

2016-11-25

Poland Syndrome (PS) is a rare congenital disorder presenting with agenesis/hypoplasia of the pectoralis major muscle variably associated with thoracic and/or upper limb anomalies. Most cases are sporadic, but familial recurrence, with different inheritance patterns, has been observed. The genetic etiology of PS remains unknown. Karyotyping and array-comparative genomic hybridization (CGH) analyses can identify genomic imbalances that can clarify the genetic etiology of congenital and neurodevelopmental disorders. We previously reported a chromosome 11 deletion in twin girls with pectoralis muscle hypoplasia and skeletal anomalies, and a chromosome six deletion in a patient presenting a complex phenotype that included pectoralis muscle hypoplasia. However, the contribution of genomic imbalances to PS remains largely unknown. To investigate the prevalence of chromosomal imbalances in PS, standard cytogenetic and array-CGH analyses were performed in 120 PS patients. Following the application of stringent filter criteria, 14 rare copy number variations (CNVs) were identified in 14 PS patients in different regions outside known common copy number variations: seven genomic duplications and seven genomic deletions, enclosing the two previously reported PS associated chromosomal deletions. These CNVs ranged from 0.04 to 4.71 Mb in size. Bioinformatic analysis of array-CGH data indicated gene enrichment in pathways involved in cell-cell adhesion, DNA binding and apoptosis processes. The analysis also provided a number of candidate genes possibly causing the developmental defects observed in PS patients, among others REV3L, a gene coding for an error-prone DNA polymerase previously associated with Möbius Syndrome with variable phenotypes including pectoralis muscle agenesis. A number of rare CNVs were identified in PS patients, and these involve genes that represent candidates for further evaluation. Rare inherited CNVs may contribute to, or represent risk factors of PS

Identification of a novel 15.5 kb SHOX deletion associated with ...

Indian Academy of Sciences (India)

ANGELOS ALEXANDROU

deletion, encompassing exons 3–6, was initially detected by array-CGH, followed by MLPA analysis. Sequencing of ... The proband, a female, second child of four children was born to ... at the age of 43 years, she had an occipitofrontal circumfer- ence (ofc) of ..... obox gene cause growth failure in idiopathic short stature and.

Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

Science.gov (United States)

Turner, Helen C; Budak, Murat T; Akinci, M A Murat; Wolosin, J Mario

2007-05-01

To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that

2018-05-20T19:10:55Z https://www.ajol.info/index.php/all/oai oai:ojs ...

African Journals Online (AJOL)

... array, Oligonucleotide array The completion of whole genome sequencing projects has led to a rapid increase in the availability of genetic information. ... of the most important technology in the field of molecular biology and transcriptomics.

A regenerated electrochemical biosensor for label-free detection of glucose and urea based on conformational switch of i-motif oligonucleotide probe

Energy Technology Data Exchange (ETDEWEB)

Gao, Zhong Feng; Chen, Dong Mei [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Lei, Jing Lei [School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China); Luo, Hong Qun, E-mail: luohq@swu.edu.cn [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Li, Nian Bing, E-mail: linb@swu.edu.cn [Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

2015-10-15

Improving the reproducibility of electrochemical signal remains a great challenge over the past decades. In this work, i-motif oligonucleotide probe-based electrochemical DNA (E-DNA) sensor is introduced for the first time as a regenerated sensing platform, which enhances the reproducibility of electrochemical signal, for label-free detection of glucose and urea. The addition of glucose or urea is able to activate glucose oxidase-catalyzed or urease-catalyzed reaction, inducing or destroying the formation of i-motif oligonucleotide probe. The conformational switch of oligonucleotide probe can be recorded by electrochemical impedance spectroscopy. Thus, the difference of electron transfer resistance is utilized for the quantitative determination of glucose and urea. We further demonstrate that the E-DNA sensor exhibits high selectivity, excellent stability, and remarkable regenerated ability. The human serum analysis indicates that this simple and regenerated strategy holds promising potential in future biosensing applications. - Highlights: • Conformational switch of i-motif is used for the detection of glucose and urea. • The sensor can be regenerated. • The proposed method is successfully applied in real sample assay. • Our method is label-free and inexpensive.

Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay.

Science.gov (United States)

Godinho, Bruno M D C; Gilbert, James W; Haraszti, Reka A; Coles, Andrew H; Biscans, Annabelle; Roux, Loic; Nikan, Mehran; Echeverria, Dimas; Hassler, Matthew; Khvorova, Anastasia

2017-12-01

Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), hold great promise for the treatment of incurable genetically defined disorders by targeting cognate toxic gene products for degradation. To achieve meaningful tissue distribution and efficacy in vivo, siRNAs must be conjugated or formulated. Clear understanding of the pharmacokinetic (PK)/pharmacodynamic behavior of these compounds is necessary to optimize and characterize the performance of therapeutic oligonucleotides in vivo. In this study, we describe a simple and reproducible methodology for the evaluation of in vivo blood/plasma PK profiles and tissue distribution of oligonucleotides. The method is based on serial blood microsampling from the saphenous vein, coupled to peptide nucleic acid hybridization assay for quantification of guide strands. Performed with minimal number of animals, this method allowed unequivocal detection and sensitive quantification without the need for amplification, or further modification of the oligonucleotides. Using this methodology, we compared plasma clearances and tissue distribution profiles of two different hydrophobically modified siRNAs (hsiRNAs). Notably, cholesterol-hsiRNA presented slow plasma clearances and mainly accumulated in the liver, whereas, phosphocholine-docosahexaenoic acid-hsiRNA was rapidly cleared from the plasma and preferably accumulated in the kidney. These data suggest that the PK/biodistribution profiles of modified hsiRNAs are determined by the chemical nature of the conjugate. Importantly, the method described in this study constitutes a simple platform to conduct pilot assessments of the basic clearance and tissue distribution profiles, which can be broadly applied for evaluation of new chemical variants of siRNAs and micro-RNAs.

Sox9 duplications are a relevant cause of Sry-negative XX sex reversal dogs.

Science.gov (United States)

Rossi, Elena; Radi, Orietta; De Lorenzi, Lisa; Vetro, Annalisa; Groppetti, Debora; Bigliardi, Enrico; Luvoni, Gaia Cecilia; Rota, Ada; Camerino, Giovanna; Zuffardi, Orsetta; Parma, Pietro

2014-01-01

Sexual development in mammals is based on a complicated and delicate network of genes and hormones that have to collaborate in a precise manner. The dark side of this pathway is represented by pathological conditions, wherein sexual development does not occur properly either in the XX and the XY background. Among them a conundrum is represented by the XX individuals with at least a partial testis differentiation even in absence of SRY. This particular condition is present in various mammals including the dog. Seven dogs characterized by XX karyotype, absence of SRY gene, and testicular tissue development were analysed by Array-CGH. In two cases the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9. The duplication contained the SOX9 coding region. In this work we provide for the first time a causative mutation for the XXSR condition in the dog. Moreover this report supports the idea that the dog represents a good animal model for the study of XXSR condition caused by abnormalities in the SOX9 locus.

Sox9 duplications are a relevant cause of Sry-negative XX sex reversal dogs.

Directory of Open Access Journals (Sweden)

Elena Rossi

Full Text Available Sexual development in mammals is based on a complicated and delicate network of genes and hormones that have to collaborate in a precise manner. The dark side of this pathway is represented by pathological conditions, wherein sexual development does not occur properly either in the XX and the XY background. Among them a conundrum is represented by the XX individuals with at least a partial testis differentiation even in absence of SRY. This particular condition is present in various mammals including the dog. Seven dogs characterized by XX karyotype, absence of SRY gene, and testicular tissue development were analysed by Array-CGH. In two cases the array-CGH analysis detected an interstitial heterozygous duplication of chromosome 9. The duplication contained the SOX9 coding region. In this work we provide for the first time a causative mutation for the XXSR condition in the dog. Moreover this report supports the idea that the dog represents a good animal model for the study of XXSR condition caused by abnormalities in the SOX9 locus.

Genomic profiling using array comparative genomic hybridization define distinct subtypes of diffuse large b-cell lymphoma: a review of the literature

Directory of Open Access Journals (Sweden)

Tirado Carlos A

2012-09-01

Full Text Available Abstract Diffuse large B-cell lymphoma (DLBCL is the most common type of non-Hodgkin Lymphoma comprising of greater than 30% of adult non-Hodgkin Lymphomas. DLBCL represents a diverse set of lymphomas, defined as diffuse proliferation of large B lymphoid cells. Numerous cytogenetic studies including karyotypes and fluorescent in situ hybridization (FISH, as well as morphological, biological, clinical, microarray and sequencing technologies have attempted to categorize DLBCL into morphological variants, molecular and immunophenotypic subgroups, as well as distinct disease entities. Despite such efforts, most lymphoma remains undistinguishable and falls into DLBCL, not otherwise specified (DLBCL-NOS. The advent of microarray-based studies (chromosome, RNA, gene expression, etc has provided a plethora of high-resolution data that could potentially facilitate the finer classification of DLBCL. This review covers the microarray data currently published for DLBCL. We will focus on these types of data; 1 array based CGH; 2 classical CGH; and 3 gene expression profiling studies. The aims of this review were three-fold: (1 to catalog chromosome loci that are present in at least 20% or more of distinct DLBCL subtypes; a detailed list of gains and losses for different subtypes was generated in a table form to illustrate specific chromosome loci affected in selected subtypes; (2 to determine common and distinct copy number alterations among the different subtypes and based on this information, characteristic and similar chromosome loci for the different subtypes were depicted in two separate chromosome ideograms; and, (3 to list re-classified subtypes and those that remained indistinguishable after review of the microarray data. To the best of our knowledge, this is the first effort to compile and review available literatures on microarray analysis data and their practical utility in classifying DLBCL subtypes. Although conventional cytogenetic methods such

Electrical manipulation of oligonucleotides grafted to charged surfaces.

Science.gov (United States)

Rant, Ulrich; Arinaga, Kenji; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard; Tornow, Marc

2006-09-21

The electrical manipulation of short DNA molecules on surfaces offers novel functionalities with fascinating possibilities in the field of bio-interfaces. Here we present systematic investigations of the electrical interactions which govern the structure of oligonucleotides on charged gold surfaces. Successively, we address influences of the applied field strength, the role of DC electrode potentials, in particular for polycrystalline surfaces, as well as screening effects of the surrounding electrolyte solution. Data obtained for single and double stranded DNA exhibit differences which can be attributed to the dissimilar flexibility of the different molecular conformations. A comparison of the experimental results with a basic model shows how the alignment of the molecules adjusts according to a balance between electrically induced ordering and stochastic thermal motions. The presented conclusions are expected to be of general relevance for the behaviour of polyelectrolytes exposed to localized electric fields at interfaces.

Clinical assessment of five patients with BRWD3 mutation at Xq21.1 gives further evidence for mild to moderate intellectual disability and macrocephaly

NARCIS (Netherlands)

Grotto, S.; Drouin-Garraud, V.; Ounap, K.; Puusepp-Benazzouz, H.; Schuurs-Hoeijmakers, J.H.; Meur, N. Le; Chambon, P.; Fehrenbach, S.; Bokhoven, H. van; Frebourg, T.; Brouwer, A.P.M. de; Saugier-Veber, P.

2014-01-01

Truncating mutations of the BRWD3 gene have been reported in two distinct families with in total four patients so far. By using array-CGH, we detected a 74 Kb de novo deletion encompassing exons 11 through 41 of BRWD3 at Xq21.1 in a 20 year old boy presenting with syndromic intellectual disability.

Pretreatment of Mice with Oligonucleotide prop5 Protects Them from Influenza Virus Infections

Directory of Open Access Journals (Sweden)

Kang Li

2014-02-01

Full Text Available Influenza A virus is a successful parasite and requires host factors to complete its life cycle. Prop5 is an antisense oligonucleotide, targeting programmed cell death protein 5 (PDCD5. In this study, we tested the antiviral activity of prop5 against mouse-adapted A/FM/1/47 strain of influenza A virus in a mouse model. Prop5 intranasally administered the mice at dosages of 10 and 20 mg/kg/d at 24 h and 30 min before infection, provided 80% and 100% survival rates and prolonged mean survival days in comparison with influenza virus-infected mice (both p < 0.01. Moreover, viral titres in mice pretreated with prop5, at dose of 10 and 20 mg/kg/d, had declined significantly on day two, four, and six post-infection compared with the yields in infected mice (p < 0.05 or p < 0.01; lung index in mice pretreated with prop5 (20 mg/kg/d had been inhibited on day six post-infection (p < 0.05. Western blotting and immunohistochemistry showed that prop5 could down-regulate the PDCD5 protein expression levels in lung tissues of infected mice. These data indicate that antisense oligonucleotide prop5 is a promising drug for prophylaxis and control influenza virus infections and provides an insight into the host-pathogen interaction.

Development of a general methodology for labelling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistry.

Science.gov (United States)

Shabanpoor, Fazel; Gait, Michael J

2013-11-11

We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.

Preliminary analysis of numerical chromosome abnormalities in reciprocal and Robertsonian translocation preimplantation genetic diagnosis cases with 24-chromosomal analysis with an aCGH/SNP microarray.

Science.gov (United States)

Xie, Yanxin; Xu, Yanwen; Wang, Jing; Miao, Benyu; Zeng, Yanhong; Ding, Chenhui; Gao, Jun; Zhou, Canquan

2018-01-01

The aim of this study was to determine whether an interchromosomal effect (ICE) occurred in embryos obtained from reciprocal translocation (rcp) and Robertsonian translocation (RT) carriers who were following a preimplantation genetic diagnosis (PGD) with whole chromosome screening with an aCGH and SNP microarray. We also analyzed the chromosomal numerical abnormalities in embryos with aneuploidy in parental chromosomes that were not involved with a translocation and balanced in involved parental translocation chromosomes. This retrospective study included 832 embryos obtained from rcp carriers and 382 embryos from RT carriers that were biopsied in 139 PGD cycles. The control group involved embryos obtained from age-matched patient karyotypes who were undergoing preimplantation genetic screening (PGS) with non-translocation, and 579 embryos were analyzed in the control group. A single blastomere at the cleavage stage or trophectoderm from a blastocyst was biopsied, and 24-chromosomal analysis with an aCGH/SNP microarray was conducted using the PGD/PGS protocols. Statistical analyses were implemented on the incidences of cumulative aneuploidy rates between the translocation carriers and the control group. Reliable results were obtained from 138 couples, among whom only one patient was a balanced rcp or RT translocation carrier, undergoing PGD testing in our center from January 2012 to June 2014. For day 3 embryos, the aneuploidy rates were 50.7% for rcp carriers and 49.1% for RT carriers, compared with the control group, with 44.8% at a maternal age < 36 years. When the maternal age was ≥ 36 years, the aneuploidy rates were increased to 61.1% for rcp carriers, 56.7% for RT carriers, and 60.3% for the control group. There were no significant differences. In day 5 embryos, the aneuploidy rates were 24.5% for rcp carriers and 34.9% for RT carriers, compared with the control group with 53.6% at a maternal age < 36 years. When the maternal age was ≥ 36�

Detection of chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes

International Nuclear Information System (INIS)

Lloyd, R.V.; Jin, L.; Fields, K.

1990-01-01

We analyzed the distribution of chromogranins A and B in normal and neoplastic endocrine tissues with secretory granules using 35 S-labeled and biotin-labeled oligonucleotide probes by in situ hybridization (ISH). Both radioactive and nonradioactive probes detected messenger RNAs (mRNAs) in frozen and paraffin tissue sections. Endocrine tissues with variable immunoreactivities for chromogranin A protein, such as small-cell lung carcinomas, neuroblastomas, insulinomas, and parathyroid adenomas, expressed the mRNA for chromogranins A and B in most cells. Some technical problems with the biotinylated probes included nonspecific nuclear staining and endogenous alkaline phosphatase, which was not completely abolished by levamisole pretreatment. A differential distribution of chromogranins A and B was seen in pituitary prolactinomas, which expressed abundant chromogranin B but not chromogranin A mRNAs, and in parathyroid adenomas, which expressed abundant chromogranin A but only small amounts of chromogranin B mRNAs. These results indicate that ISH can be used to detect chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes and that the mRNAs for chromogranin A and B are demonstrable in some tumors even when the chromogranin proteins cannot be detected by immunohistochemistry

Detection of chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes

Energy Technology Data Exchange (ETDEWEB)

Lloyd, R.V.; Jin, L.; Fields, K. (Univ. of Michigan, Ann Arbor (USA))

1990-01-01

We analyzed the distribution of chromogranins A and B in normal and neoplastic endocrine tissues with secretory granules using {sup 35}S-labeled and biotin-labeled oligonucleotide probes by in situ hybridization (ISH). Both radioactive and nonradioactive probes detected messenger RNAs (mRNAs) in frozen and paraffin tissue sections. Endocrine tissues with variable immunoreactivities for chromogranin A protein, such as small-cell lung carcinomas, neuroblastomas, insulinomas, and parathyroid adenomas, expressed the mRNA for chromogranins A and B in most cells. Some technical problems with the biotinylated probes included nonspecific nuclear staining and endogenous alkaline phosphatase, which was not completely abolished by levamisole pretreatment. A differential distribution of chromogranins A and B was seen in pituitary prolactinomas, which expressed abundant chromogranin B but not chromogranin A mRNAs, and in parathyroid adenomas, which expressed abundant chromogranin A but only small amounts of chromogranin B mRNAs. These results indicate that ISH can be used to detect chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes and that the mRNAs for chromogranin A and B are demonstrable in some tumors even when the chromogranin proteins cannot be detected by immunohistochemistry.

Oligonucleotide Length-Dependent Formation of Virus-Like Particles.

Science.gov (United States)

Maassen, Stan J; de Ruiter, Mark V; Lindhoud, Saskia; Cornelissen, Jeroen J L M

2018-05-23

Understanding the assembly pathway of viruses can contribute to creating monodisperse virus-based materials. In this study, the cowpea chlorotic mottle virus (CCMV) is used to determine the interactions between the capsid proteins of viruses and their cargo. The assembly of the capsid proteins in the presence of different lengths of short, single-stranded (ss) DNA is studied at neutral pH, at which the protein-protein interactions are weak. Chromatography, electrophoresis, microscopy, and light scattering data show that the assembly efficiency and speed of the particles increase with increasing length of oligonucleotides. The minimal length required for assembly under the conditions used herein is 14 nucleotides. Assembly of particles containing such short strands of ssDNA can take almost a month. This slow assembly process enabled the study of intermediate states, which confirmed a low cooperative assembly for CCMV and allowed for further expansion of current assembly theories. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Information Limited Oligonucleotide Amplification Assay for Affinity-Based, Parallel Detection Studies.

Directory of Open Access Journals (Sweden)

Harish Bokkasam

Full Text Available Molecular communication systems encounter similar constraints as telecommunications. In either case, channel crosstalk at the receiver end will result in information loss that statistical analysis cannot compensate. This is because in any communication channel there is a physical limit to the amount of information that can be transmitted. We present a novel and simple modified end amplification (MEA technique to generate reduced and defined amounts of specific information in form of short fragments from an oligonucleotide source that also contains unrelated and redundant information. Our method can be a valuable tool to investigate information overflow and channel capacity in biomolecular recognition systems.

Detection of autoimmune antibodies in localized scleroderma by synthetic oligonucleotide antigens

DEFF Research Database (Denmark)

Samuelsen, Simone; Jørgensen, Christian Damsgaard; Mellins, Elizabeth D

2018-01-01

In this study, we developed a series of synthetic oligonucleotides that allowed us to investigate the details on the antigen recognition by autoimmune antibodies in localized scleroderma subjects. Besides dramatically improved analytical specificity of the assay, our data suggests a potential...... linking for antibodies to DNA to the biological status of disease state in localized scleroderma. Moreover, introducing chemical modifications into short synthetic deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules completely changed the binding titers of corresponding antibodies...... and their clinical relevance. The strongest observed effect was registered for the localized scleroderma skin damage index (LoSDI) on the IgG antibodies to TC dinucleotide-rich double-stranded antigen (p

Detection of KatG Gen Mutation on Mycobacterium Tuberculosis by Means of PCR-Dot Blot Hybridization with 32P Labeled Oligonucleotide Probe Methods

International Nuclear Information System (INIS)

Maria Lina R; Budiman Bela; Andi Yasmon

2009-01-01

Handling and controlling of tuberculosis, a disease caused by Mycobacterium tuberculosis (MTB), is now complicated since there are many MTBs that are resistant against anti-tuberculosis drugs such as isoniazid. The drug resistance could occurred due to the inadequate and un-regular drug utilization that cause gene mutation of the drug target such as katG gene for isoniazid. The molecular biology techniques such as the PCR- dot blot hybridization with radioisotope ( 32 P) labeled oligonucleotide probe, has been reported as a technique that is more sensitive and rapid for detection of gene mutations related with drug resistances. Hence, the aim of this study was to apply the PCR- dot blot hybridization technique using 32 P labeled oligonucleotide probe for detection of single mutation at codon 315 of katG gene of MTBs that rise the isoniazid resistance. In this study, we used 89 sputum specimens and a standard MTB (MTB H 37 RV) as a control. DNA extractions were performed by the BOOM method and the phenol chloroform for sputum samples and standard MTB, respectively. Primers used for PCR technique were Pt8 and Pt9 and RTB59 and RTB36 for detecting tuberculosis causing Mycobacterium and the existence of katG gene, respectively. Both of the primers are specific for IS6110 region and katG gene, respectively. PCR products were detected by an agarose gel electrophoresis technique. Dot blot hybridization with 32 P-oligonucleotide probe 315mu was performed to detect mutation at codon 315 of tested samples. Results of the PCR using primer Pt8 and Pt9 showed that all sputum specimens had positive results. Mutation detection by PCR- dot blot hybridization with 32 P-oligonucleotide probe 315mu, revealed that 11 of 89 tested samples had a mutation at their codon 315 of katG gene. Based upon these results, it is concluded that PCR-dot blot hybridization with 32 P-oligonucleotide probe is a technique that is rapid and highly specific and sensitive for detection of mutation at codon

Case report of newborn with de novo partial trisomy 2q31.2–37.3 ...

Indian Academy of Sciences (India)

MAURIZIA COLANGELO

2018-02-24

Feb 24, 2018 ... Keywords. duplication of 2q31.2 and 2q37.3; monosomy 9p; array CGH. Introduction ... of gestation from a 39-year old Caucasian female. She is the second child of healthy and nonconsanguineous par- ents. She has a healthy ..... growth with intellectual disability, while duplications dis- tal to 2q33 show a ...

Plume characteristics and dynamics of UV and IR laser-desorbed oligonucleotides.

Science.gov (United States)

Merrigan, Tony L; Timson, David J; Hunniford, C Adam; Catney, Martin; McCullough, Robert W

2012-05-01

Laser desorption of dye-tagged oligonucleotides was studied using laser-induced fluorescence imaging. Desorption with ultra violet (UV) and infra-red (IR) lasers resulted in forward directed plumes of molecules. In the case of UV desorption, the initial shot desorbed approximately seven-fold more material than subsequent shots. In contrast, the initial shot in IR desorption resulted in the ejection of less material compared to subsequent shots and these plumes had a component directed along the path of the laser. Thermal equilibrium of the molecules in the plume was achieved after approximately 25 μs with a spread in molecular temperature which was described by a modified Maxwell-Boltzmann equation. Copyright © 2012 Elsevier B.V. All rights reserved.

Rapid aneuploidy diagnosis by multiplex ligation-dependent probe amplification and array comparative genomic hybridization in pregnancy with major congenital malformations

Directory of Open Access Journals (Sweden)

Chih-Ping Chen

2011-03-01

Conclusions: Prenatal diagnosis of major congenital malformations should alert one to the possibility of chromosomal abnormalities. Multiplex ligation-dependent probe amplification and aCGH have the advantage of rapid aneuploidy diagnosis of common aneuploidies in cases with major congenital malformations.

A non-covalent peptide-based strategy for ex vivo and in vivo oligonucleotide delivery.

Science.gov (United States)

Crombez, Laurence; Morris, May C; Heitz, Frederic; Divita, Gilles

2011-01-01

The dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide-nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy based on short amphiphatic peptides (MPG8/PEP3) that have been successfully applied ex vivo and in vivo for the delivery of therapeutic siRNA and PNA molecules. PEP3 and MPG8 form stable nanoparticles with PNA analogues and siRNA, respectively, and promote their efficient cellular uptake, independently of the endosomal pathway, into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. This chapter describes easy-to-handle protocols for the use of MPG-8 or PEP-3-nanoparticle technologies for PNA and siRNA delivery into adherent and suspension cell lines as well as in vivo into cancer mouse models.

Focal plane array with modular pixel array components for scalability

Science.gov (United States)

Kay, Randolph R; Campbell, David V; Shinde, Subhash L; Rienstra, Jeffrey L; Serkland, Darwin K; Holmes, Michael L

2014-12-09

A modular, scalable focal plane array is provided as an array of integrated circuit dice, wherein each die includes a given amount of modular pixel array circuitry. The array of dice effectively multiplies the amount of modular pixel array circuitry to produce a larger pixel array without increasing die size. Desired pixel pitch across the enlarged pixel array is preserved by forming die stacks with each pixel array circuitry die stacked on a separate die that contains the corresponding signal processing circuitry. Techniques for die stack interconnections and die stack placement are implemented to ensure that the desired pixel pitch is preserved across the enlarged pixel array.

Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes

DEFF Research Database (Denmark)

Astakhova, I Kira; Wengel, Jesper

2013-01-01

Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little...

Hammerhead ribozyme activity and oligonucleotide duplex stability in mixed solutions of water and organic compounds

Directory of Open Access Journals (Sweden)

Shu-ichi Nakano

2014-01-01

Full Text Available Nucleic acids are useful for biomedical targeting and sensing applications in which the molecular environment is different from that of a dilute aqueous solution. In this study, the influence of various types of mixed solutions of water and water-soluble organic compounds on RNA was investigated by measuring the catalytic activity of the hammerhead ribozyme and the thermodynamic stability of an oligonucleotide duplex. The compounds with a net neutral charge, such as poly(ethylene glycol, small primary alcohols, amide compounds, and aprotic solvent molecules, added at high concentrations changed the ribozyme-catalyzed RNA cleavage rate, with the magnitude of the effect dependent on the NaCl concentration. These compounds also changed the thermodynamic stability of RNA base pairs of an oligonucleotide duplex and its dependence on the NaCl concentration. Specific interactions with RNA molecules and reduced water activity could account for the inhibiting effects on the ribozyme catalysis and destabilizing effects on the duplex stability. The salt concentration dependence data correlated with the dielectric constant, but not with water activity, viscosity, and the size of organic compounds. This observation suggests the significance of the dielectric constant effects on the RNA reactions under molecular crowding conditions created by organic compounds.

Chemical Ligation Reactions of Oligonucleotides for Biological and Medicinal Applications.

Science.gov (United States)

Abe, Hiroshi; Kimura, Yasuaki

2018-01-01

Chemical ligation of oligonucleotides (ONs) is the key reaction for various ON-based technologies. We have tried to solve the problems of RNA interference (RNAi) technology by applying ON chemical ligation to RNAi. We designed a new RNAi system, called intracellular buildup RNAi (IBR-RNAi), where the RNA fragments are built up into active small-interference RNA (siRNA) in cells through a chemical ligation reaction. Using the phosphorothioate and iodoacetyl groups as reactive functional groups for the ligation, we achieved RNAi effects without inducing immune responses. Additionally, we developed a new chemical ligation for IBR-RNAi, which affords a more native-like structure in the ligated product. The new ligation method should be useful not only for IBR-RNAi but also for the chemical synthesis of biofunctional ONs.

Timed arrays wideband and time varying antenna arrays

CERN Document Server

Haupt, Randy L

2015-01-01

Introduces timed arrays and design approaches to meet the new high performance standards The author concentrates on any aspect of an antenna array that must be viewed from a time perspective. The first chapters briefly introduce antenna arrays and explain the difference between phased and timed arrays. Since timed arrays are designed for realistic time-varying signals and scenarios, the book also reviews wideband signals, baseband and passband RF signals, polarization and signal bandwidth. Other topics covered include time domain, mutual coupling, wideband elements, and dispersion. The auth

Chromosome 12q24.31-q24.33 deletion causes multiple dysmorphic features and developmental delay: First mosaic patient and overview of the phenotype related to 12q24qter defects

Directory of Open Access Journals (Sweden)

Sakati Nadia

2011-04-01

Full Text Available Abstract Background Genomic imbalances of the 12q telomere are rare; only a few patients having 12q24.31-q24.33 deletions were reported. Interestingly none of these were mosaic. Although some attempts have been made to establish phenotype/genotype interaction for the deletions in this region, no clear relationship has been established to date. Results We have clinically screened more than 100 patients with dysmorphic features, mental retardation and normal karyotype using high density oligo array-CGH (aCGH and identified a ~9.2 Mb hemizygous interstitial deletion at the 12q telomere (Chromosome 12: 46,XY,del(12(q24.31q24.33 in a severely developmentally retarded patient having dysmorphic features such as low set ears, microcephaly, undescended testicles, bent elbow, kyphoscoliosis, and micropenis. Parents were found to be not carriers. MLPA experiments confirmed the aCGH result. Interphase FISH revealed mosaicism in cultured peripheral blood lymphocytes. Conclusions Since conventional G-Banding technique missed the abnormality; this work re-confirms that any child with unexplained developmental delay and systemic involvement should be studied by aCGH techniques. The FISH technique, however, would still be useful to further delineate the research work and identify such rare mosaicism. Among the 52 deleted genes, P2RX2, ULK1, FZD10, RAN, NCOR2 STX2, TESC, FBXW8, and TBX3 are noteworthy since they may have a role in observed phenotype.

[Comparative genomic hybridisation as a first option in genetic diagnosis: 1,000 cases and a cost-benefit analysis].

Science.gov (United States)

Castells-Sarret, Neus; Cueto-González, Anna M; Borregan, Mar; López-Grondona, Fermina; Miró, Rosa; Tizzano, Eduardo; Plaja, Alberto

2017-09-25

Conventional cytogenetics diagnoses 3-5% of patients with unexplained developmental delay/intellectual disability and/or multiple congenital anomalies. The Multiplex Ligation-dependent Probe Amplification increases diagnostic rates from between 2.4 to 5.8%. Currently the comparative genomic hybridisation array or aCGH is the highest performing diagnostic tool in patients with developmental delay/intellectual disability, congenital anomalies and autism spectrum disorders. Our aim is to evaluate the efficiency of the use of aCGH as first-line test in these and other indications (epilepsy, short stature). A total of 1000 patients referred due to one or more of the abovementioned disorders were analysed by aCGH. Pathogenic genomic imbalances were detected in 14% of the cases, with a variable distribution of diagnosis according to the phenotypes: 18.9% of patients with developmental delay/intellectual disability; 13.7% of multiple congenital anomalies, 9.76% of psychiatric pathologies, 7.02% of patients with epilepsy, and 13.3% of patients with short stature. Within the multiple congenital anomalies, central nervous system abnormalities and congenital heart diseases accounted for 14.9% and 10.6% of diagnoses, respectively. Among the psychiatric disorders, patients with autism spectrum disorders accounted for 8.9% of the diagnoses. Our results demonstrate the effectiveness and efficiency of the use of aCGH as the first line test in genetic diagnosis of patients suspected of genomic imbalances, supporting its inclusion within the National Health System. Copyright © 2017. Publicado por Elsevier España, S.L.U.

Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy

Science.gov (United States)

Shabanpoor, Fazel; Hammond, Suzan M; Abendroth, Frank; Hazell, Gareth; Wood, Matthew J.A.

2017-01-01

Splice-switching antisense oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood–brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141–150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases. PMID:28118087

New approach to the determination phosphorothioate oligonucleotides by ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry.

Science.gov (United States)

Studzińska, Sylwia; Mounicou, Sandra; Szpunar, Joanna; Łobiński, Ryszard; Buszewski, Bogusław

2015-01-15

This text presents a novel method for the separation and detection of phosphorothioate oligonucleotides with the use of ion pair ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry The research showed that hexafluoroisopropanol/triethylamine based mobile phases may be successfully used when liquid chromatography is coupled with such elemental detection. However, the concentration of both HFIP and TEA influences the final result. The lower concentration of HFIP, the lower the background in ICP-MS and the greater the sensitivity. The method applied for the analysis of serum samples was based on high resolution inductively coupled plasma mass spectrometry. Utilization of this method allows determination of fifty times lower quantity of phosphorothioate oligonucleotides than in the case of quadrupole mass analyzer. Monitoring of (31)P may be used to quantify these compounds at the level of 80 μg L(-1), while simultaneous determination of sulfur is very useful for qualitative analysis. Moreover, the results presented in this paper demonstrate the practical applicability of coupling LC with ICP-MS in determining phosphorothioate oligonucleotides and their metabolites in serum within 7 min with a very good sensitivity. The method was linear in the concentration range between 0.2 and 3 mg L(-1). The limit of detection was in the range of 0.07 and 0.13 mg L(-1). Accuracy varied with concentration, but was in the range of 3%. Copyright © 2014 Elsevier B.V. All rights reserved.

Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

Science.gov (United States)

Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Natkanska, Urszula; Kwiatkowska, Aleksandra; Rawska, Ewa; Potocki, Leszek; Kuna, Ewelina; Panek, Anita; Lewinska, Anna; Wnuk, Maciej

2016-09-01

The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement.

ArrayBridge: Interweaving declarative array processing with high-performance computing

Energy Technology Data Exchange (ETDEWEB)

Xing, Haoyuan [The Ohio State Univ., Columbus, OH (United States); Floratos, Sofoklis [The Ohio State Univ., Columbus, OH (United States); Blanas, Spyros [The Ohio State Univ., Columbus, OH (United States); Byna, Suren [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Prabhat, Prabhat [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Wu, Kesheng [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Brown, Paul [Paradigm4, Inc., Waltham, MA (United States)

2017-05-04

Scientists are increasingly turning to datacenter-scale computers to produce and analyze massive arrays. Despite decades of database research that extols the virtues of declarative query processing, scientists still write, debug and parallelize imperative HPC kernels even for the most mundane queries. This impedance mismatch has been partly attributed to the cumbersome data loading process; in response, the database community has proposed in situ mechanisms to access data in scientific file formats. Scientists, however, desire more than a passive access method that reads arrays from files. This paper describes ArrayBridge, a bi-directional array view mechanism for scientific file formats, that aims to make declarative array manipulations interoperable with imperative file-centric analyses. Our prototype implementation of ArrayBridge uses HDF5 as the underlying array storage library and seamlessly integrates into the SciDB open-source array database system. In addition to fast querying over external array objects, ArrayBridge produces arrays in the HDF5 file format just as easily as it can read from it. ArrayBridge also supports time travel queries from imperative kernels through the unmodified HDF5 API, and automatically deduplicates between array versions for space efficiency. Our extensive performance evaluation in NERSC, a large-scale scientific computing facility, shows that ArrayBridge exhibits statistically indistinguishable performance and I/O scalability to the native SciDB storage engine.

[Inheritable phenotypic normalization of rodent cells transformed by simian adenovirus SA7 E1 oncogenes by singled-stranded oligonucleotides complementary to a long region of integrated oncogenes].

Science.gov (United States)

Grineva, N I; Borovkova, T V; Sats, N V; Kurabekova, R M; Rozhitskaia, O S; Solov'ev, G Ia; Pantin, V I

1995-08-01

G11 mouse cells and SH2 rat cells transformed with simian adenovirus SA7 DNA showed inheritable oncogen-specific phenotypic normalization when treated with sense and antisense oligonucleotides complementary to long RNA sequences, plus or minus strands of the integrated adenovirus oncogenes E1A and E1B. Transitory treatment of the cells with the oligonucleotides in the absence of serum was shown to cause the appearance of normalized cell lines with fibroblastlike morphology, slower cell proliferation, and lack of ability to form colonies in soft agar. Proliferative activity and adhesion of the normalized cells that established cell lines were found to depend on the concentration of growth factors in the cultural medium. In some of the cell lines, an inhibition of transcription of the E1 oncogenes was observed. The normalization also produced cells that divided 2 - 5 times and died and cells that reverted to a transformed phenotype in 2 - 10 days. The latter appeared predominantly upon the action of the antisense oligonucleotides.

Feasibility of SPECT-CT imaging to study the pharmacokinetics of antisense oligonucleotides in a mouse model of Duchenne muscular dystrophy

NARCIS (Netherlands)

Steeg, E. van de; Läppchen, T.; Aguilera, B.; Jansen, H.T.; Muilwijk, D.; Vermue, R.; Hoorn, J.W. van der; Donato, K.; Rossin, R.; Visser, P.C. de; Vlaming, M.L.H.

2017-01-01

Antisense oligonucleotides (AONs) are promising candidates for treatment of Duchenne muscular dystrophy (DMD), a severe and progressive disease resulting in premature death. However, more knowledge on the pharmacokinetics of new AON drug candidates is desired for effective application in the clinic.

Comprehensive genetic assessment of the human embryo: can empiric application of microarray comparative genomic hybridization reduce multiple gestation rate by single fresh blastocyst transfer?

Science.gov (United States)

Sills, Eric Scott; Yang, Zhihong; Walsh, David J; Salem, Shala A

2012-09-01

The unacceptable multiple gestation rate currently associated with in vitro fertilization (IVF) would be substantially alleviated if the routine practice of transferring more than one embryo were reconsidered. While transferring a single embryo is an effective method to reduce the clinical problem of multiple gestation, rigid adherence to this approach has been criticized for negatively impacting clinical pregnancy success in IVF. In general, single embryo transfer is viewed cautiously by IVF patients although greater acceptance would result from a more effective embryo selection method. Selection of one embryo for fresh transfer on the basis of chromosomal normalcy should achieve the dual objective of maintaining satisfactory clinical pregnancy rates and minimizing the multiple gestation problem, because embryo aneuploidy is a major contributing factor in implantation failure and miscarriage in IVF. The initial techniques for preimplantation genetic screening unfortunately lacked sufficient sensitivity and did not yield the expected results in IVF. However, newer molecular genetic methods could be incorporated with standard IVF to bring the goal of single embryo transfer within reach. Aiming to make multiple embryo transfers obsolete and unnecessary, and recognizing that array comparative genomic hybridization (aCGH) will typically require an additional 12 h of laboratory time to complete, we propose adopting aCGH for mainstream use in clinical IVF practice. As aCGH technology continues to develop and becomes increasingly available at lower cost, it may soon be considered unusual for IVF laboratories to select a single embryo for fresh transfer without regard to its chromosomal competency. In this report, we provide a rationale supporting aCGH as the preferred methodology to provide a comprehensive genetic assessment of the single embryo before fresh transfer in IVF. The logistics and cost of integrating aCGH with IVF to enable fresh embryo transfer are also

Efficient assessment of modified nucleoside stability under conditions of automated oligonucleotide synthesis: characterization of the oxidation and oxidative desulfurization of 2-thiouridine.

Science.gov (United States)

Sochacka, E

2001-01-01

In order to efficiently assess the chemical stability of modified nucleosides to the reagents and conditions of automated oligonucleotide synthesis, we designed, developed and tested a scheme in which the modified nucleoside, directly attached to a solid support, is exposed to the cyclic chemistry of the instrument. Stability of 2-thiouridine against different oxidizers was investigated. Tertbutyl hydroperoxide (1 M) in anhydrous acetonitrile was a more effective oxidizer for the incorporation of 2-thiouridine into oligonucleotide chains than the same oxidizer in methylene chloride. Carbon tetrachloride/water in the presence of a basic catalyst was superior in maintaining the thiocarbonyl function, but its utility for RNA synthesis has yet to be fully tested, whereas 2-phenylsulfonyloxaziridine was a very efficient reagent for oxidative desulfurization of 2-thiouridine.

A New Achiral Linker Reagent for the Incorporation of Multiple Amino Groups Into Oligonucleotides

DEFF Research Database (Denmark)

1997-01-01

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......-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, texas red, tetramethyl rhodamine, 7-nitrobenzo-2-oxa-1-diazole (NBD), or pyrene. The present invention also relates to a solid phase support, e.g. a Controlled Pore Glass (CPG), immobilized linker reagent...

Association Mapping of Malting Quality Quantitative Trait Loci in Winter Barley: Positive Signals from Small Germplasm Arrays

Directory of Open Access Journals (Sweden)

Lucía Gutiérrez

2011-11-01

Full Text Available Malting quality comprises one of the most economically relevant set of traits in barley ( L.. It is a complex phenotype, expensive and difficult to measure, that would benefit from a marker-assisted selection strategy. Malting quality is a target of the U.S. Barley Coordinated Agricultural Project (CAP and development of winter habit malting barley varieties is a key objective of the U.S. barley research community. The objective of this work was to detect quantitative trait loci (QTL for malting quality traits in a winter breeding program that is a component of the U.S. Barley CAP. We studied the association between five malting quality traits and 3072 single nucleotide polymorphisms (SNPs from the barley oligonucleotide pool assay (BOPA 1 and 2, assayed in advanced inbred lines from the Oregon State University (OSU breeding program from three germplasm arrays (CAP I, CAP II, and CAP III. After comparing 16 models we selected a structured association model with posterior probabilities inferred from software STRUCTURE (QK approach to use on all germplasm arrays. Most of the marker-trait associations are germplasm- and environment-specific and close to previously mapped genes and QTL relevant for malt and beer quality. We found alleles fixed by random genetic drift, novel unmasked alleles, and genetic-background interaction. In a relatively small population size study we provide strong evidence for detecting true QTL.

Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization

Directory of Open Access Journals (Sweden)

Oikawa Masahiro

2011-12-01

Full Text Available Abstract Background It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN, which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH. Methods Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. Results The mean of the derivative log ratio spread (DLRSpread, which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05. The concordance of results between aCGH and fluorescence in situ hybridization (FISH for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively. The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15. Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40. Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005 independent factor which was associated with larger total length of CNA of breast cancers. Conclusions Thus, archival FFPE tissues from A-bomb survivors are useful for

Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization

International Nuclear Information System (INIS)

Oikawa, Masahiro; Yoshiura, Koh-ichiro; Kondo, Hisayoshi; Miura, Shiro; Nagayasu, Takeshi; Nakashima, Masahiro

2011-01-01

It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH). Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. The mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers. Thus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A

Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization.

Science.gov (United States)

Oikawa, Masahiro; Yoshiura, Koh-ichiro; Kondo, Hisayoshi; Miura, Shiro; Nagayasu, Takeshi; Nakashima, Masahiro

2011-12-07

It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH). Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. The mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers. Thus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A

Advancements of antisense oligonucleotides in treatment of breast cancer

Institute of Scientific and Technical Information of China (English)

YANGShuan-Ping; SONGSan-Tai; 等

2003-01-01

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 interference.one 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.

Application of a cholesterol stationary phase in the analysis of phosphorothioate oligonucleotides by means of ion pair chromatography coupled with tandem mass spectrometry.

Science.gov (United States)

Studzińska, Sylwia; Krzemińska, Katarzyna; Szumski, Michał; Buszewski, Bogusław

2016-07-01

The main aim of this study was the investigation of the influence of several ion pair reagents towards both the retention and the mass spectrometry sensitivity of phosphorothioate oligonucleotides. A cholesterol stationary phase was applied for the first time in the analysis of this group of compounds. The mobile phase composition was modified by changing the concentration and the type of amines and acetates or 1,1,1,3,3,3-hexafluoroisopropanol. It has been shown that the increase of amines concentration results in the retention factor increase for each oligonucleotide, on each adsorbent. The only exception was the mobile phase composed of triethylamine and 1,1,1,3,3,3-hexafluoroisopropanol. This is a consequence of interactions taking place between a cholesterol molecule and an alcohol. This effect was convenient when the mass spectrometry detection was applied, since it allowed an increase in the sensitivity. Moreover, optimization of the mobile phase composition and its impact on the efficiency of ionization process and on the sensitivity in mass spectrometry were also presented. The optimization of this new method, based on cholesterol stationary phase coupled with mass spectrometry detection, was finally applied for the determination of phosphorothioate oligonucleotides impurity in a real sample. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of Genetic Alterations in Breast Sentinel Lymph Node by Array-CGH

National Research Council Canada - National Science Library

Cavalli, Luciane R

2005-01-01

The sentinel lymph node (SLN) is the first node in the mammary gland to harbor malignant cells in breast tumors with metastasis, and SLN positivity is an indication for axillary lymph node dissection...

Detection of Genetic Alterations in Breast Sentinel Lymph Node by Array-CGH

National Research Council Canada - National Science Library

Cavalli, Luciane R

2006-01-01

The sentinel lymph node (SLN) is the first node in the mammary gland to harbor malignant cells in breast tumors with metastasis, and SLN positivity is an indication for axillary lymph node dissection...

The detection of platelet derived growth factor using decoupling of quencher-oligonucleotide from aptamer/quantum dot bioconjugates

International Nuclear Information System (INIS)

Kim, Gang-Il; Sung, Yun-Mo; Kim, Kyung-Woo; Oh, Min-Kyu

2009-01-01

High-sensitivity, high-specificity detection of platelet derived growth factor (PDGF)-BB was realized using the change in fluorescence resonance energy transfer (FRET) occurring between quantum dot (QD) donors and black hole quencher (BHQ) acceptors. CdSe/ZnS QD/mercaptoacetic acid (MAA)/PDGF aptamer bioconjugates were successfully synthesized using ligand exchange. Black hole quencher (BHQ)-bearing oligonucleotide molecules showing partial sequence matching to PDGF aptamer were attached to PDGF aptamers and photoluminescence (PL) quenching was obtained through FRET. By adding target PDGF-BB to the bioconjugates containing BHQs, PL recovery was detected due to detachment of BHQ-bearing oligonucleotide from the PDGF aptamer as a result of the difference in affinity to the PDGF aptamer. The detection limit of the sensor was ∼0.4 nM and the linearity was maintained up to 1.6 nM in the PL intensity versus concentration curve. Measurement of PL recovery was suggested as a strong tool for high-sensitivity detection of PDGF-BB. Epidermal growth factor (EGF), the negative control molecule, did not contribute to PL recovery due to lack of binding affinity to the PDGF aptamers, which demonstrates the selectivity of the biosensor.

Efficacy and Safety Profile of Tricyclo-DNA Antisense Oligonucleotides in Duchenne Muscular Dystrophy Mouse Model

Directory of Open Access Journals (Sweden)

Karima Relizani

2017-09-01

Full Text Available Antisense oligonucleotides (AONs hold promise for therapeutic splice-switching correction in many genetic diseases. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake and sufficient therapeutic efficacy is still difficult to achieve. A novel class of AONs made of tricyclo-DNA (tcDNA is considered very promising for the treatment of Duchenne muscular dystrophy (DMD, a neuromuscular disease typically caused by frameshifting deletions or nonsense mutations in the gene-encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, and respiratory failure in addition to cognitive impairment. Herein, we report the efficacy and toxicology profile of a 13-mer tcDNA in mdx mice. We show that systemic delivery of 13-mer tcDNA allows restoration of dystrophin in skeletal muscles and to a lower extent in the brain, leading to muscle function improvement and correction of behavioral features linked to the emotional/cognitive deficiency. More importantly, tcDNA treatment was generally limited to minimal glomerular changes and few cell necroses in proximal tubules, with only slight variation in serum and urinary kidney toxicity biomarker levels. These results demonstrate an encouraging safety profile for tcDNA, albeit typical of phosphorothiate AONs, and confirm its therapeutic potential for the systemic treatment of DMD patients. Keywords: antisense oligonucleotides, Duchenne muscular dystrophy, preclinical, splice switching, tcDNA-AONs

Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: predictive thermodynamic model.

Science.gov (United States)

Moreira, Bernardo G; You, Yong; Owczarzy, Richard

2015-03-01

Cyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5' end of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ΔG°, Tm). Both Cy dyes enhance duplex formation by 1.2 kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidine-purine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications. Copyright © 2015. Published by Elsevier B.V.

Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray.

Science.gov (United States)

Wang, Zheng; Vora, Gary J; Stenger, David A

2004-07-01

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.

The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

Directory of Open Access Journals (Sweden)

Negin Saffarzadeh

2014-10-01

Full Text Available Objective(s: Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs and 1,2-dioleoyl-sn-glycero-3-phosphoethanol​amine (DOPE-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium nitrate and diammonium phosphate were used for the synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene glycol (PEG, DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then, hydroxyapatite NPs and DOPE-modified hydroxyapatite NPs were incubated 48 hours with cervical cancer cells and their uptakes were evaluated by fluorescent microscopy. Results: The hydroxyapatite NPs had different shapes and some agglomeration with average size of 100 nm. The results showed DOPE-modified hydroxyapatite NPs had higher uptake than hydroxyapatite NPs (P

Oligonucleotides as probes for studying polymerization reactions in dilute aqueous solution

Science.gov (United States)

Kolb, V.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

1994-01-01

We have prepared a [32P]-labled oligonucleotide probe carrying a free primary amine at its 3'-terminus. This probe is used to initiate polymerization of aziridine (ethyleneimine) in aqueous solution. The nature of the oligomeric products and the kinetics of their formation are then monitored by gel electrophoresis. Our results are generally consistent with those obtained using conventional techniques. We have also investigated the effect of polyanionic templates on the rate of oligomerization of aziridine. We find that water-soluble polyanions generally accelerate the polymerization. The sodium salt of polymethacrylic acid is the most effective of the templates that we studied. The methods introduced in this paper should be applicable to a variety of polymerization reactions in aqueous solution. They should greatly simplify the screening of potentially prebiotic polymerization reactions.

The Spectral Properties and Photostability of DNA, RNA and Oligonucleotides

International Nuclear Information System (INIS)

Kudrya, V.Yu.; Yashchuk, V.M.

2012-01-01

The present work discusses the results of comparative investigations of the optical absorption, luminescence, and photostability of the biomacromolecules (DNA, RNA), as well as synthetic poly- and oligonucleotides. The separate nucleotides in DNA and RNA are examined as almost independent absorbing centers. It is confirmed that the main triplet excitons traps responsible for the DNA phosphorescence emission are AT-complexes in DNA. In contrast to DNA, the main triplet excitons traps in RNA are adenosine bases. These bases are the most photostable against UV-irradiation as compared with all other nucleotides in both DNA and RNA. The fact of the photostability of adenosine bases and the AT-complex provides the existence of the DNA/RNA self-protection mechanisms against a damage caused by UV-irradiation. It is found the deoxyribonucleotides are more photostable than the corresponding ribonucleotides. So, the results presented here show that DNA is more photostable than RNA.

Functional oligonucleotide recognition nanomodules for electrochemical DNA biosensors

OpenAIRE

Campàs i Homs, Mònica

2002-01-01

El objetivo de esta tesis ha sido diseñar, caracterizar y optimizar un array de sensores de ADN electroquímico. Para el estudio de la inmovilización de las sondas de oligonucleótidos y la detección de la hibridación se realizaron experimentos preliminares con un sistema simplificado. Dicho sistema demostró que las monocapas auto-ensambladas (SAMs) en superficies de oro eran apropiadas como método de inmovilización. Debido al rápido desarrollo de los sensores de ADN hacia los arrays de ADN, se...

SNP Arrays

Directory of Open Access Journals (Sweden)

Jari Louhelainen

2016-10-01

Full Text Available The papers published in this Special Issue “SNP arrays” (Single Nucleotide Polymorphism Arrays focus on several perspectives associated with arrays of this type. The range of papers vary from a case report to reviews, thereby targeting wider audiences working in this field. The research focus of SNP arrays is often human cancers but this Issue expands that focus to include areas such as rare conditions, animal breeding and bioinformatics tools. Given the limited scope, the spectrum of papers is nothing short of remarkable and even from a technical point of view these papers will contribute to the field at a general level. Three of the papers published in this Special Issue focus on the use of various SNP array approaches in the analysis of three different cancer types. Two of the papers concentrate on two very different rare conditions, applying the SNP arrays slightly differently. Finally, two other papers evaluate the use of the SNP arrays in the context of genetic analysis of livestock. The findings reported in these papers help to close gaps in the current literature and also to give guidelines for future applications of SNP arrays.

A novel method to design sparse linear arrays for ultrasonic phased array.

Science.gov (United States)

Yang, Ping; Chen, Bin; Shi, Ke-Ren

2006-12-22

In ultrasonic phased array testing, a sparse array can increase the resolution by enlarging the aperture without adding system complexity. Designing a sparse array involves choosing the best or a better configuration from a large number of candidate arrays. We firstly designed sparse arrays by using a genetic algorithm, but found that the arrays have poor performance and poor consistency. So, a method based on the Minimum Redundancy Linear Array was then adopted. Some elements are determined by the minimum-redundancy array firstly in order to ensure spatial resolution and then a genetic algorithm is used to optimize the remaining elements. Sparse arrays designed by this method have much better performance and consistency compared to the arrays designed only by a genetic algorithm. Both simulation and experiment confirm the effectiveness.

Gene Expression Analysis of Circulating Hormone Refractory Prostate Cancer Micrometastases

National Research Council Canada - National Science Library

Rosenberg, Jonathan

2007-01-01

... as well as chemotherapy resistance. Oligonucleotide array comparative genomic hybridization allows the assessment of genetic changes that may occur in the process of metastasis and chemotherapy resistance...

Gene Expression Analysis of Circulating Hormone Refractory Prostate Cancer Micrometastases

National Research Council Canada - National Science Library

Rosenberg, Jonathan

2008-01-01

... as well as chemotherapy resistance. Oligonucleotide array comparative genomic hybridization allows the assessment of genetic changes that may occur in the process of metastasis and chemotherapy resistance...

Synthesis, hybridization characteristics, and fluorescence properties of oligonucleotides modified with nucleobase-functionalized locked nucleic acid adenosine and cytidine monomers.

Science.gov (United States)

Kaura, Mamta; Kumar, Pawan; Hrdlicka, Patrick J

2014-07-03

Conformationally restricted nucleotides such as locked nucleic acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing modifications in oligonucleotide chemistry to produce probes for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. Considerable efforts have been devoted in recent years to optimize the biophysical properties of LNA through additional modification of the sugar skeleton. We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically more straightforward approach to improve the biophysical properties of LNA. In the present work, we set out to test the generality of this concept by studying the characteristics of oligonucleotides modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines is indeed a viable approach for improving the binding affinity, target specificity, and/or enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is detrimental to binding affinity and specificity. These insights will impact the future design of conformationally restricted nucleotides for nucleic acid targeting applications.

ATRX mutation in two adult brothers with non-specific moderate intellectual disability identified by exome sequencing

OpenAIRE

Moncini, S.; Bedeschi, M.F.; Castronovo, P.; Crippa, M.; Calvello, M.; Garghentino, R.R.; Scuvera, G.; Finelli, P.; Venturin, M.

2013-01-01

In this report, we describe two adult brothers affected by moderate non-specific intellectual disability (ID). They showed minor facial anomalies, not clearly ascribable to any specific syndromic patterns, microcephaly, brachydactyly and broad toes. Both brothers presented seizures. Karyotype, subtelomeric and FMR1 analysis were normal in both cases. We performed array-CGH analysis that revealed no copy-number variations potentially associated with ID. Subsequent exome sequence analysis allow...

Epithelial Plasticity in Castration-Resistant Prostate Cancer: Biology of the Lethal Phenotype

Science.gov (United States)

2014-07-01

34·- ... _ ···-·--.. ~ .... _.... ..... , - .... -.==-:; ...... 0(1"𔃻""!:* Figure 6. Analysis of the genomic locus of MYCN ( chromosome 2) by arrayCGH in...uncoupled from mutations and chromosomal anomalies. This metastatic dis- semination leads to pathological fractures, anemia, bone mar- row failure...emergence of active bone lesions over time in the same regions, indicating persistent viable tumor despite the disappearance on scans. We hypothesize

Whole-cell hybridization of Methanosarcina cells with two new oligonucleotide probes

DEFF Research Database (Denmark)

Sørensen, A.H.; Torsvik, V.L.; Torsvik, T.

1997-01-01

Two new oligonucleotide probes targeting the 16S rRNA of the methanogenic genus Methanosarcina were developed. The probes have the following sequences (Escherichia coli numbering): probe SARCI551, 5'-GAC CCAATAATCACGATCAC-3', and probe SARCI645, 5'-TCCCGGTTCCAAGTCTGGC-3'. In situ hybridization...... with the fluorescently labelled probes required several modifications of standard procedures. Cells of Methanosarcina mazeii S-6 were found to lyse during the hybridization step if fixed in 3% formaldehyde and stored in 50% ethanol. Lysis was, however, not observed with cells fixed and stored in 1.6% formaldehyde-0.......85% NaCl. Extensive autofluorescence of the cells was found upon hybridization in the presence of 5 mM EDTA, but successful hybridization could be obtained without addition of this compound. The mounting agent Citifluor AF1, often used in conjugation with the fluorochrome fluorescein, was found to wash...

Staphylococcus aureus detection in blood samples by silica nanoparticle-oligonucleotides conjugates.

Science.gov (United States)

Borsa, Baris A; Tuna, Bilge G; Hernandez, Frank J; Hernandez, Luiza I; Bayramoglu, Gulay; Arica, M Yakup; Ozalp, V Cengiz

2016-12-15

A fast, specific and sensitive homogeneous assay for Staphylococcus aureus detection was developed by measuring the activity of secreted nuclease from the bacteria via a modified DNA oligonucleotide. As biosensor format, an effective system, Nanokeepers as previously reported, were used for triggered release of confined fluorophores, and hence specific detection of S. aureus on nuclease activity was obtained. The interference from blood components for fluorescent quantification was eliminated by a pre-purification by aptamer-functionalized silica magnetic nanoparticles. The reported assay system was exclusively formed by nucleic acid oligos and magnetic or mesoporous silica nanoparticles, that can be used on blood samples in a stepwise manner. The assay was successfully used as a sensing platform for the specific detection of S. aureus cells as low as 682 CFU in whole blood. Copyright © 2016 Elsevier B.V. All rights reserved.

Interaction of the E. coli DNA G:T-mismatch endonuclease (vsr protein) with oligonucleotides containing its target sequence.

Science.gov (United States)

Turner, D P; Connolly, B A

2000-12-15

The Escherichia coli vsr endonuclease recognises G:T base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The enzyme shows a preference for G:T mismatches within a particular sequence context, derived from the recognition site of the E. coli dcm DNA-methyltransferase (CC[A/T]GG). Thus, the preferred substrate for the vsr protein is (CT[A/T]GG), where the underlined T is opposed by a dG base. This paper provides quantitative data for the interaction of the vsr protein with a number of oligonucleotides containing G:T mismatches. Evaluation of specificity constant (k(st)/K(D); k(st)=rate constant for single turnover, K(D)=equilibrium dissociation constant) confirms vsr's preference for a G:T mismatch within a hemi-methylated dcm sequence, i.e. the best substrate is a duplex (both strands written in the 5'-3' orientation) composed of CT[A/T]GG and C(5Me)C[T/A]GG. Conversion of the mispaired T (underlined) to dU or the d(5Me)C to dC gave poorer substrates. No interaction was observed with oligonucleotides that lacked a G:T mismatch or did not possess a dcm sequence. An analysis of the fraction of active protein, by "reverse-titration" (i.e. adding increasing amounts of DNA to a fixed amount of protein followed by gel-mobility shift analysis) showed that less than 1% of the vsr endonuclease was able to bind to the substrate. This was confirmed using "competitive titrations" (where competitor oligonucleotides are used to displace a (32)P-labelled nucleic acid from the vsr protein) and burst kinetic analysis. This result is discussed in the light of previous in vitro and in vivo data which indicate that the MutL protein may be needed for full vsr activity. Copyright 2000 Academic Press.

Identification, purification and partial characterisation of an oligonucleotide receptor in membranes of HepG2 cells

OpenAIRE

Diesbach, Philippe de; Berens, Catherine; N’Kuli, Francisca; Monsigny, Michel; Sonveaux, Etienne; Wattiez, Ruddy; Courtoy, Pierre J.

2000-01-01

The low and unpredictable uptake and cytosolic transfer of oligonucleotides (ODN) is a major reason for their limited benefit. Improving the ODN potential for therapy and research requires a better understanding of their receptor-mediated endocytosis. We have undertaken to identify a membrane ODN receptor on HepG2 cells by ligand blotting of cell extracts with [(125)I]ODN and by photolabelling of living cells with a [(125)I]ODN-benzophenone conjugate. A major band at 66 kDa was identified by ...

Autism-specific copy number variants further implicate the phosphatidylinositol signaling pathway and the glutamatergic synapse in the etiology of the disorder.

Science.gov (United States)

Cuscó, Ivon; Medrano, Andrés; Gener, Blanca; Vilardell, Mireia; Gallastegui, Fátima; Villa, Olaya; González, Eva; Rodríguez-Santiago, Benjamín; Vilella, Elisabet; Del Campo, Miguel; Pérez-Jurado, Luis A

2009-05-15

Autism spectrum disorders (ASDs) constitute a group of severe neurodevelopmental conditions with complex multifactorial etiology. In order to explore the hypothesis that submicroscopic genomic rearrangements underlie some ASD cases, we have analyzed 96 Spanish patients with idiopathic ASD after extensive clinical and laboratory screening, by array comparative genomic hybridization (aCGH) using a homemade bacterial artificial chromosome (BAC) array. Only 13 of the 238 detected copy number alterations, ranging in size from 89 kb to 2.4 Mb, were present specifically in the autistic population (12 out of 96 individuals, 12.5%). Following validation by additional molecular techniques, we have characterized these novel candidate regions containing 24 different genes including alterations in two previously reported regions of chromosome 7 associated with the ASD phenotype. Some of the genes located in ASD-specific copy number variants act in common pathways, most notably the phosphatidylinositol signaling and the glutamatergic synapse, both known to be affected in several genetic syndromes related with autism and previously associated with ASD. Our work supports the idea that the functional alteration of genes in related neuronal networks is involved in the etiology of the ASD phenotype and confirms a significant diagnostic yield for aCGH, which should probably be included in the diagnostic workup of idiopathic ASD.

Prenatal Diagnosis of 4p and 4q Subtelomeric Microdeletion in De Novo Ring Chromosome 4

Directory of Open Access Journals (Sweden)

Halit Akbas

2013-01-01

Full Text Available Ring chromosomes are unusual abnormalities that are observed in prenatal diagnosis. A 23-year-old patient (gravida 1, para 0 referred for amniocentesis due to abnormal maternal serum screening result in the 16th week of second pregnancy. Cytogenetic analysis of cultured amniyotic fluid cells revealed out ring chromosome 4. Both maternal and paternal karyotypes were normal. Terminal deletion was observed in both 4p and 4q arms of ring chromosome 4 by fluorescence in situ hybridization (FISH. However deletion was not observed in the WHS critical region of both normal and ring chromosome 4 by an additional FISH study. These results were confirmed by means of array-CGH showing terminal deletions on 4p16.3 (130 kb and 4q35.2 (2.449 Mb. In the 21th week of pregnancy, no gross anomalia, except two weeks symmetric growth retardation, was present in the fetal ultrasonographic examination. According to our review of literature, this is the first prenatal case with 4p and 4q subtelomeric deletion of ring chromosome 4 without the involvement of WHS critical region. Our report describes the prenatal case with a ring chromosome 4 abnormality completely characterized by array-CGH which provided complementary data for genetic counseling of prenatal diagnosis.

Prenatal diagnosis of 4p and 4q subtelomeric microdeletion in de novo ring chromosome 4.

Science.gov (United States)

Akbas, Halit; Cine, Naci; Erdemoglu, Mahmut; Atay, Ahmet Engin; Simsek, Selda; Turkyilmaz, Aysegul; Fidanboy, Mehmet

2013-01-01

Ring chromosomes are unusual abnormalities that are observed in prenatal diagnosis. A 23-year-old patient (gravida 1, para 0) referred for amniocentesis due to abnormal maternal serum screening result in the 16th week of second pregnancy. Cytogenetic analysis of cultured amniyotic fluid cells revealed out ring chromosome 4. Both maternal and paternal karyotypes were normal. Terminal deletion was observed in both 4p and 4q arms of ring chromosome 4 by fluorescence in situ hybridization (FISH). However deletion was not observed in the WHS critical region of both normal and ring chromosome 4 by an additional FISH study. These results were confirmed by means of array-CGH showing terminal deletions on 4p16.3 (130 kb) and 4q35.2 (2.449 Mb). In the 21th week of pregnancy, no gross anomalia, except two weeks symmetric growth retardation, was present in the fetal ultrasonographic examination. According to our review of literature, this is the first prenatal case with 4p and 4q subtelomeric deletion of ring chromosome 4 without the involvement of WHS critical region. Our report describes the prenatal case with a ring chromosome 4 abnormality completely characterized by array-CGH which provided complementary data for genetic counseling of prenatal diagnosis.

Discrimination of p53 immunohistochemistry-positive tumors by its staining pattern in gastric cancer

International Nuclear Information System (INIS)

Ando, Koji; Oki, Eiji; Saeki, Hiroshi; Yan, Zhao; Tsuda, Yasuo; Hidaka, Gen; Kasagi, Yuta; Otsu, Hajime; Kawano, Hiroyuki; Kitao, Hiroyuki; Morita, Masaru; Maehara, Yoshihiko

2015-01-01

Immunohistochemistry staining of p53 is a cheap and simple method to detect aberrant function of p53. However, there are some discrepancies between the result of immunohistochemistry staining and mutation analysis. This study attempted to find a new definition of p53 staining by its staining pattern. Immunohistochemistry staining of p53 and TP53 gene mutation analysis were performed in 148 gastric cancer patients. Also SNP-CGH array analysis was conducted to four cases. Positive staining of p53 was observed in 88 (59.5%) tumors. Tumors with positive p53 staining showed malignant features compared to negative tumors. Mutation of TP53 gene was observed in 29 (19.6%) tumors with higher age and differentiated type. In positive p53 tumors, two types could be distinguished; aberrant type and scattered type. With comparison to TP53 gene mutation analysis, all the scattered type had wild-type TP53 gene (P = 0.0003). SNP-CGH array showed that scattered-type tumors had no change in the structure of chromosome 17. P53-scattered-type staining tumors may reflect a functionally active nonmutated TP53 gene. In interpretation of p53 immunohistochemistry staining, distinguishing p53-positive tumors by their staining pattern may be important in gastric cancer

Novel mouse model recapitulates genome and transcriptome alterations in human colorectal carcinomas.

Science.gov (United States)

McNeil, Nicole E; Padilla-Nash, Hesed M; Buishand, Floryne O; Hue, Yue; Ried, Thomas

2017-03-01

Human colorectal carcinomas are defined by a nonrandom distribution of genomic imbalances that are characteristic for this disease. Often, these imbalances affect entire chromosomes. Understanding the role of these aneuploidies for carcinogenesis is of utmost importance. Currently, established transgenic mice do not recapitulate the pathognonomic genome aberration profile of human colorectal carcinomas. We have developed a novel model based on the spontaneous transformation of murine colon epithelial cells. During this process, cells progress through stages of pre-immortalization, immortalization and, finally, transformation, and result in tumors when injected into immunocompromised mice. We analyzed our model for genome and transcriptome alterations using ArrayCGH, spectral karyotyping (SKY), and array based gene expression profiling. ArrayCGH revealed a recurrent pattern of genomic imbalances. These results were confirmed by SKY. Comparing these imbalances with orthologous maps of human chromosomes revealed a remarkable overlap. We observed focal deletions of the tumor suppressor genes Trp53 and Cdkn2a/p16. High-level focal genomic amplification included the locus harboring the oncogene Mdm2, which was confirmed by FISH in the form of double minute chromosomes. Array-based global gene expression revealed distinct differences between the sequential steps of spontaneous transformation. Gene expression changes showed significant similarities with human colorectal carcinomas. Pathways most prominently affected included genes involved in chromosomal instability and in epithelial to mesenchymal transition. Our novel mouse model therefore recapitulates the most prominent genome and transcriptome alterations in human colorectal cancer, and might serve as a valuable tool for understanding the dynamic process of tumorigenesis, and for preclinical drug testing. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Coupling in reflector arrays

DEFF Research Database (Denmark)

Appel-Hansen, Jørgen

1968-01-01

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......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...

Isolation and characterization of human glycophorin A cDNA clones by a synthetic oligonucleotide approach: nucleotide sequence and mRNA structure

International Nuclear Information System (INIS)

Siebert, P.D.; Fukuda, M.

1986-01-01

In an effort to understand the relationships among and the regulation of human glycophorins, the authors have isolated and characterized several glycophorin A-specific cDNA clones obtained from a human erythroleukemic K562 cell cDNA library. This was accomplished by using mixed synthetic oligonucleotides, corresponding to various regions of the known amino acid sequence, to prime the synthesis of the cDNA as well as to screen the cDNA library. They also used synthetic oligonucleotides to sequence the largest of the glycophorin cDNAs. The nucleotide sequence obtained suggests the presence of a potential leader peptide, consistent with the membrane localization of this glycoprotein. Examination of the structure of glycophorin mRNA by blot hybridization revealed the existence of several electrophoretically distinct mRNAs numbering three or four, depending on the size of the glycophorin cDNA used as a hybridization probe. The smaller cDNA hybridized to three mRNAs of approximately 2.8, 1.7, and 1.0 kilobases. In contrast, the larger cDNA hybridized to an additional mRNA of approximately 0.6 kilobases. Further examination of the relationships between these multiple mRNAs by blot hybridization was conducted with the use of exact-sequence oligonucleotide probes constructed from various regions of the cDNA representing portions of the amino acid sequence of glycophorin A with or without known homology with glycophorin B. In total, the results obtained are consistent with the hypothesis that the three larger mRNAs represent glycophorin A gene transcripts and that the smallest (0.6 kilobase) mRNA may be specific for glycophorin B

XX males SRY negative: a confirmed cause of infertility.

Science.gov (United States)

Vetro, Annalisa; Ciccone, Roberto; Giorda, Roberto; Patricelli, Maria Grazia; Della Mina, Erika; Forlino, Antonella; Zuffardi, Orsetta

2011-10-01

SOX9 is a widely expressed transcription factor playing several relevant functions during development and essential for testes differentiation. It is considered to be the direct target gene of the protein encoded by SRY and its overexpression in an XX murine gonad can lead to male development in the absence of Sry. Recently, a family was reported with a 178 kb duplication in the gene desert region ending about 500 kb upstream of SOX9 in which 46,XY duplicated persons were completely normal and fertile whereas the 46,XX ones were males who came to clinical attention because of infertility. We report a family with two azoospermic brothers, both 46,XX, SRY negative, having a 96 kb triplication 500 kb upstream of SOX9. Both subjects have been analyzed trough oligonucleotide array-CGH and the triplication was confirmed and characterised through qPCR, defining the minimal region of amplification upstream of SOX9 associated with 46,XX infertile males, SRY negative. Our results confirm that even in absence of SRY, complete male differentiation may occur, possibly driven by overexpression of SOX9 in the gonadal ridge, as a consequence of the amplification of a gene desert region. We hypothesize that this region contains gonadal specific long-range regulation elements whose alteration may impair the normal sex development. Our data show that normal XX males, with alteration in copy number or, possibly, in the critical sequence upstream to SOX9 are a new category of infertility inherited in a dominant way with expression limited to the XX background.

Characterization of adjacent breast tumors using oligonucleotide microarrays

International Nuclear Information System (INIS)

Unger, Meredith A; Rishi, Mazhar; Clemmer, Virginia B; Hartman, Jennifer L; Keiper, Elizabeth A; Greshock, Joel D; Chodosh, Lewis A; Liebman, Michael N; Weber, Barbara L

2001-01-01

Current methodology often cannot distinguish second primary breast cancers from multifocal disease, a potentially important distinction for clinical management. In the present study we evaluated the use of oligonucleotide-based microarray analysis in determining the clonality of tumors by comparing gene expression profiles. Total RNA was extracted from two tumors with no apparent physical connection that were located in the right breast of an 87-year-old woman diagnosed with invasive ductal carcinoma (IDC). The RNA was hybridized to the Affymetrix Human Genome U95A Gene Chip ® (12,500 known human genes) and analyzed using the Gene Chip Analysis Suite ® 3.3 (Affymetrix, Inc, Santa Clara, CA, USA) and JMPIN ® 3.2.6 (SAS Institute, Inc, Cary, NC, USA). Gene expression profiles of tumors from five additional patients were compared in order to evaluate the heterogeneity in gene expression between tumors with similar clinical characteristics. The adjacent breast tumors had a pairwise correlation coefficient of 0.987, and were essentially indistinguishable by microarray analysis. Analysis of gene expression profiles from different individuals, however, generated a pairwise correlation coefficient of 0.710. Transcriptional profiling may be a useful diagnostic tool for determining tumor clonality and heterogeneity, and may ultimately impact on therapeutic decision making

Sieve-based device for MALDI sample preparation. I. Influence of sample deposition conditions in oligonucleotide analysis to achieve significant increases in both sensitivity and resolution.

Science.gov (United States)

Molin, Laura; Cristoni, Simone; Crotti, Sara; Bernardi, Luigi Rossi; Seraglia, Roberta; Traldi, Pietro

2008-11-01

Spraying of oligonucleotide-matrix solutions through a stainless steel (ss) sieve (38 microm, 450 mesh) leads to the formation, on the matrix-assisted laser desorption/ionization (MALDI) sample holder, of uniformly distributed microcrystals, well separated from each other. When the resulting sample holder surface is irradiated by laser, abundant molecular species form, with a clear increase in both intensity and resolution with respect to values obtained by 'Dried Droplet', 'Double Layer', and 'Sandwich' deposition methods. In addition, unlike the usual situation, the sample is perfectly homogeneous, and identical spectra are obtained by irradiating different areas. On one hand, the data indicate that this method is highly effective for oligonucleotide MALDI analysis, and on the other, that it can be validly employed for fully automated MALDI procedures.

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

Science.gov (United States)

Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.

2002-01-01

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659

Integral computer-generated hologram via a modified Gerchberg-Saxton algorithm

International Nuclear Information System (INIS)

Wu, Pei-Jung; Lin, Bor-Shyh; Chen, Chien-Yue; Huang, Guan-Syun; Deng, Qing-Long; Chang, Hsuan T

2015-01-01

An integral computer-generated hologram, which modulates the phase function of an object based on a modified Gerchberg–Saxton algorithm and compiles a digital cryptographic diagram with phase synthesis, is proposed in this study. When the diagram completes position demultiplexing decipherment, multi-angle elemental images can be reconstructed. Furthermore, an integral CGH with a depth of 225 mm and a visual angle of ±11° is projected through the lens array. (paper)

Developmental delay and connective tissue disorder in four patients sharing a common microdeletion at 6q13-14

OpenAIRE

2010-01-01

Abstract Interstitial deletions of the long arm of chromosome 6 are rare, and most of the reported cases represent large, cytogenetically detectable deletions. The implementation of array-CGH in the diagnostic work-up of patients presenting with congenital disorders including developmental delay has enabled the identification of many patients with smaller chromosomal imbalances. Here we present 4 patients with a de novo interstitial deletion of chromosome 6q13-14, resulting in a c...

Enzymatic production of single-molecule FISH and RNA capture probes.

Science.gov (United States)

Gaspar, Imre; Wippich, Frank; Ephrussi, Anne

2017-10-01

Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed. © 2017 Gaspar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.