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Sample records for automated dna mutation

  1. Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

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    Wu Bai-Lin

    2009-10-01

    Full Text Available Abstract Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other

  2. Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA

    International Nuclear Information System (INIS)

    The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. The authors have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. They also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN

  3. Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Gibbs, R.A. (Baylor College of Medicine, Houston, TX (USA)); Nguyen, Phinga (Howard Hughes Medical Institute, Houston, TX (USA)); McBride, L.J.; Koepf, S.M. (Applied Biosystems, Foster City, CA (USA)); Caskey, C.T. (Baylor College of Medicine, Houston, TX (USA) Howard Hughes Medical Institute, Houston, TX (USA))

    1989-03-01

    The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. The authors have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. They also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.

  4. Correlation of the UV-induced mutational spectra and the DNA damage distribution of the human HPRT gene: Automating the analysis

    International Nuclear Information System (INIS)

    Automated DNA sequencers can be readily adapted for various types of sequence-based nucleic acid analysis: more recently it was determined the distribution of UV photoproducts in the E. coli laci gene using techniques developed for automated fluorescence-based analysis. We have been working to improve the automated approach of damage distribution. Our current method is more rigorous. We have new software that integrates the area under the individual peaks, rather than measuring the height of the curve. In addition, we now employ an internal standard. The analysis can also be partially automated. Detection limits for both major types of UV-photoproducts (cyclobutane dimers and pyrimidine (6-4) pyrimidone photoproducts) are reported. The UV-induced damage distribution in the hprt gene is compared to the mutational spectra in human and rodents cells

  5. Minisequencing mitochondrial DNA pathogenic mutations

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    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  6. BRAF Mutation Testing in Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using a Rapid, Automated Molecular Diagnostics System.

    Science.gov (United States)

    Janku, Filip; Huang, Helen J; Claes, Bart; Falchook, Gerald S; Fu, Siqing; Hong, David; Ramzanali, Nishma M; Nitti, Giovanni; Cabrilo, Goran; Tsimberidou, Apostolia M; Naing, Aung; Piha-Paul, Sarina A; Wheler, Jennifer J; Karp, Daniel D; Holley, Veronica R; Zinner, Ralph G; Subbiah, Vivek; Luthra, Rajyalakshmi; Kopetz, Scott; Overman, Michael J; Kee, Bryan K; Patel, Sapna; Devogelaere, Benoit; Sablon, Erwin; Maertens, Geert; Mills, Gordon B; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-06-01

    Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAF(V600) status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAF(V600) mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63-0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60-0.83) and specificity of 98% (95% CI, 0.93-1.00). A higher percentage, but not concentration, of BRAF(V600) cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAF(V600) cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAF(V600) mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAF(V600) in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397-404. ©2016 AACR. PMID:27207774

  7. Automated Extraction of DNA from clothing

    OpenAIRE

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas; Hansen, Anders Johannes; Morling, Niels

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles.

  8. Automated Extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas;

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing the...... amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles....

  9. Mutator phenotypes due to DNA replication infidelity

    OpenAIRE

    Arana, Mercedes E.; Kunkel, Thomas A.

    2010-01-01

    This article considers the fidelity of DNA replication performed by eukaryotic DNA polymerases involved in replicating the nuclear genome. DNA replication fidelity can vary widely depending on the DNA polymerase, the composition of the error, the flanking sequence, the presence of DNA damage and the ability to correct errors. As a consequence, defects in processes that determine DNA replication fidelity can confer strong mutator phenotypes whose specificity can help determine the molecular na...

  10. j5 DNA assembly design automation software.

    Science.gov (United States)

    Hillson, Nathan J; Rosengarten, Rafael D; Keasling, Jay D

    2012-01-20

    Recent advances in Synthetic Biology have yielded standardized and automatable DNA assembly protocols that enable a broad range of biotechnological research and development. Unfortunately, the experimental design required for modern scar-less multipart DNA assembly methods is frequently laborious, time-consuming, and error-prone. Here, we report the development and deployment of a web-based software tool, j5, which automates the design of scar-less multipart DNA assembly protocols including SLIC, Gibson, CPEC, and Golden Gate. The key innovations of the j5 design process include cost optimization, leveraging DNA synthesis when cost-effective to do so, the enforcement of design specification rules, hierarchical assembly strategies to mitigate likely assembly errors, and the instruction of manual or automated construction of scar-less combinatorial DNA libraries. Using a GFP expression testbed, we demonstrate that j5 designs can be executed with the SLIC, Gibson, or CPEC assembly methods, used to build combinatorial libraries with the Golden Gate assembly method, and applied to the preparation of linear gene deletion cassettes for E. coli. The DNA assembly design algorithms reported here are generally applicable to broad classes of DNA construction methodologies and could be implemented to supplement other DNA assembly design tools. Taken together, these innovations save researchers time and effort, reduce the frequency of user design errors and off-target assembly products, decrease research costs, and enable scar-less multipart and combinatorial DNA construction at scales unfeasible without computer-aided design. PMID:23651006

  11. Natural radioactivity and human mitochondrial DNA mutations

    OpenAIRE

    Forster, Lucy; Forster, Peter; Lutz-Bonengel, Sabine; Willkomm, Horst; Brinkmann, Bernd

    2002-01-01

    Radioactivity is known to induce tumors, chromosome lesions, and minisatellite length mutations, but its effects on the DNA sequence have not previously been studied. A coastal peninsula in Kerala (India) contains the world's highest level of natural radioactivity in a densely populated area, offering an opportunity to characterize radiation-associated DNA mutations. We sampled 248 pedigrees (988 individuals) in the high-radiation peninsula and in nearby low-radiation islands as a control pop...

  12. Mitochondrial DNA mutations in gynecological cancers

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    Kinga Księżakowska

    2011-12-01

    Full Text Available Mitochondria are metabolic organelles inherited only from the mother and possessing their own genome(mtDNA. The mt DNA is a circular, double-stranded molecule of 16.569 bp length containing 37 genes coding13 polypeptides, 2 genes of rRNA (12S, 16S, and 22 genes of tRNA. All of these proteins are subunits of the oxidativephosphorylation system (OXPHO localized at the mitochondrial inner membrane. Human mitochondrialdysfunctions have been linked to various metabolic diseases and cancer development. So far we have knownseveral of the inherited and somatic mtDNA mutations predisposing to tumor development, occurring in bothnon-coding and coding regions. The genetic alternations in the mtDNA include point mutations, deletions, insertions,mtMSI (mitochondrial microsatellite instability. Most of mtDNA mutations in gynecological cancersare observed in the D-loop region. Studies suggest that both mtDNA polymorphism and classes of inherited haplogroupsin the human population may be correlated with the risk of cancer development. Mitochondrial DNAmutation and polymorphism analysis may enable to identify individuals with high risk of cancer development,establish early detection or monitor the progression of cancer.

  13. [Diseases caused by mutations in mitochondrial DNA].

    Science.gov (United States)

    Wojewoda, Marta; Zabłocki, Krzysztof; Szczepanowska, Joanna

    2011-01-01

    Mitochondrial diseases associated with mutations within mitochondrial genome are a subgroup of metabolic disorders since their common consequence is reduced metabolic efficiency caused by impaired oxidative phophorylation and shortage of ATP. Although the vast majority of mitochondrial proteins (approximately 1500) is encoded by nuclear genome, mtDNA encodes 11 subunits of respiratory chain complexes, 2 subunits of ATP synthase, 22 tRNAs and 2 rRNAs. Up to now, more than 250 pathogenic mutations have been described within mtDNA. The most common are point mutations in genes encoding mitochondrial tRNAs such as 3243A-->G and 8344T-->G that cause, respectively, MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) or MIDD (maternally-inherited diabetes and deafness) and MERRF (myoclonic epilepsy with ragged red fibres) syndromes. There have been also found mutations in genes encoding subunits of ATP synthase such as 8993T-->G substitution associated with NARP (neuropathy, ataxia and retinitis pigmentosa) syndrome. It is worth to note that mitochondrial dysfunction can also be caused by mutations within nuclear genes coding for mitochondrial proteins. PMID:21913424

  14. Detection and quantification of the age-related point mutation A189G in the human mitochondrial DNA.

    OpenAIRE

    Thèves, Catherine; Keyser-Tracqui, Christine; Crubézy, Eric; Pierre Salles, Jean; Ludes, Bertrand; Telmon, Norbert

    2006-01-01

    Mutation analysis in the mitochondrial DNA (mtDNA) control region is widely used in population genetic studies as well as in forensic medicine. Among the difficulties linked to the mtDNA analysis, one can find the detection of heteroplasmy, which can be inherited or somatic. Recently, age-related point mutation A189G was described in mtDNA and shown to accumulate with age in muscles. We carried out the detection of this 189 heteroplasmic point mutation using three technologies: automated DNA ...

  15. A novel fully automated molecular diagnostic system (AMDS for colorectal cancer mutation detection.

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    Shiro Kitano

    Full Text Available BACKGROUND: KRAS, BRAF and PIK3CA mutations are frequently observed in colorectal cancer (CRC. In particular, KRAS mutations are strong predictors for clinical outcomes of EGFR-targeted treatments such as cetuximab and panitumumab in metastatic colorectal cancer (mCRC. For mutation analysis, the current methods are time-consuming, and not readily available to all oncologists and pathologists. We have developed a novel, simple, sensitive and fully automated molecular diagnostic system (AMDS for point of care testing (POCT. Here we report the results of a comparison study between AMDS and direct sequencing (DS in the detection of KRAS, BRAF and PI3KCA somatic mutations. METHODOLOGY/PRINCIPAL FINDING: DNA was extracted from a slice of either frozen (n = 89 or formalin-fixed and paraffin-embedded (FFPE CRC tissue (n = 70, and then used for mutation analysis by AMDS and DS. All mutations (n = 41 among frozen and 27 among FFPE samples detected by DS were also successfully (100% detected by the AMDS. However, 8 frozen and 6 FFPE samples detected as wild-type in the DS analysis were shown as mutants in the AMDS analysis. By cloning-sequencing assays, these discordant samples were confirmed as true mutants. One sample had simultaneous "hot spot" mutations of KRAS and PIK3CA, and cloning assay comfirmed that E542K and E545K were not on the same allele. Genotyping call rates for DS were 100.0% (89/89 and 74.3% (52/70 in frozen and FFPE samples, respectively, for the first attempt; whereas that of AMDS was 100.0% for both sample sets. For automated DNA extraction and mutation detection by AMDS, frozen tissues (n = 41 were successfully detected all mutations within 70 minutes. CONCLUSIONS/SIGNIFICANCE: AMDS has superior sensitivity and accuracy over DS, and is much easier to execute than conventional labor intensive manual mutation analysis. AMDS has great potential for POCT equipment for mutation analysis.

  16. Mitochondrial DNA mutations in single human blood cells.

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    Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S

    2015-09-01

    Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. PMID:26149767

  17. Implications of mitochondrial DNA mutations and mitochondrial dysfunction in tumorigenesis

    Institute of Scientific and Technical Information of China (English)

    Jianxin Lu; Lokendra Kumar Sharma; Yidong Bai

    2009-01-01

    Alterations in oxidative phosphorylation resulting from mitochondrial dysfunction have long been hypothesized to be involved in tumorigenesis. Mitochondria have recently been shown to play an important role in regulating both programmed cell death and cell proliferation. Furthermore, mitochondrial DNA (mtDNA) mutations have been found in various cancer cells. However, the role of these mtDNA mutations in tumorigenesis remains largely unknown. This review focuses on basic mitochondrial genetics, mtDNA mutations and consequential mitochondrial dysfunction associated with cancer. The potential molecular mechanisms, mediating the pathogenesis from mtDNA mutations and mitochondrial dysfunction to tumorigenesis are also discussed.

  18. Novel dnaG mutation in a dnaP mutant of Escherichia coli.

    OpenAIRE

    Murakami, Y.; Nagata, T; Schwarz, W.; Wada, C.; Yura, T

    1985-01-01

    Reexamination of the dnaP18 mutant strain of Escherichia coli revealed that the mutation responsible for the arrest of DNA replication and cell growth at high temperatures resides in the dnaG gene rather than in the dnaP locus as previously thought; this mutation has been designated dnaG2903.

  19. Mitochondrial DNA mutation in essential hypertension

    Institute of Scientific and Technical Information of China (English)

    Yuqi Liu; Shiwen Wang

    2008-01-01

    Essential hypertension (EH) is an escalating problem for developed and developing countries.It is currently seen as a 'complex' genetic trait caused by multiple susceptibility genes which are modulated by gene-environment and gene-gene interactions.Over the past 10 years,mitochondrial defects have been implicated in a wide variety of degenerative diseases,aging,and cancer.Recently several studies showed that human essential hypertension has excess maternal transmission which suggests a possible mitochondrial involvement.However,the exact pathophysiology of mitochondrial DNA mutation (mtDNA) in essential hypertension still remains perplexing.With the application of a variety of imaging approaches and successive mouse model of mitochonddal diseases we convince that these problems will be resolved in the near future.(J Geriatr Cardiol 2008;5(1):60-64)

  20. Pathogenic Mitochondrial DNA Mutations Are Common in the General Population

    OpenAIRE

    Elliott, Hannah R.; Samuels, David C.; Eden, James A.; Relton, Caroline L; Chinnery, Patrick F

    2008-01-01

    Mitochondrial DNA (mtDNA) mutations are a major cause of genetic disease, but their prevalence in the general population is not known. We determined the frequency of ten mitochondrial point mutations in 3168 neonatal-cord-blood samples from sequential live births, analyzing matched maternal-blood samples to estimate the de novo mutation rate. mtDNA mutations were detected in 15 offspring (0.54%, 95% CI = 0.30–0.89%). Of these live births, 0.00107% (95% CI = 0.00087–0.0127) harbored a mutation...

  1. Capacity of DNA Data Embedding Under Substitution Mutations

    CERN Document Server

    Balado, Félix

    2011-01-01

    A number of methods have been proposed over the last decade for encoding information using deoxyribonucleic acid (DNA), giving rise to the emerging area of DNA data embedding. Since a DNA sequence is conceptually equivalent to a sequence of quaternary symbols (bases), DNA data embedding (diversely called DNA watermarking or DNA steganography) can be seen as a digital communications problem where channel errors are tantamount to mutations of DNA bases. Depending on the use of coding or noncoding DNA hosts, which, respectively, denote DNA segments that can or cannot be translated into proteins, DNA data embedding is essentially a problem of communications with or without side information at the encoder. In this paper the Shannon capacity of DNA data embedding is obtained for the case in which DNA sequences are subject to substitution mutations modelled using the Kimura model from molecular evolution studies. Inferences are also drawn with respect to the biological implications of some of the results presented.

  2. The timing of mitochondrial DNA mutations in aging

    OpenAIRE

    KHRAPKO, KONSTANTIN

    2011-01-01

    Somatic mutations in mitochondrial DNA build up in aging tissues and are thought to contribute to physiological aging. Surprisingly, it is not known if these mutations occur early or late in life. A new study looks at mechanisms of accelerated mitochondrial aging in HIV-infected individuals treated with nucleoside analog anti-retroviral drugs and offers support for an early origin of mitochondrial DNA mutations.

  3. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

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    Amie J Radenbaugh

    Full Text Available The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis, a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84% and very high precision (98% and 99% for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA.

  4. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

    Science.gov (United States)

    Radenbaugh, Amie J; Ma, Singer; Ewing, Adam; Stuart, Joshua M; Collisson, Eric A; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84%) and very high precision (98% and 99%) for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA. PMID:25405470

  5. Mitochondrial DNA Mutations Regulate Metastasis of Human Breast Cancer Cells

    OpenAIRE

    Hirotake Imanishi; Keisuke Hattori; Reiko Wada; Kaori Ishikawa; Sayaka Fukuda; Keizo Takenaga; Kazuto Nakada; Jun-ichi Hayashi

    2011-01-01

    Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously e...

  6. Somatic mitochondrial DNA mutations in Chinese patients with osteosarcoma.

    Science.gov (United States)

    Yu, Man; Wan, Yanfang; Zou, Qinghua

    2013-04-01

    Somatic mutations in mitochondrial DNA (mtDNA) have been long proposed to drive the pathogenesis and progression of human malignancies. Previous investigations have revealed a high frequency of somatic mutations in the D-loop control region of mtDNA in osteosarcoma. However, little is known with regard to whether or not somatic mutations also occur in the coding regions of mtDNA in osteosarcoma. To test this possibility, in the present study we screened somatic mutations over the full-length mitochondrial genome of 31 osteosarcoma tumour tissue samples, and corresponding peripheral blood samples from the same cohort of patients. We detected a sum of 11 somatic mutations in the mtDNA coding regions in our series. Nine of them were missense or frameshift mutations that have the potential to hamper mitochondrial respiratory function. In combination with our earlier observations on the D-loop fragment, 71.0% (22/31) of patients with osteosarcoma carried at least one somatic mtDNA mutation, and a total of 40 somatic mutations were identified. Amongst them, 29 (72.5%) were located in the D-loop region, two (5%) were in the sequences of the tRNA genes, two (5%) were in the mitochondrial ATP synthase subunit 6 gene and seven (17.5%) occurred in genes encoding components of the mitochondrial respiratory complexes. In addition, somatic mtDNA mutation was not closely associated with the clinicopathological characteristics of osteosarcoma. Together, these findings suggest that somatic mutations are highly prevalent events in both coding and non-coding regions of mtDNA in osteosarcoma. Some missense and frameshift mutations are putatively harmful to proper mitochondrial activity and might play vital roles in osteosarcoma carcinogenesis. PMID:23441585

  7. A Fast Determination of DNA Mutation Induced by Ultraviolet Radiation

    Institute of Scientific and Technical Information of China (English)

    LuFeng; LiuLili; ZhangXiaofang; WuYutian

    2001-01-01

    Electrophoresis, chromatography, immunoassay, sequencing and other time consuming ap-proaches have been developed to determine DNA base mismatching, oxidative lesion or strand breaks. Sometimes,however, only qualitative information is enough to decide whether mutation has happened to DNA and its extent.Convolution spectrometry (CS), a new technique to discover ultrafme difference on ultraviolet (UV) absorption ofdifferent substances, is originally employed to find out any subtle mutation of DNA induced by UV radiation. Muta-tive DNA is compared with ego criteria based on the spectra of the former DNA, any difference is quantitatively ex-pressed by dispersion (5). Visible changes cannot be observed on second -derivative spectra until the mutation gets 5up to 11.48%. Dimethyl sulfoxide is an intensifier of UV 254 nm induced DNA mutation and protector at 365 nm,which is simply confirmed by increasing and decreasing 5. Every convolution procedure takes less than 1 min. Convolution spectrometry provides a fast, simple, sensitive and inexpensive alternative to determine DNA mutation, andto screen anti-mutational medicines.

  8. Performance of mitochondrial DNA mutations detecting early stage cancer

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    Wagner Paul D

    2008-10-01

    Full Text Available Abstract Background Mutations in the mitochondrial genome (mtgenome have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy. The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites. Methods We determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid from each cancer patient and two matched specimens (blood and sputum from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region. Results Using this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17% contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors. Conclusion Our findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with

  9. Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice

    OpenAIRE

    Safdar, Adeel; Khrapko, Konstantin; Flynn, James M.; Saleem, Ayesha; De Lisio, Michael; Johnston, Adam P. W.; Kratysberg, Yevgenya; Samjoo, Imtiaz A.; Kitaoka, Yu; Ogborn, Daniel I.; Little, Jonathan P.; Raha, Sandeep; Parise, Gianni; Akhtar, Mahmood; Bart P Hettinga

    2016-01-01

    Background Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. Results Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pat...

  10. Mitochondrial DNA mutations in oxyphilic and chief cell parathyroid adenomas

    Directory of Open Access Journals (Sweden)

    Roth Sanford I

    2007-10-01

    Full Text Available Abstract Background The potential pathogenetic significance of mitochondrial DNA (mtDNA mutations in tumorigenesis is controversial. We hypothesized that benign tumorigenesis of a slowly replicating tissue like the human parathyroid might constitute an especially fertile ground on which a selective advantage conferred by mtDNA mutation could be manifested and might contribute to the oxyphilic phenotype observed in a subset of parathyroid tumors. Methods We sought acquired mitochondrial DNA mutations by sequencing the entire 16.6 kb mitochondrial genome of each of thirty sporadic parathyroid adenomas (18 chief cell and 12 oxyphil cell, eight independent, polyclonal, parathyroid primary chief cell hyperplasias plus corresponding normal control samples, five normal parathyroid glands, and one normal thyroid gland. Results Twenty-seven somatic mutations were identified in 15 of 30 (9 of 12 oxyphil adenomas, 6 of 18 chief cell parathyroid adenomas studied. No somatic mutations were observed in the hyperplastic parathyroid glands. Conclusion Features of the somatic mutations suggest that they may confer a selective advantage and contribute to the molecular pathogenesis of parathyroid adenomas. Importantly, the statistically significant differences in mutation prevalence in oxyphil vs. chief cell adenomas also suggest that mtDNA mutations may contribute to the oxyphil phenotype.

  11. Automated protein-DNA interaction screening of Drosophila regulatory elements

    OpenAIRE

    Hens, Korneel; Feuz, Jean-Daniel; Isakova, Alina; Iagovitina, Antonina; Massouras, Andreas; Bryois, Julien; Callaerts, Patrick; Celniker, Susan E.; Deplancke, Bart

    2011-01-01

    Drosophila melanogaster has one of the best characterized metazoan genomes in terms of functionally annotated regulatory elements. To explore how these elements contribute to gene regulation in the context of gene regulatory networks, we need convenient tools to identify the proteins that bind to them. Here, we present the development and validation of a highly automated protein-DNA interaction detection method, enabling the high-throughput yeast one-hybrid-based screening of DNA elements ver...

  12. DNA-directed mutations. Leading and lagging strand specificity

    Science.gov (United States)

    Sinden, R. R.; Hashem, V. I.; Rosche, W. A.

    1999-01-01

    The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

  13. Summary Statistics of Neutral Mutations in Longitudinal DNA Samples

    OpenAIRE

    Liu, Xiaoming; Fu, Yun-Xin

    2008-01-01

    Longitudinal samples of DNA sequences are the DNA sequences sampled from the same population at different time points. For fast evolving organisms, e.g. RNA virus, these kind of samples have increasingly been used to study the evolutionary process in action. Longitudinal samples provide some interesting new summary statistics of genetic variation, such as the frequency of mutation of size i in one sample and size j in another, the average number of mutations accumulated since the common ances...

  14. Automation of a single-DNA molecule stretching device

    DEFF Research Database (Denmark)

    Sørensen, Kristian Tølbøl; Lopacinska, Joanna M.; Tommerup, Niels;

    2015-01-01

    We automate the manipulation of genomic-length DNA in a nanofluidic device based on real-time analysis of fluorescence images. In our protocol, individual molecules are picked from a microchannel and stretched with pN forces using pressure driven flows. The millimeter-long DNA fragments free......, we demonstrate how to estimate the length of molecules by continuous real-time image stitching and how to increase the effective resolution of a pressure controller by pulse width modulation. The sequence of image-processing steps addresses the challenges of genomic-length DNA visualization; however...

  15. Accumulation of point mutations in mitochondrial DNA of aging mice

    Energy Technology Data Exchange (ETDEWEB)

    Khaidakov, Magomed; Heflich, Robert H.; Manjanatha, Mugimane G.; Myers, Meagan B.; Aidoo, Anane

    2003-05-15

    Mitochondrial DNA (mtDNA) exists in a highly genotoxic environment created by exposure to reactive oxygen species, somewhat deficient DNA repair, and the relatively low fidelity of polymerase gamma. Given the severity of the environment, it was anticipated that mutation accumulation in the mtDNA of aging animals should exceed that of nuclear genes by several orders of magnitude. We have analyzed fragments amplified from the D-loop region of mtDNA from 2 to 22-month-old mice. The amplified 432 bp fragments were cloned into plasmid vectors, and plasmid DNAs from individual clones were purified and sequenced. None of 110 fragments from young mice contained a mutation, while 9 of 87 clones originating from old animals contained base substitutions (chi square = 11.9, P<0.001). The estimated mutation frequency in mtDNA from old mice was 11.6{+-}2.7 or 25.4{+-}7.8 per 10{sup 5} nucleotides (depending on assumptions of clonality), which exceeds existing estimates for mutation frequencies for nuclear genes by approximately 1000-fold. Our data suggest that at 22 months of age, which roughly corresponds to 3/4 of the mouse natural life span, most mtDNA molecules carry multiple point mutations.

  16. Automated preparation of DNA sequences for publication.

    OpenAIRE

    Shapiro, M B; Senapathy, P

    1986-01-01

    A computer program which draws DNA sequences is described. A simple method is used which enables the user to highlight or annotate specific parts of a sequence. The sizes of the characters in the sequence to be drawn are specified by the user. In addition, vertical spacing between lines and horizontal spacing between characters can be specified. Sequences can be prepared and high quality output produced on a plotter in a short period of time, making the program advantageous to use over typing...

  17. Automated Image Processing for the Analysis of DNA Repair Dynamics

    CERN Document Server

    Riess, Thorsten; Tomas, Martin; Ferrando-May, Elisa; Merhof, Dorit

    2011-01-01

    The efficient repair of cellular DNA is essential for the maintenance and inheritance of genomic information. In order to cope with the high frequency of spontaneous and induced DNA damage, a multitude of repair mechanisms have evolved. These are enabled by a wide range of protein factors specifically recognizing different types of lesions and finally restoring the normal DNA sequence. This work focuses on the repair factor XPC (xeroderma pigmentosum complementation group C), which identifies bulky DNA lesions and initiates their removal via the nucleotide excision repair pathway. The binding of XPC to damaged DNA can be visualized in living cells by following the accumulation of a fluorescent XPC fusion at lesions induced by laser microirradiation in a fluorescence microscope. In this work, an automated image processing pipeline is presented which allows to identify and quantify the accumulation reaction without any user interaction. The image processing pipeline comprises a preprocessing stage where the ima...

  18. Exploring the common molecular basis for the universal DNA mutation bias: Revival of Loewdin mutation model

    International Nuclear Information System (INIS)

    Highlights: → There exists a universal G:C → A:T mutation bias in three domains of life. → This universal mutation bias has not been sufficiently explained. → A DNA mutation model proposed by Loewdin 40 years ago offers a common explanation. -- Abstract: Recently, numerous genome analyses revealed the existence of a universal G:C → A:T mutation bias in bacteria, fungi, plants and animals. To explore the molecular basis for this mutation bias, we examined the three well-known DNA mutation models, i.e., oxidative damage model, UV-radiation damage model and CpG hypermutation model. It was revealed that these models cannot provide a sufficient explanation to the universal mutation bias. Therefore, we resorted to a DNA mutation model proposed by Loewdin 40 years ago, which was based on inter-base double proton transfers (DPT). Since DPT is a fundamental and spontaneous chemical process and occurs much more frequently within GC pairs than AT pairs, Loewdin model offers a common explanation for the observed universal mutation bias and thus has broad biological implications.

  19. Automation of cDNA Synthesis and Labelling Improves Reproducibility

    Directory of Open Access Journals (Sweden)

    Daniel Klevebring

    2009-01-01

    Full Text Available Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

  20. Accumulation of point mutations in mitochondrial DNA of aging mice

    International Nuclear Information System (INIS)

    Mitochondrial DNA (mtDNA) exists in a highly genotoxic environment created by exposure to reactive oxygen species, somewhat deficient DNA repair, and the relatively low fidelity of polymerase gamma. Given the severity of the environment, it was anticipated that mutation accumulation in the mtDNA of aging animals should exceed that of nuclear genes by several orders of magnitude. We have analyzed fragments amplified from the D-loop region of mtDNA from 2 to 22-month-old mice. The amplified 432 bp fragments were cloned into plasmid vectors, and plasmid DNAs from individual clones were purified and sequenced. None of 110 fragments from young mice contained a mutation, while 9 of 87 clones originating from old animals contained base substitutions (chi square = 11.9, P5 nucleotides (depending on assumptions of clonality), which exceeds existing estimates for mutation frequencies for nuclear genes by approximately 1000-fold. Our data suggest that at 22 months of age, which roughly corresponds to 3/4 of the mouse natural life span, most mtDNA molecules carry multiple point mutations

  1. POINT MUTATIONS ON MITOCHONDRIAL DNA IN IRANIAN PATIENTS WITH FRIEDREICH’S ATAXIA

    Directory of Open Access Journals (Sweden)

    S. Etemad Ahari

    2008-11-01

    Full Text Available ObjectiveMitochondrial DNA (mtDNA is considered a candidate modifier factor for neuro-degenerative disorders. The most common type of ataxia is Friedreich's ataxia (FA. The aim of this study was to investigate different parts of mtDNA in 20 Iranian FA patients and 80 age-matched controls by polymerase chain reaction (PCR and automated DNA sequencing methods to find any probable point mutations involved in the pathogenesis of FA.Materials and MethodsWe identified 13 nucleotide substitutions including A3505G, T3335C, G3421A, G8251A, A8563G, A8563G, G8584A, T8614C, T8598C, C8684T, A8701G, G8994A and A9024G.ResultsTwelve of 13 nucleotide substitutions had already been reported as polymorphism. One of the nucleotide substitutions (A9024G had not been reported before. The A9024G nucleotide substitution does not change its amino acid. The controls were also investigated for this polymorphism which was found in two of them (2.5%.ConclusionNone of the mutations found in this study can affect the clinical manifestations of FA. This survey also provides evidence that the mtDNA A9024G allele is a new nonpathogenic polymorphism. We suggest follow-up studies for this polymorphism in different populations.Keywords:Mitochondrial DNA, Friedreich's Ataxia

  2. Prospects for DNA methods to measure human heritable mutation rates

    International Nuclear Information System (INIS)

    A workshop cosponsored by ICPEMC and the US Department of Energy was held in Alta, Utah, December 9-13, 1984 to examine the extent to which DNA-oriented methods might provide new approaches to the important but intractable problem of measuring mutation rates in control and exposed human populations. The workshop identified and analyzed six DNA methods for detection of human heritable mutation, including several created at the meeting, and concluded that none of the methods combine sufficient feasibility and efficiency to be recommended for general application. 8 refs

  3. Marcus model of spontaneous point mutation in DNA

    Science.gov (United States)

    Turaeva, N.; Brown-Kennerly, V.

    2015-11-01

    The theoretical model of Löwdin's mechanism of spontaneous mutation based on 2D Marcus theory of DPT has been proposed in this work. The equation for the kinetics of DPT during DNA replication has been established, and the expression for the probability of spontaneous mutation has been received. The probability of spontaneous mutation formation has been estimated for tautomeric G∗-C∗ complexes, which is in the range of experimental results. The probability of spontaneous mutation as a function of temperature, replication rate, and solvent effect has been discussed. It increases with temperature and decreases with replication rate. The solvent and pH effects on the probability of spontaneous mutation can also be discussed within the framework of the model.

  4. Mutational properties of the primary aflatoxin B1-DNA adduct.

    OpenAIRE

    Bailey, E A; Iyer, R S; Stone, M. P.; Harris, T M; Essigmann, J M

    1996-01-01

    The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo. An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct was inserted into the single-stranded genome of bacteriophage M13. Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFB1-N7-Gua of 4%. The predominant mutation was G --> T, identical to the principal mutation in human liver tumors believ...

  5. Length Mutations in Human Mitochondrial DNA

    OpenAIRE

    Cann, R. L.; Wilson, A. C.

    1983-01-01

    By high-resolution, restriction mapping of mitochondrial DNAs purified from 112 human individuals, we have identified 14 length variants caused by small additions and deletions (from about 6 to 14 base pairs in length). Three of the 14 length differences are due to mutations at two locations within the D loop, whereas the remaining 11 occur at seven sites that are probably within other noncoding sequences and at junctions between coding sequences. In five of the nine regions of length polymor...

  6. RADIA: RNA and DNA Integrated Analysis for Somatic Mutation Detection

    OpenAIRE

    Radenbaugh, Amie J.; Ma, Singer; Ewing, Adam; Stuart, Joshua M.; Collisson, Eric A.; Zhu, Jingchun; Haussler, David

    2014-01-01

    The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA) to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis), a method that combines the patient-matched normal and tumor DNA with the tumor RN...

  7. Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations.

    Science.gov (United States)

    Itsara, Leslie S; Kennedy, Scott R; Fox, Edward J; Yu, Selina; Hewitt, Joshua J; Sanchez-Contreras, Monica; Cardozo-Pelaez, Fernando; Pallanck, Leo J

    2014-02-01

    The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5)), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations. PMID:24516391

  8. Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations.

    Directory of Open Access Journals (Sweden)

    Leslie S Itsara

    2014-02-01

    Full Text Available The accumulation of somatic mitochondrial DNA (mtDNA mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5, an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine, did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.

  9. Absence of pathogenic mitochondrial DNA mutations in mouse brain tumors

    International Nuclear Information System (INIS)

    Somatic mutations in the mitochondrial genome occur in numerous tumor types including brain tumors. These mutations are generally found in the hypervariable regions I and II of the displacement loop and unlikely alter mitochondrial function. Two hypervariable regions of mononucleotide repeats occur in the mouse mitochondrial genome, i.e., the origin of replication of the light strand (OL) and the Arg tRNA. In this study we examined the entire mitochondrial genome in a series of chemically induced brain tumors in the C57BL/6J strain and spontaneous brain tumors in the VM mouse strain. The tumor mtDNA was compared to that of mtDNA in brain mitochondrial populations from the corresponding syngeneic mouse host strain. Direct sequencing revealed a few homoplasmic base pair insertions, deletions, and substitutions in the tumor cells mainly in regions of mononucleotide repeats. A heteroplasmic mutation in the 16srRNA gene was detected in a spontaneous metastatic VM brain tumor. None of the mutations were considered pathogenic, indicating that mtDNA somatic mutations do not likely contribute to the initiation or progression of these diverse mouse brain tumors

  10. Absence of pathogenic mitochondrial DNA mutations in mouse brain tumors

    Directory of Open Access Journals (Sweden)

    Seyfried Thomas N

    2005-08-01

    Full Text Available Abstract Background Somatic mutations in the mitochondrial genome occur in numerous tumor types including brain tumors. These mutations are generally found in the hypervariable regions I and II of the displacement loop and unlikely alter mitochondrial function. Two hypervariable regions of mononucleotide repeats occur in the mouse mitochondrial genome, i.e., the origin of replication of the light strand (OL and the Arg tRNA. Methods In this study we examined the entire mitochondrial genome in a series of chemically induced brain tumors in the C57BL/6J strain and spontaneous brain tumors in the VM mouse strain. The tumor mtDNA was compared to that of mtDNA in brain mitochondrial populations from the corresponding syngeneic mouse host strain. Results Direct sequencing revealed a few homoplasmic base pair insertions, deletions, and substitutions in the tumor cells mainly in regions of mononucleotide repeats. A heteroplasmic mutation in the 16srRNA gene was detected in a spontaneous metastatic VM brain tumor. Conclusion None of the mutations were considered pathogenic, indicating that mtDNA somatic mutations do not likely contribute to the initiation or progression of these diverse mouse brain tumors.

  11. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile. PMID:26409535

  12. A semi-automated protocol for Archaea DNA extraction from stools.

    OpenAIRE

    Khelaifia, Saber; Ramonet, Pierre-Yves; Bedotto Buffet, Marielle; Drancourt, Michel

    2013-01-01

    BACKGROUND: The PCR-based detection of archaea DNA in human specimens relies on efficient DNA extraction. We previously designed one such protocol involving only manual steps. In an effort to reduce the workload involved, we compared this manual protocol to semi-automated and automated protocols for archaea DNA extraction from human specimens. FINDINGS: We tested 110 human stool specimens using each protocol. An automated protocol using the EZ1 Advanced XL extractor with the V 1.066069118 Qia...

  13. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    OpenAIRE

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J.; Morling, Niels

    2013-01-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cell...

  14. The influence of DNA repair inhibitors on the mutation rate

    International Nuclear Information System (INIS)

    The simultaneous influence of gamma-radiation and DNA-repair inhibiting substances on the mutation frequency of mice was investigated in vivo with the micronucleus test. The detergens Tween 80, vitamin A, and the antiphlogisticum phenylbutazone were used as DNA-repair inhibiting substances. Using the same irradiation doses, a statistic significant increase of mutagenicity respectively micronucleus frequency was found in high concentrations of Tween 80 and in all used dosages of vitamin A, but not in phenylbutazone and in low concentrations of tween. (auth.)

  15. Development of an Automated DNA Detection System Using an Electrochemical DNA Chip Technology

    Science.gov (United States)

    Hongo, Sadato; Okada, Jun; Hashimoto, Koji; Tsuji, Koichi; Nikaido, Masaru; Gemma, Nobuhiro

    A new compact automated DNA detection system Genelyzer™ has been developed. After injecting a sample solution into a cassette with a built-in electrochemical DNA chip, processes from hybridization reaction to detection and analysis are all operated fully automatically. In order to detect a sample DNA, electrical currents from electrodes due to an oxidization reaction of electrochemically active intercalator molecules bound to hybridized DNAs are detected. The intercalator is supplied as a reagent solution by a fluid supply unit of the system. The feasibility test proved that the simultaneous typing of six single nucleotide polymorphisms (SNPs) associated with a rheumatoid arthritis (RA) was carried out within two hours and that all the results were consistent with those by conventional typing methods. It is expected that this system opens a new way to a DNA testing such as a test for infectious diseases, a personalized medicine, a food inspection, a forensic application and any other applications.

  16. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)

    N R Jena

    2012-07-01

    DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and DNA–protein crosslinks can also affect the structure of DNA significantly. These structural modifications are involved in mutation, cancer and many other diseases. As it has the least oxidation potential among all the DNA bases, guanine is frequently attacked by reactive species, producing a plethora of lethal lesions. Fortunately, living cells are evolved with intelligent enzymes that continuously protect DNA from such damages. This review provides an overview of different guanine lesions formed due to reactions of guanine with different reactive species. Involvement of these lesions in inter- and intra-strand crosslinks, DNA–protein crosslinks and mutagenesis are discussed. How certain enzymes recognize and repair different guanine lesions in DNA are also presented.

  17. Common Mitochondrial DNA Mutations Generated through DNA-Mediated Charge Transport

    OpenAIRE

    Merino, Edward J.; Davis, Molly L.; Barton, Jacqueline K.

    2009-01-01

    Mutation sites that arise in human mitochondrial DNA as a result of oxidation by a rhodium photooxidant have been identified. HeLa cells were incubated with [Rh(phi)2bpy]Cl3 (phi is 9,10-phenanthrenequinone diimine), an intercalating photooxidant, to allow the complex to enter the cell and bind mitochondrial DNA. Photoexcitation of DNA-bound [Rh(phi)2bpy]3+ can promote the oxidation of guanine from a distance through DNA-mediated charge transport. After two rounds of photolysis and growth of ...

  18. Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

    Energy Technology Data Exchange (ETDEWEB)

    Lushaj, Entela B., E-mail: lushaj@surgery.wisc.edu [Division of Cardiothoracic Surgery, Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53792 (United States); Lozonschi, Lucian; Barnes, Maria; Anstadt, Emily; Kohmoto, Takushi [Division of Cardiothoracic Surgery, Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53792 (United States)

    2012-06-01

    We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.

  19. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    International Nuclear Information System (INIS)

    Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis

  20. Summary statistics of neutral mutations in longitudinal DNA samples.

    Science.gov (United States)

    Liu, Xiaoming; Fu, Yun-Xin

    2008-08-01

    Longitudinal samples of DNA sequences are the DNA sequences sampled from the same population at different time points. For fast evolving organisms, e.g. RNA virus, these kind of samples have increasingly been used to study the evolutionary process in action. Longitudinal samples provide some interesting new summary statistics of genetic variation, such as the frequency of mutation of size i in one sample and size j in another, the average number of mutations accumulated since the common ancestor of two sequences each from a different sample, and number of private, shared and fixed mutations within samples. To make the results more applicable, we used in this study a general two-sample model, which assumes two longitudinal samples were taken from the same measurably evolving population. Inspired by the HIV study, we also studied a two-sample-two-stage model, which is a special case of two-sample model and assumes a treatment after the first sampling instantaneously changes the population size. We derived the formulas for calculating statistical properties, e.g. expectations, variances and covariances, of these new summary statistics under the two models. Potential applications of these results were discussed. PMID:18547598

  1. Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences.

    Science.gov (United States)

    Paul, Moumita; Murray, Vincent

    2012-03-01

    The determination of the sequence selectivity of DNA-damaging agents is very important in elucidating the mechanism of action of anti-tumour drugs. The development of automated capillary DNA sequencers with fluorescent labelling has enabled a more precise method for DNA sequence specificity analysis. In this work we utilized the ABI 3730 capillary sequencer with laser-induced fluorescence to examine the sequence selectivity of cisplatin with purified DNA sequences. The use of this automated machine enabled a higher degree of precision of both position and intensity of cisplatin-DNA adducts than previously possible with manual and automated slab gel procedures. A problem with artefact bands was overcome by ethanol precipitation. It was found that cisplatin strongly formed adducts with telomeric DNA sequences. PMID:21678458

  2. Simultaneous DNA and RNA mapping of somatic mitochondrial mutations across diverse human cancers

    DEFF Research Database (Denmark)

    Stewart, James B.; Alaei-Mahabadi, Babak; Radhakrishnan, Sabarinathan; Samuelsson, Tore; Gorodkin, Jan; Gustafsson, Claes M; Larsson, Erik

    2015-01-01

    Somatic mutations in the nuclear genome are required for tumor formation, but the functional consequences of somatic mitochondrial DNA (mtDNA) mutations are less understood. Here we identify somatic mtDNA mutations across 527 tumors and 14 cancer types, using an approach that takes advantage of e...... mitochondrial tRNA biogenesis that are difficult to address in controlled experimental systems.......Somatic mutations in the nuclear genome are required for tumor formation, but the functional consequences of somatic mitochondrial DNA (mtDNA) mutations are less understood. Here we identify somatic mtDNA mutations across 527 tumors and 14 cancer types, using an approach that takes advantage of......, demonstrating that correct tRNA folding is a major determinant for processing of polycistronic mitochondrial transcripts. Additionally, the data suggest that tRNA clusters are preferably processed in the 3' to 5' direction. Our study gives insights into mtDNA function in cancer and answers questions regarding...

  3. Mutations in the D-loop region of mitochondrial DNA in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Yibing Zhao; Hongyu Yang; Guoyu Chen

    2005-01-01

    Objective: To investigate the mutations in the D-loop region of mitochondrial DNA (mtDNA) in gastric cancer.Methods: The mtDNA of D-loop region was amplified by PCR and sequenced in 20 samples from gastric cancer tissue and adjacent normal membrane. Results: There were 7/20(35% ) mutations in the mtDNA of D-loop region in gastric cancer patients. There were four microsatellite instabilities among the 18 mutations. Nine new polymorphisms were identified in 20 patients. Conclusion: The mtDNA of Dloop region might be highly polymorphoric and the mutation rate is high in patients with gastric cancer.

  4. Mitochondrial cardiomyopathies: how to identify candidate pathogenic mutations by mitochondrial DNA sequencing, MITOMASTER and phylogeny

    OpenAIRE

    Zaragoza, Michael V; Brandon, Martin C; Diegoli, Marta; Arbustini, Eloisa; Wallace, Douglas C.

    2010-01-01

    Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause cardiomyopathy and heart failure. Owing to a high mutation rate, mtDNA defects may occur at any nucleotide in its 16 569 bp sequence. Complete mtDNA sequencing may detect pathogenic mutations, which can be difficult to interpret because of normal ethnic/geographic-associated haplogroup variation. Our goal is to show how to identify candidate mtDNA mutations by sorting out polymorphisms using readily ...

  5. Evaluation of Automated and Manual Commercial DNA Extraction Methods for Recovery of Brucella DNA from Suspensions and Spiked Swabs ▿

    OpenAIRE

    Dauphin, Leslie A.; Hutchins, Rebecca J.; Bost, Liberty A.; Bowen, Michael D.

    2009-01-01

    This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit...

  6. Mutations in the mitochondrial DNA D-loop region are frequent in cervical cancer

    OpenAIRE

    Jain Sunesh; Sharma Chandresh; Singh Archna; Sharma Himani; Singh Neeta

    2005-01-01

    Abstract Background Mitochondrial DNA (mtDNA) is known for high mutation rates caused by lack of protective histones, inefficient DNA repair systems, and continuous exposure to mutagenic effects of oxygen radicals. Alterations in the non-coding displacement (D) loop of mitochondrial DNA are present in many cancers. It has been suggested that the extent of mitochondrial DNA mutations might be useful in the prognosis of cancer outcome and/or the response to certain therapies. In order to invest...

  7. Role of DNA deletion length in mutation and cell survival

    International Nuclear Information System (INIS)

    A model is presented which is based on the assumption that malignant transformation, mutation, chromosome aberration, and reproductive death of cells are all manifestations of radiation induced deletions in the DNA of the cell, and that the size of the deletion in relation to the spacing of essential genes determines the consequences of that deletion. It is assumed that two independent types of potentially lethal lesions can result in DNA deletions, and that the relative numbers of these types of damage is dependent on radiation quality. The repair of the damage reduces the length of a deletion, but does not always eliminate it. The predictions of this model are in good agreement with a wide variety of experimental evidence. (author)

  8. Electrophile and oxidant damage of mitochondrial DNA leading to rapid evolution of homoplasmic mutations

    OpenAIRE

    Mambo, Elizabeth; Gao, Xiangqun; Cohen, Yoram; Guo, Zhongmin; Talalay, Paul; Sidransky, David

    2003-01-01

    mtDNA mutations occur in a wide variety of degenerative diseases and cancer. mtDNA seems to be more susceptible to DNA damage and consequently sustains higher rates of mutation than does nuclear DNA (nDNA). Many of the somatic mtDNA mutations in human cancers are located in the displacement loop (D-loop) and in particular in a polycytidine stretch (C-tract) termed D310. The D310 region exhibits polymorphic length variation among individuals and has been described as a “hot spot” for somatic m...

  9. Mitochondrial DNA mutations in preneoplastic lesions of the gastrointestinal tract: A biomarker for the early detection of cancer

    Directory of Open Access Journals (Sweden)

    Montgomery Elizabeth A

    2006-12-01

    Full Text Available Abstract Background Somatic mutations of mitochondrial DNA (mtDNA are common in many human cancers. We have described an oligonucleotide microarray ("MitoChip" for rapid sequencing of the entire mitochondrial genome (Zhou et al, J Mol Diagn 2006, facilitating the analysis of mtDNA mutations in preneoplastic lesions. We examined 14 precancerous lesions, including seven Barrett esophagus biopsies, with or without associated dysplasia; four colorectal adenomas; and three inflammatory colitis-associated dysplasia specimens. In all cases, matched normal tissues from the corresponding site were obtained as germline control. MitoChip analysis was performed on DNA obtained from cryostat-embedded specimens. Results A total of 513,639 bases of mtDNA were sequenced in the 14 samples, with 490,224 bases (95.4% bases assigned by the automated genotyping software. All preneoplastic lesions examined demonstrated at least one somatic mtDNA sequence alteration. Of the 100 somatic mtDNA alterations observed in the 14 cases, 27 were non-synonymous coding region mutations (i.e., resulting in an amino acid change, 36 were synonymous, and 37 involved non-coding mtDNA. Overall, somatic alterations most commonly involved the COI, ND4 and ND5 genes. Notably, somatic mtDNA alterations were observed in preneoplastic lesions of the gastrointestinal tract even in the absence of histopathologic evidence of dysplasia, suggesting that the mitochondrial genome is susceptible at the earliest stages of multistep cancer progression. Conclusion Our findings further substantiate the rationale for exploring the mitochondrial genome as a biomarker for the early diagnosis of cancer, and confirm the utility of a high-throughput array-based platform for this purpose from a clinical applicability standpoint.

  10. Studies of human mutation rates: Progress report

    International Nuclear Information System (INIS)

    Progress was recorded between January 1 and July 1, 1987 on a project entitled ''Studies of Human Mutation Rates''. Studies underway include methodology for studying mutation at the DNA level, algorithms for automated analyses of two-dimensional polyacrylamide DNA gels, theoretical and applied population genetics, and studies of mutation frequency in A-bomb survivors

  11. Review: Clinical aspects of hereditary DNA Mismatch repair gene mutations.

    Science.gov (United States)

    Sijmons, Rolf H; Hofstra, Robert M W

    2016-02-01

    Inherited mutations of the DNA Mismatch repair genes MLH1, MSH2, MSH6 and PMS2 can result in two hereditary tumor syndromes: the adult-onset autosomal dominant Lynch syndrome, previously referred to as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and the childhood-onset autosomal recessive Constitutional Mismatch Repair Deficiency syndrome. Both conditions are important to recognize clinically as their identification has direct consequences for clinical management and allows targeted preventive actions in mutation carriers. Lynch syndrome is one of the more common adult-onset hereditary tumor syndromes, with thousands of patients reported to date. Its tumor spectrum is well established and includes colorectal cancer, endometrial cancer and a range of other cancer types. However, surveillance for cancers other than colorectal cancer is still of uncertain value. Prophylactic surgery, especially for the uterus and its adnexa is an option in female mutation carriers. Chemoprevention of colorectal cancer with aspirin is actively being investigated in this syndrome and shows promising results. In contrast, the Constitutional Mismatch Repair Deficiency syndrome is rare, features a wide spectrum of childhood onset cancers, many of which are brain tumors with high mortality rates. Future studies are very much needed to improve the care for patients with this severe disorder. PMID:26746812

  12. Random point mutations with major effects on protein-coding genes are the driving force behind premature aging in mtDNA mutator mice.

    NARCIS (Netherlands)

    Edgar, D.; Shabalina, I.; Camara, Y.; Wredenberg, A.; Calvaruso, M.A.; Nijtmans, L.G.J.; Nedergaard, J.; Cannon, B.; Larsson, N.G.; Trifunovic, A.

    2009-01-01

    The mtDNA mutator mice have high levels of point mutations and linear deletions of mtDNA causing a progressive respiratory chain dysfunction and a premature aging phenotype. We have now performed molecular analyses to determine the mechanism whereby these mtDNA mutations impair respiratory chain fun

  13. Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication.

    Science.gov (United States)

    Shinbrot, Eve; Henninger, Erin E; Weinhold, Nils; Covington, Kyle R; Göksenin, A Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M; Gibbs, Richard A; Sander, Chris; Pursell, Zachary F; Wheeler, David A

    2014-11-01

    Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication. PMID:25228659

  14. The m.13051G>A mitochondrial DNA mutation results in variable neurology and activated mitophagy

    OpenAIRE

    Dombi, E.; Diot, A.; Morten, K.; Carver, J; Lodge, T.; Fratter, C.; Ng, Y.S.; Liao, C.; Muir, R; Blakely, E.L.; Hargreaves, I; Al-Dosary, M.; Sarkar, G; Hickman, S. J.; Downes, S M

    2016-01-01

    Maternally inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber hereditary optic neuropathy (LHON) in -1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects1 and there is no proven prophylactic treatment.

  15. Relationship between mutations of mitochondrial DNA ND1 gene and type 2 diabetes

    Institute of Scientific and Technical Information of China (English)

    于珮; 于德民; 刘德敏; 王琨; 汤新之

    2004-01-01

    Background Recent studies have indicated that many mutations in mitochondrial (mt)DNA NDI gene region are related to diabetes mellitus. In this study we explored the relationship between various mtDNA ND1 gene mutations and type 2 diabetes mellitus (DM) among Chinese. Methods Using PCR restriction fragment length polymorphism (PCR-RFLP) analysis and gene sequencing, 4 spots of mtDNA (nt3243, nt3316, nt3394, nt3426) were screened in 478 diabetics and 430 non-diabetic subjects.Results In diabetic group, there were 13 carriers (2.72%)of 3316 G→A mutation,12 (2.51%) of 3394 T→C mutation and 2 (0.42%) of 3426A→G mutation. In controls, only 3394 T→C mutation was observed in 2 subjects (0.47%). There was significant difference in the frequency of 3316 and 3394 mutation between two groups (P<0.05, respectively). More subjects with mitochondrial DNA ND1 gene mutations had DM family history and greater tendency of maternal inheritance when compared to those patients without mutation in diabetic group(P<0.01). A 3426 mutation diabetic pedigree was studied, and we found 12 maternal members in the family had the same mutation. Conclusion mtDNA ND1 gene mutations at nt3316 (G→A), nt3394 (T→C) and 3426 (A→G) might contribute to the pathogenesis of DM with other genetic factors and environment factors.

  16. Phylogenetic analysis of mitochondrial DNA:detection of mutations in patients with occipital stroke

    OpenAIRE

    Finnilä, S. (Saara)

    2000-01-01

    Abstract A mitochondrial disorder may be one of the rare aetiologies of occipital stroke. Clinical and molecular analysis has suggested that 10% of young patients with occipital stroke have a mitochondrial disorder and 6% harbour the mutation 3243A>G in mitochondrial DNA (mtDNA), causing the MELAS syndrome. To identify other possible mtDNA mutations involved, we studied mtDNA genotypes in patients who had suffered an occipital stroke and in whom the common pathogenic m...

  17. Deoxyribonucleic acid initiation mutation dnaB252 is suppressed by elevated dnaC+ gene dosage.

    OpenAIRE

    Sclafani, R A; Wechsler, J A

    1981-01-01

    The Escherichia coli dnaB252 allele is the only dnaB mutation which confers a deoxyribonucleic acid initiation-defective phenotype on the cell. The presence of a multicopy hybrid plasmid containing the dnaC+ gene in a dnaB252 strain completely suppressed the temperature-sensitive phenotype. It is suggested that at high temperature the dnaB252 protein has a lowered affinity for dnaC protein, and that the formation of a dnaB-dnaC complex is mandatory for initiation.

  18. Significance of somatic mutations and content alteration of mitochondrial DNA in esophageal cancer

    International Nuclear Information System (INIS)

    The roles of mitochondria in energy metabolism, the generation of ROS, aging, and the initiation of apoptosis have implicated their importance in tumorigenesis. In this study we aim to establish the mutation spectrum and to understand the role of somatic mtDNA mutations in esophageal cancer. The entire mitochondrial genome was screened for somatic mutations in 20 pairs (18 esophageal squamous cell carcinomas, one adenosquamous carcinoma and one adenocarcinoma) of tumor/surrounding normal tissue of esophageal cancers, using temporal temperature gradient gel electrophoresis (TTGE), followed by direct DNA sequencing to identify the mutations. Fourteen somatic mtDNA mutations were identified in 55% (11/20) of tumors analyzed, including 2 novel missense mutations and a frameshift mutation in ND4L, ATP6 subunit, and ND4 genes respectively. Nine mutations (64%) were in the D-loop region. Numerous germline variations were found, at least 10 of them were novel and five were missense mutations, some of them occurred in evolutionarily conserved domains. Using real-time quantitative PCR analysis, the mtDNA content was found to increase in some tumors and decrease in others. Analysis of molecular and other clinicopathological findings does not reveal significant correlation between somatic mtDNA mutations and mtDNA content, or between mtDNA content and metastatic status. Our results demonstrate that somatic mtDNA mutations in esophageal cancers are frequent. Some missense and frameshift mutations may play an important role in the tumorigenesis of esophageal carcinoma. More extensive biochemical and molecular studies will be necessary to determine the pathological significance of these somatic mutations

  19. Significance of somatic mutations and content alteration of mitochondrial DNA in esophageal cancer

    Directory of Open Access Journals (Sweden)

    Wang Yu-Fen

    2006-04-01

    Full Text Available Abstract Background The roles of mitochondria in energy metabolism, the generation of ROS, aging, and the initiation of apoptosis have implicated their importance in tumorigenesis. In this study we aim to establish the mutation spectrum and to understand the role of somatic mtDNA mutations in esophageal cancer. Methods The entire mitochondrial genome was screened for somatic mutations in 20 pairs (18 esophageal squamous cell carcinomas, one adenosquamous carcinoma and one adenocarcinoma of tumor/surrounding normal tissue of esophageal cancers, using temporal temperature gradient gel electrophoresis (TTGE, followed by direct DNA sequencing to identify the mutations. Results Fourteen somatic mtDNA mutations were identified in 55% (11/20 of tumors analyzed, including 2 novel missense mutations and a frameshift mutation in ND4L, ATP6 subunit, and ND4 genes respectively. Nine mutations (64% were in the D-loop region. Numerous germline variations were found, at least 10 of them were novel and five were missense mutations, some of them occurred in evolutionarily conserved domains. Using real-time quantitative PCR analysis, the mtDNA content was found to increase in some tumors and decrease in others. Analysis of molecular and other clinicopathological findings does not reveal significant correlation between somatic mtDNA mutations and mtDNA content, or between mtDNA content and metastatic status. Conclusion Our results demonstrate that somatic mtDNA mutations in esophageal cancers are frequent. Some missense and frameshift mutations may play an important role in the tumorigenesis of esophageal carcinoma. More extensive biochemical and molecular studies will be necessary to determine the pathological significance of these somatic mutations.

  20. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura;

    2013-01-01

    from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore......, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already......Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction...

  1. Streamlining DNA barcoding protocols: automated DNA extraction and a new cox1 primer in arachnid systematics.

    Directory of Open Access Journals (Sweden)

    Nina Vidergar

    Full Text Available BACKGROUND: DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences--mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1--are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1 improving an automated DNA extraction protocol, (2 testing the performance of commonly used primer combinations, and (3 developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses. METHODOLOGY: We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor-an automated high throughput DNA extraction system-and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs. RESULTS: The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198 that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93% matched that of C1-J-2183. CONCLUSIONS: The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding.

  2. Rapid Detection of Mutation in RRDR of rpo B Gene for Rifampicin Resistance in MDR-Pulmonary Tuberculosis by DNA Sequencing

    OpenAIRE

    Patra, Surajeet Kumar; Jain, Anju; B L Sherwal; Khanna, Ashwani

    2010-01-01

    To detect the site of mutation in RRDR of rpo B gene for rifampicin resistance in MDR-TB by DNA sequencing. 50 MDR-TB patients were enrolled in our study after informed written consent. Mycobacterial DNA was extracted from sputum samples by Universal Sample Processing (USP) method and RRDR of rpo B gene was amplified by PCR using primers RP4T and RP8T and then sequenced by automated DNA sequencing. The nucleotide sequences of RRDR of rpo B gene were compared with the reference sequence. We ob...

  3. Scar-less multi-part DNA assembly design automation

    Energy Technology Data Exchange (ETDEWEB)

    Hillson, Nathan J.

    2016-06-07

    The present invention provides a method of a method of designing an implementation of a DNA assembly. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which to assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding flanking homology sequences to each of the DNA oligos. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which to assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding optimized overhang sequences to each of the DNA oligos.

  4. Direct estimation of the mitochondrial DNA mutation rate in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Cathy Haag-Liautard

    2008-08-01

    Full Text Available Mitochondrial DNA (mtDNA variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA. We detected a total of 28 point mutations and eight insertion-deletion (indel mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 x 10(-8 per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G-->A mutations on the major strand (the sense strand for the majority of mitochondrial genes. These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10x higher than the nuclear mutation rate, but the mitochondrial major strand G-->A mutation rate is about 70x higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base

  5. An automated annotation tool for genomic DNA sequences using GeneScan and BLAST

    Indian Academy of Sciences (India)

    Andrew M. Lynn; Chakresh Kumar Jain; K. Kosalai; Pranjan Barman; Nupur Thakur; Harish Batra; Alok Bhattacharya

    2001-04-01

    Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated annotation of genome DNA sequences.

  6. Multi-center evaluation of the novel fully-automated PCR-based Idylla™ BRAF Mutation Test on formalin-fixed paraffin-embedded tissue of malignant melanoma.

    Science.gov (United States)

    Melchior, Linea; Grauslund, Morten; Bellosillo, Beatriz; Montagut, Clara; Torres, Erica; Moragón, Ester; Micalessi, Isabel; Frans, Johan; Noten, Veerle; Bourgain, Claire; Vriesema, Renske; van der Geize, Robert; Cokelaere, Kristof; Vercooren, Nancy; Crul, Katrien; Rüdiger, Thomas; Buchmüller, Diana; Reijans, Martin; Jans, Caroline

    2015-12-01

    The advent of BRAF-targeted therapies led to increased survival in patients with metastatic melanomas harboring a BRAF V600 mutation (implicated in 46-48% of malignant melanomas). The Idylla(™) System (Idylla(™)), i.e., the real-time-PCR-based Idylla(™) BRAF Mutation Test performed on the fully-automated Idylla(™) platform, enables detection of the most frequent BRAF V600 mutations (V600E/E2/D, V600K/R/M) in tumor material within approximately 90 min and with 1% detection limit. Idylla(™) performance was determined in a multi-center study by analyzing BRAF mutational status of 148 archival formalin-fixed paraffin-embedded (FFPE) tumor samples from malignant melanoma patients, and comparing Idylla(™) results with assessments made by commercial or in-house routine diagnostic methods. Of the 148 samples analyzed, Idylla(™) initially recorded 7 insufficient DNA input calls and 15 results discordant with routine method results. Further analysis learned that the quality of 8 samples was insufficient for Idylla(™) testing, 1 sample had an invalid routine test result, and Idylla(™) results were confirmed in 10 samples. Hence, Idylla(™) identified all mutations present, including 7 not identified by routine methods. Idylla(™) enables fully automated BRAF V600 testing directly on FFPE tumor tissue with increased sensitivity, ease-of-use, and much shorter turnaround time compared to existing diagnostic tests, making it a tool for rapid, simple and highly reliable analysis of therapeutically relevant BRAF mutations, in particular for diagnostic units without molecular expertise and infrastructure. PMID:26407762

  7. Study in mutation of alfalfa genome DNA due to low energy N+ implantation using RAPD

    International Nuclear Information System (INIS)

    After implanted by various dosage N+ beams, germination rate of alfalfa seeds appears to be saddle line with dosage increasing. The authors have studied in mutation of genome DNA due to low energy N+ implantation, and concluded that 30 differential DNA fragments have been amplified by 8 primers (S41, S42, S45, S46, S50, S52, S56, S58) in 100 primers, moreover, number of differential DNA fragments between CK and treatments increases with dosage. Consequently, low energy ion implantation can cause mutation of alfalfa genome DNA. The more dosage it is, the more mutation alfalfa will be

  8. Previous Estimates of Mitochondrial DNA Mutation Level Variance Did Not Account for Sampling Error: Comparing the mtDNA Genetic Bottleneck in Mice and Humans

    OpenAIRE

    Wonnapinij, Passorn; Chinnery, Patrick F.; Samuels, David C

    2010-01-01

    In cases of inherited pathogenic mitochondrial DNA (mtDNA) mutations, a mother and her offspring generally have large and seemingly random differences in the amount of mutated mtDNA that they carry. Comparisons of measured mtDNA mutation level variance values have become an important issue in determining the mechanisms that cause these large random shifts in mutation level. These variance measurements have been made with samples of quite modest size, which should be a source of concern becaus...

  9. Mutations in the mitochondrial DNA D-loop region are frequent in cervical cancer

    Directory of Open Access Journals (Sweden)

    Jain Sunesh

    2005-12-01

    Full Text Available Abstract Background Mitochondrial DNA (mtDNA is known for high mutation rates caused by lack of protective histones, inefficient DNA repair systems, and continuous exposure to mutagenic effects of oxygen radicals. Alterations in the non-coding displacement (D loop of mitochondrial DNA are present in many cancers. It has been suggested that the extent of mitochondrial DNA mutations might be useful in the prognosis of cancer outcome and/or the response to certain therapies. In order to investigate whether a high incidence of mutations exist in mitochondrial DNA of cervical cancer patients, we examined the frequency of mutations in the D-loop region in 19 patients of cervical cancer. Results Mutations, often multiple, were detected in 18 of 19 (95% patients. The presence of mutations correlated with Human Papilloma Virus (HPV infection in these patients. Mutations were also detected in normal samples and lymphocytes obtained from cervical cancer patients, but their frequency of occurrence was much lower as compared to the cervical cancer tissues. Conclusion Our findings indicate that D-loop alterations are frequent in cervical cancers and are possibly caused by HPV infection. There was no association of mtDNA D-loop mutations with the histopathological grade and tumor staging.

  10. Limited clinical relevance of mitochondrial DNA mutation and gene expression analyses in ovarian cancer

    OpenAIRE

    Rachinger Andrea; Bartnik Ewa; Kupryjanczyk Jolanta; Bragoszewski Piotr; Ostrowski Jerzy

    2008-01-01

    Abstract Background In recent years, numerous studies have investigated somatic mutations in mitochondrial DNA in various tumours. The observed high mutation rates might reflect mitochondrial deregulation; consequently, mutation analyses could be clinically relevant. The purpose of this study was to determine if mutations in the mitochondrial D-loop region and/or the level of mitochondrial gene expression could influence the clinical course of human ovarian carcinomas. Methods We sequenced a ...

  11. Deciphering the spectrum of somatic mutations in the entire mitochondrial DNA genome.

    Science.gov (United States)

    Chen, X Z; Fang, Y; Shi, Y H; Cui, J H; Li, L Y; Xu, Y C; Ling, B

    2015-01-01

    The mitochondrion is a crucial intracellular organelle responsible for regulating cellular energy metabolism, producing free radicals, initiating and executing the apoptotic pathways. Previous studies have shown that somatic mutations in mitochondrial DNA are associated with various tumors, which may be involved during carcinogenesis and tumor progression. To examine the mutation pattern in cancer, 625 reported somatic mutations in the mitochondrial DNA genome were analyzed. We found that, except for deletions and insertions, most somatic mutations were point mutations, accounting for 89.44% of somatic mutations. Transition was the predominant form of somatic mutation in the entire mitochondrial DNA genome, accounting for 87.12% of point mutations, most of which were homoplastic. Frequency statistics analysis of point mutations indicated that, except for 3 tRNA genes, the mutations were distributed on all resting genes and in the D-loop region, with the latter showing the highest frequency of somatic mutation (19.34%), followed by the tRNA leucine 2 gene and non-coding regions between base pairs 5892 and 5903, while 13 coding-region genes and 2 rRNA genes showed a relatively lower frequency of somatic point mutations. Nonsynonymous mutations and terminal amino acid changes were the primary point somatic mutations detected from 13 coding-region genes, which may cause mitochondrial dysfunction in cancer cells. We found that the somatic mutations may affect the mitochondrial DNA genome; the non-coding region should be examined to identify somatic mutations as potential diagnostic biomarkers for early detection of cancer. PMID:25966205

  12. Frequency and phenotypic implications of mitochondrial DNA mutations in human squamous cell cancers of the head and neck

    OpenAIRE

    Zhou, Shaoyu; Kachhap, Sushant; Sun, Wenyue; Wu, Guojun; Chuang, Alice; Poeta, Luana; Grumbine, Lawson; Mithani, Suhail K.; Chatterjee, Aditi; Koch, Wayne; Westra, William H.; Maitra, Anirban; Glazer, Chad; Carducci, Michael; Sidransky, David

    2007-01-01

    Mitochondrial genomic mutations are found in a variety of human cancers; however, the frequency of mitochondrial DNA (mtDNA) mutations in coding regions remains poorly defined, and the functional effects of mitochondrial mutations found in primary human cancers are not well described. Using MitoChip, we sequenced the whole mitochondrial genome in 83 head and neck squamous cell carcinomas. Forty-one of 83 (49%) tumors contained mtDNA mutations. Mutations occurred within noncoding (D-loop) and ...

  13. Applications of capillary electrophoresis in DNA mutation analysis of genetic disorders.

    OpenAIRE

    Le, H; Fung, D.; Trent, R.J.

    1997-01-01

    AIM: To facilitate DNA mutation analysis by use of capillary electrophoresis. METHODS: The usefulness and applications of capillary electrophoresis in DNA fragment sizing and sequencing were evaluated. RESULTS: DNA mutation testing in disorders such as cystic fibrosis, Huntington disease, alpha thalassaemia, and hereditary fructose intolerance were undertaken effectively. However, sizing the (CAG)n repeat in the case of Huntington disease was a potential problem when using capillary electroph...

  14. Mitochondrial DNA mutation screening of male patients with obstructive sleep apnea-hypopnea syndrome

    OpenAIRE

    HUANG Xiao-ying; Li, Hong; XU, XIAO-MEI; Wang, Liang-xing

    2014-01-01

    The aim of the present study was to analyze the differences between the genes of the mitochondrial DNA (mtDNA) displacement loop (D-loop) region and the Cambridge Reference sequence, in order to screen the mutation sites and investigate the correlation between mutations, clinical parameters and complications associated with obstructive sleep apnea-hypopnea syndrome (OSAHS). mtDNA was obtained from male patients with OSAHS in the Zhejiang Province. In total, 60 male patients with OSAHS and 102...

  15. A Modified Extraction Method of Circulating Free DNA for Epidermal Growth Factor Receptor Mutation Analysis

    OpenAIRE

    Yuan, Haihua; Zhu, Zhong-Zheng; Lu, Yachao; Feng LIU; Zhang, Wenying; Huang, Gang; Zhu, Guanshan; Jiang, Bin

    2011-01-01

    Purpose Circulating free DNA (cfDNA) in plasma is promising to be a surrogate for tumor tissue DNA. However, not all epidermal growth factor receptor (EGFR) mutations in tumor tissue DNA has been detected in matched cfDNA, at least partly due to inefficient cfDNA extraction method. The purpose of this study was to establish an efficient plasma cfDNA extraction protocol. Materials and Methods The yield of plasma cfDNA extracted by our modified phenol-chloroform (MPC) method from non-small-cell...

  16. Helicobacter pylori induces mitochondrial DNA mutation and reactive oxygen species level in AGS cells

    Directory of Open Access Journals (Sweden)

    Xue-Wen Huang, Rui-Hua Luo, Qi Zhao, Zhong-Ze Shen, Li-Li Huang, Xian-Yuan An, Lan-Jing Zhao, Jie Wang, Yu-Zheng Huang

    2011-01-01

    Full Text Available To investigate the role of ROS in the helicobacter pylori (Hp induced mtDNA mutations, AGS cells were treated by extracts of Hp11638 or Hp11638M. The ROS levels, cytochrome C reductions, and intracellular ATP levels were measured. The coding region and the D-Loop region were amplified and sequenced. Results showed the ROS levels, cytochrome C reduction and mtDNA mutations were markedly increased and cell viability decreased after treatment with both Hp extracts, and 616 mutations were detected in D-Loop region and 3 heteroplasmic point mutations in the Cytb gene. No mutations were found in the coding region. The mutation rates of mtDNA D-Loop region were positively correlated with the ROS levels and negatively to the ATP levels.

  17. Uracil-DNA glycosylase-deficient yeast exhibit a mitochondrial mutator phenotype

    OpenAIRE

    Chatterjee, Aditi; Keshav K Singh

    2001-01-01

    Mutations in mitochondrial DNA (mtDNA) have been reported in cancer and are involved in the pathogenesis of many mitochondrial diseases. Uracil-DNA glycosylase, encoded by the UNG1 gene in Saccharomyces cerevisiae, repairs uracil in DNA formed due to deamination of cytosine. Our study demonstrates that inactivation of the UNG1 gene leads to at least a 3-fold increased frequency of mutations in mtDNA compared with the wild-type. Using a Ung1p–green fluorescent protein (GFP) fusion construct, w...

  18. Automated extraction of DNA from biological stains on fabric from crime cases. A comparison of a manual and three automated methods

    OpenAIRE

    Stangegaard, Michael; Hjort, Benjamin B; Hansen, Thomas N.; Hoflund, Anders; Mogensen, Helle S; Hansen, Anders J.; Morling, Niels

    2013-01-01

    The presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. DNA extraction from fabric for forensic genetic purposes may be challenging due to the occasional presence of PCR inhibitors that may be co-extracted with the DNA. Using 120 forensic trace evidence samples consisting of various types of fabric, we compared three automated DNA extraction methods based on magnetic beads (Pr...

  19. PIK3CA mutation detection in metastatic biliary cancer using cell-free DNA

    OpenAIRE

    Kim, Seung Tae; Lira, Maruja; Deng, Shibing; Lee, Sujin; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Mao, Mao; Heo, Jin Seok; Kwon, Wooil; Jang, Kee-Taek; Lee, Jeeyun; Park, Joon Oh

    2015-01-01

    PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad's PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, an...

  20. mtDNA mutation C1494T, haplogroup A, and hearing loss in Chinese

    International Nuclear Information System (INIS)

    Mutation C1494T in mitochondrial 12S rRNA gene was recently reported in two large Chinese families with aminoglycoside-induced and nonsyndromic hearing loss (AINHL) and was claimed to be pathogenic. This mutation, however, was first reported in a sample from central China in our previous study that was aimed to reconstruct East Asian mtDNA phylogeny. All these three mtDNAs formed a subclade defined by mutation C1494T in mtDNA haplogroup A. It thus seems that mutation C1494T is a haplogroup A-associated mutation and this matrilineal background may contribute a high risk for the penetrance of mutation C1494T in Chinese with AINHL. To test this hypothesis, we first genotyped mutation C1494T in 553 unrelated individuals from three regional Chinese populations and performed an extensive search for published complete or near-complete mtDNA data sets (>3000 mtDNAs), we then screened the C1494T mutation in 111 mtDNAs with haplogroup A status that were identified from 1823 subjects across China. The search for published mtDNA data sets revealed no other mtDNA besides the above-mentioned three carrying mutation C1494T. None of the 553 randomly selected individuals and the 111 haplogroup A mtDNAs was found to bear this mutation. Therefore, our results suggest that C1494T is a very rare event. The mtDNA haplogroup A background in general is unlikely to play an active role in the penetrance of mutation C1494T in AINHL

  1. Mitochondrial DNA mutation screening of male patients with obstructive sleep apnea-hypopnea syndrome.

    Science.gov (United States)

    Huang, Xiao-Ying; Li, Hong; Xu, Xiao-Mei; Wang, Liang-Xing

    2014-08-01

    The aim of the present study was to analyze the differences between the genes of the mitochondrial DNA (mtDNA) displacement loop (D-loop) region and the Cambridge Reference sequence, in order to screen the mutation sites and investigate the correlation between mutations, clinical parameters and complications associated with obstructive sleep apnea-hypopnea syndrome (OSAHS). mtDNA was obtained from male patients with OSAHS in the Zhejiang Province. In total, 60 male patients with OSAHS and 102 healthy adults were assessed to determine the levels of fasting blood glucose, total cholesterol, triglyceride (TG) and high-density and low-density lipoproteins (LDL). Furthermore, peripheral mtDNA was extracted and bidirectional sequencing was conducted to enable mutation screening. In the mtDNA D-loop region, 178 mutation sites were identified, of which 115 sites were present in the two groups. The number of non-common sites in the OSAHS group was significantly higher compared with the control group (Pmutations among the mild, moderate and severe OSAHS groups (P>0.05). A total of 21 cases in the severe OSAHS group exhibited mutation rates of >10%. In the control group, there were 24 cases where the np73A-G and np263A-G mutations were predominant. The np303-np315 region was identified to be the highly variable region and various mutation forms were observed. Statistically significant differences were observed in the neck perimeter, TG and LDL levels among the OSAHS-no-mutation subgroups (Pmutation in the OSAHS group. Numerous polymorphic mutation sites were identified in the mtDNA D-loop region of the OSAHS group. Therefore, mtDNA mutation sites may be closely associated with the clinical manifestations and complications of OSAHS. PMID:25009612

  2. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Highlights: → Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. → This process is independent of endogenous ROS production. → Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O2) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  3. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kukat, Alexandra [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Edgar, Daniel [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Bratic, Ivana [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Maiti, Priyanka [Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Trifunovic, Aleksandra, E-mail: aleksandra.trifunovic@ki.se [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany)

    2011-06-10

    Highlights: {yields} Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. {yields} This process is independent of endogenous ROS production. {yields} Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O{sub 2}) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  4. Automated serial extraction of DNA and RNA from biobanked tissue specimens

    Science.gov (United States)

    2013-01-01

    Background With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. Results 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. Conclusions The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage. PMID:23957867

  5. SERS-melting: a new method for discriminating mutations in dna sequences

    OpenAIRE

    Mahajan, Sumeet; Richardson, James; Brown, Tom; Bartlett, Philip N

    2008-01-01

    The reliable discrimination of mutations, single nucleotide polymorphisms (SNPs), and other differences in genomic sequence is an essential part of DNA diagnostics and forensics. It is commonly achieved using fluorescently labeled DNA probes and thermal gradients to distinguish between the matched and mismatched DNA. Here, we describe a novel method that uses surface enhanced (resonance) Raman spectroscopy (SER(R)S) to follow denaturation of dsDNA attached to a structured gold surface. T...

  6. Mitochondria DNA mutations cause sex-dependent development of hypertension and alterations in cardiovascular function

    OpenAIRE

    Golob, Mark J.; Tian, Lian; Wang, Zhijie; Zimmerman, Todd A.; Caneba, Christine A.; Hacker, Timothy A.; Song, Guoqing; Chesler, Naomi C.

    2014-01-01

    Aging is associated with conduit artery stiffening that is a risk factor for and can precede hypertension and ventricular dysfunction. Increases in mitochondria DNA (mtDNA) frequency have been correlated with aging. Mice with a mutation in the encoding domain (D257A) of a proof-reading deficient version of mtDNA polymerase-γ (POLG) have musculoskeletal features of premature aging and a shortened lifespan. However, few studies using these mice have investigated the effects of mtDNA mutations o...

  7. Differential DNA mismatch repair underlies mutation rate variation across the human genome

    Science.gov (United States)

    Supek, Fran; Lehner, Ben

    2015-01-01

    Cancer genome sequencing has revealed considerable variation in somatic mutation rates across the human genome, with mutation rates elevated in heterochromatic late replicating regions and reduced in early replicating euchromatin1-5. Multiple mechanisms have been suggested to underlie this2,6-10, but the actual cause is unknown. Here we identify variable DNA mismatch repair (MMR) as the basis of this variation. Analysing ~17 million single nucleotide variants from the genomes of 652 tumours, we show that regional autosomal mutation rates at megabase resolution are largely stable across cancer types, with differences related to changes in replication timing and gene expression. However, mutations arising after the inactivation of MMR are no longer enriched in early replicating euchromatin relative to late replicating heterochromatin. Thus, differential DNA repair and not differential mutation supply is the primary cause of the large-scale regional mutation rate variation across the human genome. PMID:25707793

  8. Automated extraction of DNA from biological stains on fabric from crime cases. A comparison of a manual and three automated methods

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin B; Hansen, Thomas N;

    2013-01-01

    The presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. DNA extraction from fabric for forensic genetic purposes may be challenging due to the occasional presence of PCR inhibitors...... that may be co-extracted with the DNA. Using 120 forensic trace evidence samples consisting of various types of fabric, we compared three automated DNA extraction methods based on magnetic beads (PrepFiler Express Forensic DNA Extraction Kit on an AutoMate Express, QIAsyphony DNA Investigator kit...... either with the sample pre-treatment recommended by Qiagen or an in-house optimized sample pre-treatment on a QIAsymphony SP) and one manual method (Chelex) with the aim of reducing the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable STR-profiles. A total of 480...

  9. Mitochondrial DNA mutations in renal cell carcinomas revealed no general impact on energy metabolism

    OpenAIRE

    Meierhofer, D.; Mayr, J. A.; Fink, K.; Schmeller, N.; Kofler, B; Sperl, W.

    2006-01-01

    Previously, renal cell carcinoma tissues were reported to display a marked reduction of components of the respiratory chain. To elucidate a possible relationship between tumourigenesis and alterations of oxidative phosphorylation, we screened for mutations of the mitochondrial DNA (mtDNA) in renal carcinoma tissues and patient-matched normal kidney cortex. Seven of the 15 samples investigated revealed at least one somatic heteroplasmic mutation as determined by denaturating HPLC analysis (DHP...

  10. Significance of somatic mutations and content alteration of mitochondrial DNA in esophageal cancer

    OpenAIRE

    Wang Yu-Fen; Bai Ren-Kui; Liu Ling-Ling; Chang Julia; Tan Duan-Jun; Yeh Kun-Tu; Wong Lee-Jun C

    2006-01-01

    Abstract Background The roles of mitochondria in energy metabolism, the generation of ROS, aging, and the initiation of apoptosis have implicated their importance in tumorigenesis. In this study we aim to establish the mutation spectrum and to understand the role of somatic mtDNA mutations in esophageal cancer. Methods The entire mitochondrial genome was screened for somatic mutations in 20 pairs (18 esophageal squamous cell carcinomas, one adenosquamous carcinoma and one adenocarcinoma) of t...

  11. Mitochondrial DNA Complex I and III Mutations Associated with Leber's Hereditary Optic Neuropathy

    OpenAIRE

    Brown, M D; Voljavec, A. S.; Lott, M T; Torroni, A.; Yang, C. C.; Wallace, D C

    1992-01-01

    Four new missense mutations have been identified through restriction analysis and sequencing of the mitochondrial DNAs (mtDNA) from Leber's hereditary optic neuropathy (LHON) patients who lacked the previously identified 11778 mutation. Each altered a conserved amino acid and correlated with the LHON phenotype in population and phylogenetic analyses. The nucleotide pair (np) 13708 mutation (G to A, ND5 gene) changed an alanine to a threonine and was found in 6/25 (24%) of non-11778 LHON pedig...

  12. Automated extraction of DNA and PCR setup using a Tecan Freedom EVO® liquid handler

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Frøslev, Tobias G.; Frank-Hansen, Rune;

    2009-01-01

    We have implemented and validated automated methods for DNA extraction and PCR setup developed for a Tecan Freedom EVO« liquid handler mounted with a Te-MagS(TM) magnetic separation device. The DNA was extracted using the Qiagen MagAttract« DNA Mini M48 kit. The DNA was amplified using Amp...

  13. Instrument-free, Automation and multi-platform ready unmodified Iron Oxide based DNA isolation system

    Directory of Open Access Journals (Sweden)

    Rajani Kanth Vangala

    2012-04-01

    Full Text Available The majority of molecular genetic tests start with the extraction of DNA, indicating the great importance of a suitable and reliable method for this purpose. Next to many in-house standard operating procedures (SOPs, a lot of commercial extraction kits have been developed. These methods are generally accepted and have proven their validity with good results. However, it is obvious that the automation and simplification of the extraction procedure could have major benefits; i.e. a higher throughput, more reliable and reproducible processing of the samples, improved traceability and possible streamlining of the entire DNA extraction procedure. To make the process of DNA isolation robust and possible for automation on any automation machine available (example., automated liquid handler, there are several roadblocks like centrifugation. Therefore, we have developed a simple, yet robust isolation technology which can be used on any automation machine and also can be used outside the lab environment at the point of sample collection by anyone. 

  14. Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability

    DEFF Research Database (Denmark)

    Tatton-Brown, Katrina; Seal, Sheila; Ruark, Elise; Harmer, Jenny; Ramsay, Emma; Del Vecchio Duarte, Silvana; Zachariou, Anna; Hanks, Sandra; O'Brien, Eleanor; Aksglaede, Lise; Baralle, Diana; Dabir, Tabib; Gener, Blanca; Goudie, David; Homfray, Tessa; Kumar, Ajith; Pilz, Daniela T; Selicorni, Angelo; Temple, I Karen; Van Maldergem, Lionel; Yachelevich, Naomi; van Montfort, Robert; Rahman, Nazneen

    2014-01-01

    and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for...

  15. Nitrative and oxidative DNA damage caused by K-ras mutation in mice

    Energy Technology Data Exchange (ETDEWEB)

    Ohnishi, Shiho [Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science (Japan); Saito, Hiromitsu; Suzuki, Noboru [Department of Animal Genomics, Mie University Life Science Research Center (Japan); Ma, Ning [Faculty of Health Science, Suzuka University of Medical Science (Japan); Hiraku, Yusuke; Murata, Mariko [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine (Japan); Kawanishi, Shosuke, E-mail: kawanisi@suzuka-u.ac.jp [Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science (Japan)

    2011-09-23

    Highlights: {yields} Mutated K-ras in transgenic mice caused nitrative DNA damage, 8-nitroguanine. {yields} The mutagenic 8-nitroguanine seemed to be generated by iNOS via Ras-MAPK signal. {yields} Mutated K-ras produces additional mutagenic lesions, as a new oncogenic role. -- Abstract: Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-{kappa}B, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.

  16. [Mutation detection of mitochondrial DNA D-loop region in bone marrow cells of acute leukemia].

    Science.gov (United States)

    Han, Hui; Li, Da-Qi; Chen, Ping; Shao, Jian-Hua; Zhao, Hong-Yu; Dong, Xue-Bin; Gu, Lin-Ping

    2013-02-01

    This study was aimed to detect the mutations and microsatellite instability (mtMSI) in mitochondrial DNA (mtDNA) D-loop region in bone marrow cells of acute leukemia (AL) patients, and to analyze their relationship with the pathogenesis of AL. 19 cases of newly diagnosed AL were enrolled in this study. Through extracting mtDNA, the D-loop region was amplified by polymerase chain reaction (PCR), the sequences of PCR products were detected by the pros- and cons-direct sequencing methods. The sequencing results were compared with the revised Cambridge reference sequence (rCRS) and the relevant database (MITOMAP database, GenBank database, mtDB database). The results showed that the mutation rate of mtDNA D-loop region in AL was 79% (15/19). 215 variations (35 mutations, 180 SNP) and a kind of mtMSI in the D-loop region were detected. A new type of mutation nt150 C-CT was found. Also, there was no significant difference in the number of mutations between patients with different ages and different types of AL (AML, B-ALL). It is concluded that there is high frequency of mutations in the mtDNA D-loop, and the mutations may be associated with the pathogenesis of AL. PMID:23484686

  17. Automated extraction of DNA and PCR setup using a Tecan Freedom EVO® liquid handler

    DEFF Research Database (Denmark)

    Frøslev, Tobias Guldberg; Hansen, Anders Johannes; Stangegaard, Michael;

    We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO® liquid handler mounted with the TeMagS magnetic separation device. The methods were validated for accredited, forensic genetic work according to ISO 17025 using the Qiagen Mag....... With the Identifiler kit, the number of full DNA profiles was approximately 20% higher with DNA prepared with the robot compared to that obtained with DNA prepared manually with the Chelex method. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for...... accredited, forensic genetic DNA typing can be implemented on a simple robot leading to the reduction of manual work as well as increased quality and throughput....

  18. Helicobacter pylori induces mitochondrial DNA mutation and reactive oxygen species level in AGS cells

    OpenAIRE

    Xue-Wen Huang, Rui-Hua Luo, Qi Zhao, Zhong-Ze Shen, Li-Li Huang, Xian-Yuan An, Lan-Jing Zhao, Jie Wang, Yu-Zheng Huang

    2011-01-01

    To investigate the role of ROS in the helicobacter pylori (Hp) induced mtDNA mutations, AGS cells were treated by extracts of Hp11638 or Hp11638M. The ROS levels, cytochrome C reductions, and intracellular ATP levels were measured. The coding region and the D-Loop region were amplified and sequenced. Results showed the ROS levels, cytochrome C reduction and mtDNA mutations were markedly increased and cell viability decreased after treatment with both Hp extracts, and 616 mutations were detect...

  19. Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast

    International Nuclear Information System (INIS)

    HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination

  20. Absence of mtDNA mutations in leukocytes of CADASIL patients

    Directory of Open Access Journals (Sweden)

    Hellani Ali

    2008-05-01

    Full Text Available Abstract Background Ultrastructural and biochemical abnormalities of mitochondria have been reported in skeletal muscle biopsies of CADASIL patients with mutations in the NOTCH3 nuclear gene. Additionally, it was proposed that NOTCH3 gene mutations may predispose the mitochondrial DNA (mtDNA to mutations. Methods We sequenced the entire mitochondrial genome in five Arab patients affected by CADASIL. Results The mean number of mtDNA sequence variants (synonymous and nonsynonymous in CADASIL patients was not statistically significantly different from that in controls (p = 0.378. After excluding haplogroup specific single nucleotide polymorphisms (SNPs and proved silent polymorphisms, no known or novel pathologic mtDNA mutation(s could be detected in any patient. Additionally, there was no difference in the prevalence of different mitochondrial haplogroups between patients and controls. Conclusion Our study group is too small for any valid conclusion to be made. However, if our observation is confirmed in larger study group, then mtDNA mutations or mitochondrial haplogroups may not be important in the pathogenesis of CADASIL.

  1. Mutation patterns of mtDNA: Empirical inferences for the coding region

    Directory of Open Access Journals (Sweden)

    Alvarez Luis

    2008-06-01

    Full Text Available Abstract Background Human mitochondrial DNA (mtDNA has been extensively used in population and evolutionary genetics studies. Thus, a valid estimate of human mtDNA evolutionary rate is important in many research fields. The small number of estimations performed for the coding region of the molecule, showed important differences between phylogenetic and empirical approaches. We analyzed a portion of the coding region of mtDNA (tRNALeu, ND1 and tRNAIle genes, using individuals belonging to extended families from the Azores Islands (Portugal with the main aim of providing empirical estimations of the mutation rate of the coding region of mtDNA under different assumptions, and hence to better understand the mtDNA evolutionary process. Results Heteroplasmy was detected in 6.5% (3/46 of the families analyzed. In all of the families the presence of mtDNA heteroplasmy resulted from three new point mutations, and no cases of insertions or deletions were identified. Major differences were found in the proportion and type of heteroplasmy found in the genes studied when compared to those obtained in a previous report for the D-loop. Our empirical estimation of mtDNA coding region mutation rate, calculated taking into account the sex of individuals carrying new mutations, the probability of intra-individual fixation of mutations present in heteroplasmy and, to the possible extent, the effect of selection, is similar to that obtained using phylogenetic approaches. Conclusion Based on our results, the discrepancy previously reported between the human mtDNA coding region mutation rates observed along evolutionary timescales and estimations obtained using family pedigrees can be resolved when correcting for the previously cited factors.

  2. RAPD analysis of alfalfa DNA mutation via N+ implantation

    Institute of Scientific and Technical Information of China (English)

    LI Yu-Feng; HUANG Qun-Ce; LIANG Yun-Zhang; YU Zeng-Liang

    2003-01-01

    Germination capacity of alfalfa seeds under low energy N+ implantation manifests oscillations goingdown with dose strength. From analyzing alfalfa genome DNA under low energy N+ implantation by RAPD (RandomAmplified Polymorphous DNA), it is recommended that 30 polymorphic DNA fragments be amplified with 8 primersin total 100 primers, and fluorescence intensity of the identical DNA fragments amplified by RAPD is different be-tween CK and treatments. Number of different polymorphic DNA fragments between treatment and CK via N+ im-plantation manifests going up with dose strength.

  3. RAPD analysis of alfalfa DNA mutation via N+ implantation

    International Nuclear Information System (INIS)

    Germination capacity of alfalfa seeds under low energy N+ implantation manifests oscillations going down with dose strength. From analyzing alfalfa genome DNA under low energy N+ implantation by RAPD (Random Amplified Polymorphous DNA), it is recommended that 30 polymorphic DNA fragments be amplified with 8 primers in total 100 primers, and fluorescence intensity of the identical DNA fragment amplified by RAPD is different between CK and treatments. Number of different polymorphic DNA fragments between treatment and CK via N+ implantation manifests going up with dose strength

  4. PIK3CA mutation detection in metastatic biliary cancer using cell-free DNA

    Science.gov (United States)

    Deng, Shibing; Lee, Sujin; Park, Young Suk; Lim, Ho Yeong; Kang, Won Ki; Mao, Mao; Heo, Jin Seok; Kwon, Wooil; Jang, Kee-Taek; Lee, Jeeyun; Park, Joon Oh

    2015-01-01

    PIK3CA mutation is considered a good candidate for targeted therapies in cancers, especially biliary tract cancer (BTC). We evaluated the utility of cell free DNA (cfDNA) from serum by using droplet digital PCR (ddPCR) as an alternative source for PIK3CA mutation analysis. To identify matching archival tumour specimens from serum samples of advanced BTC patients, mutation detection using ddPCR with Bio-Rad's PrimePCR mutation and wild type assays were performed for PIK3CA p.E542K, p.E545K, and p.H1047R. Thirty-eight patients with metastatic BTC were enrolled. Only one (BTC 29T) sample (n = 38) was positive for PIK3CA p.E542K and another (BTC 27T) for p.H1047R mutation; none was positive for PIK3CA p.E545K. Matched serum sample (BTC 29P) was positive for PIK3CA p.E542K with 28 mutant copies detected, corresponding to 48 copies/ml of serum and an allelic prevalence of 0.3%. Another matched serum sample (BTC 27P) was positive for PIK3CA p.H1047R with 10 mutant copies detected, i.e. 18 copies/ml and an allelic frequency of 0.2%. High correlation was noted in the PIK3CA mutation status between tumour gDNA and serum cfDNA. Low-level PIK3CA mutations were detectable in the serum indicating the utility of cfDNA as a DNA source to detect cancer-derived mutations in metastatic biliary cancers. PMID:26498688

  5. The Mitochondrial DNA Mutation at Position 11778 in Chinese Families with Leber's Hereditary Optic Neuropathy

    Institute of Scientific and Technical Information of China (English)

    1994-01-01

    We amplified the 340 bp of mitochondrial DMA (mtDNA) by PCR including the recognized sequence of restriction enzyme of SfaN I . After amplification and digestion of SfaN I , two bands of 190 bp and 150 bp appeared in the mtDNA of four normal individuals but only one band of 340 bp appeared in the mtDNA with the mutation of G to A at the site of the nucleotide 11778 because such mutation destroyed the recognized sequence of SfaN I . We studied the mtDNAs of the patients with Leber's hereditary optic neur...

  6. Genetic suppression of a dnaG mutation in Escherichia coli.

    OpenAIRE

    Katayama, T; Murakami, Y.; Wada, C.; Ohmori, H; Yura, T; Nagata, T

    1989-01-01

    Escherichia coli strains with a temperature-sensitive mutation, dnaG2903, in the primase-encoding gene spontaneously reverted to the temperature-insensitive phenotype at a high frequency. Many of the reversions were caused by extragenic sdg suppressors. About 100 independently isolated sdg suppressors were analyzed. They fall into two classes. The sdgA mutations were genetically mapped very close to and upstream of the dnaG gene and were found to be cis dominant. DNA sequencing of two of them...

  7. Hair Dye–DNA Interaction: Plausible Cause of Mutation

    Directory of Open Access Journals (Sweden)

    Swati Maiti

    2015-09-01

    Full Text Available Hair dye is one of the most popular cosmetic products which are used more widely and frequently to improve an individual’s appearance. Although the genotoxic effects of dye ingredients are widely reported, hair dye in its usable form is not reported extensively. In this contribution, we report the possible mode of interaction of hair dye with DNA which leads to genotoxicity. The effect of dye DNA interaction was studied on the most popular and globally used hair dye with Calf Thymus DNA and plasmid DNA. This interaction of dye DNA was studied by spectroscopic analyses and gel electrophoresis. The result had shown positive interaction of dye with DNA. Gel electrophoresis study confirms the binding of dye with DNA which results in linearization and fragmentation of the plasmid DNA. Dye–DNA interaction causes fragmentation and oxidation of DNA in absence of any catalyst, implies high toxicity of commercial hair dyes. Thus, it can be deduced from the present studies that hair dye in its usable form may lead to its penetration through skin affecting genomic DNA possesses genotoxic property and can be treated as one of the most common mutagen.

  8. Blocking DNA Repair in Advanced BRCA-Mutated Cancer

    Science.gov (United States)

    In this trial, patients with relapsed or refractory advanced cancer and confirmed BRCA mutations who have not previously been treated with a PARP inhibitor will be given BMN 673 by mouth once a day in 28-day cycles.

  9. Control region mutations and the 'common deletion' are frequent in the mitochondrial DNA of patients with esophageal squamous cell carcinoma

    OpenAIRE

    Tang Ze-Zong; Hu Nan; McKenney Keith; Armistead David; Carrera Ana; Huppi Konrad; Abnet Christian C; Taylor Philip R; Dawsey Sanford M

    2004-01-01

    Abstract Background North central China has some of the highest rates of esophageal squamous cell carcinoma in the world with cumulative mortality surpassing 20%. Mitochondrial DNA (mtDNA) accumulates more mutations than nuclear DNA and because of its high abundance has been proposed as a early detection device for subjects with cancer at various sites. We wished to examine the prevalence of mtDNA mutation and polymorphism in subjects from this high risk area of China. Methods We used DNA sam...

  10. Automated centrifugal-microfluidic platform for DNA purification using laser burst valve and coriolis effect.

    Science.gov (United States)

    Choi, Min-Seong; Yoo, Jae-Chern

    2015-04-01

    We report a fully automated DNA purification platform with a micropored membrane in the channel utilizing centrifugal microfluidics on a lab-on-a-disc (LOD). The microfluidic flow in the LOD, into which the reagents are injected for DNA purification, is controlled by a single motor and laser burst valve. The sample and reagents pass successively through the micropored membrane in the channel when each laser burst valve is opened. The Coriolis effect is used by rotating the LOD bi-directionally to increase the purity of the DNA, thereby preventing the mixing of the waste and elution solutions. The total process from the lysed sample injection into the LOD to obtaining the purified DNA was finished within 7 min with only one manual step. The experimental result for Salmonella shows that the proposed microfluidic platform is comparable to the existing devices in terms of the purity and yield of DNA. PMID:25737025

  11. Somatic mutations in stilbene estrogen-induced Syrian hamster kidney tumors identified by DNA fingerprinting

    Directory of Open Access Journals (Sweden)

    Roy Deodutta

    2004-01-01

    Full Text Available Abstract Kidney tumors from stilbene estrogen (diethylstilbestrol-treated Syrian hamsters were screened for somatic genetic alterations by Random Amplified Polymorphic DNA-polymerase chain-reaction (RAPD-PCR fingerprinting. Fingerprints from tumor tissue were generated by single arbitrary primers and compared with fingerprints for normal tissue from the same animal, as well as normal and tumor tissues from different animals. Sixty one of the arbitrary primers amplified 365 loci that contain approximately 476 kbp of the hamster genome. Among these amplified DNA fragments, 44 loci exhibited either qualitative or quantitative differences between the tumor tissues and normal kidney tissues. RAPD-PCR loci showing decreased and increased intensities in tumor tissue DNA relative to control DNA indicate that loci have undergone allelic losses and gains, respectively, in the stilbene estrogen-induced tumor cell genome. The presence or absence of the amplified DNA fragments indicate homozygous insertions or deletions in the kidney tumor DNA compared to the age-matched normal kidney tissue DNA. Seven of 44 mutated loci also were present in the kidney tissues adjacent to tumors (free of macroscopic tumors. The presence of mutated loci in uninvolved (non-tumor surrounding tissue adjacent to tumors from stilbene estrogen-treated hamsters suggests that these mutations occurred in the early stages of carcinogenesis. The cloning and sequencing of RAPD amplified loci revealed that one mutated locus had significant sequence similarity with the hamster Cyp1A1 gene. The results show the ability of RAPD-PCR to detect and isolate, in a single step, DNA sequences representing genetic alterations in stilbene estrogen-induced cancer cells, including losses of heterozygosity, and homozygous deletion and insertion mutations. RAPD-PCR provides an alternative molecular approach for studying cancer cytogenetics in stilbene estrogen-induced tumors in humans and experimental

  12. Mutational analysis of whole mitochondrial DNA in patients with MELAS and MERRF diseases.

    Science.gov (United States)

    Choi, Byung-Ok; Hwang, Jung Hee; Cho, Eun Min; Jeong, Eun Hye; Hyun, Young Se; Jeon, Hyeon Jeong; Seong, Ki Min; Cho, Nam Soo; Chung, Ki Wha

    2010-06-30

    Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make the exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mtDNA mutations in 61 Korean unrelated families (or isolated patients) with MELAS or MERRF. In particular, the mtDNA sequences were completely determined for 49 patients. From the mutational analysis of mtDNA obtained from blood, 5 confirmed pathogenic mutations were identified in 17 families, and 4 unreported pathogenically suspected mutations were identified in 4 families. The m.3243A>G in the tRNA(Leu(UUR))was predominantly observed in 10 MELAS families, and followed by m.8344A>G in the tRNA(Lys) of 4 MERRF families. Most pathogenic mutations showed heteroplasmy, and the rates were considerably different within the familial members. Patients with a higher rate of mutations showed a tendency of having more severe clinical phenotypes, but not in all cases. This study will be helpful for the molecular diagnosis of mitochondrial diseases, as well as establishment of mtDNA database in Koreans. PMID:20440095

  13. GenePublisher: automated analysis of DNA microarray data

    DEFF Research Database (Denmark)

    Knudsen, Steen; Workman, Christopher; Sicheritz-Ponten, T.;

    2003-01-01

    GenePublisher, a system for automatic analysis of data from DNA microarray experiments, has been implemented with a web interface at http://www.cbs.dtu.dk/services/GenePublisher. Raw data are uploaded to the server together with aspecification of the data. The server performs normalization......, statistical analysis and visualization of the data. The results are run against databases of signal transduction pathways, metabolic pathways and promoter sequences in order to extract more information. The results of the entire analysis are summarized in report form and returned to the user....

  14. Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments.

    Science.gov (United States)

    Chusacultanachai, Sudsanguan; Yuthavong, Yongyuth

    2004-01-01

    The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP). These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity. The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step. After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication. The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations. This strategy is suitable for generation of random mutations with low frequency in a large target DNA. Error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained. Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries. DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides. This strategy uses two chemically synthesized degenerate oligonucleotides as primers. By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to

  15. Increased transmission of mutations by low-condition females: evidence for condition-dependent DNA repair.

    Directory of Open Access Journals (Sweden)

    Aneil F Agrawal

    2008-02-01

    Full Text Available Evidence is mounting that mutation rates are sufficiently high for deleterious alleles to be a major evolutionary force affecting the evolution of sex, the maintenance of genetic variation, and many other evolutionary phenomena. Though point estimates of mutation rates are improving, we remain largely ignorant of the biological factors affecting these rates at the individual level. Of special importance is the possibility that mutation rates are condition-dependent with low-condition individuals experiencing more mutation. Theory predicts that such condition dependence would dramatically increase the rate at which populations adapt to new environments and the extent to which populations suffer from mutation load. Despite its importance, there has been little study of this phenomenon in multicellular organisms. Here, we examine whether DNA repair processes are condition-dependent in Drosophila melanogaster. In this species, damaged DNA in sperm can be repaired by maternal repair processes after fertilization. We exposed high- and low-condition females to sperm containing damaged DNA and then assessed the frequency of lethal mutations on paternally derived X chromosomes transmitted by these females. The rate of lethal mutations transmitted by low-condition females was 30% greater than that of high-condition females, indicating reduced repair capacity of low-condition females. A separate experiment provided no support for an alternative hypothesis based on sperm selection.

  16. Gene mutations in the D-loop region of mitochondrial DNA in oral squamous cell carcinoma.

    Science.gov (United States)

    Yuan, Rong-Tao; Sun, Yang; Bu, Ling-Xue; Jia, Mu-Yun

    2015-06-01

    The present study aimed to investigate gene mutations in the displacement‑loop (D‑loop) region of mitochondrial DNA (mtDNA) in patients with oral squamous cell carcinoma (OSCC) in order to examine the role of gene mutation in mtDNA in OSCC tumorigenesis. mtDNA was obtained from cancer tissues, paracancerous tissues and normal mucosal tissues of thirty patients with OSCC. The D‑loop region of the mtDNA was amplified using polymerase chain reaction, sequenced and then analyzed by Chromas software and BLAST to identify the mutation sites. Mutations in the D‑loop region were observed in the cancer tissue samples of eight out of thirty cases with OSCC, with a mutation rate of 27%. There were nine mutations in total, including one point mutation, two base deletions, three insertion mutations and three heterozygous mutations. In these mutations, base deletions were different from each other and heterozygous mutations did not have the same mutation form; however, the three insertion mutations were the same, consisting of an insertion of a C base. One case contained a T/A heterozygous mutation as well as base insertion of C. The eight cases with mutations in the D‑loop region consisted of three cases of tongue cancer, two cases of soft palate cancer, one case of floor of the mouth cancer, one case of oropharyngeal cancer and one case of lip cancer. This study demonstrated mutations in the mtDNA D‑loop region in OSCC cells; however, the association between occurrences of OSCC and mtDNA mutations requires further investigation. PMID:25625701

  17. Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer.

    Science.gov (United States)

    Ju, Young Seok; Alexandrov, Ludmil B; Gerstung, Moritz; Martincorena, Inigo; Nik-Zainal, Serena; Ramakrishna, Manasa; Davies, Helen R; Papaemmanuil, Elli; Gundem, Gunes; Shlien, Adam; Bolli, Niccolo; Behjati, Sam; Tarpey, Patrick S; Nangalia, Jyoti; Massie, Charles E; Butler, Adam P; Teague, Jon W; Vassiliou, George S; Green, Anthony R; Du, Ming-Qing; Unnikrishnan, Ashwin; Pimanda, John E; Teh, Bin Tean; Munshi, Nikhil; Greaves, Mel; Vyas, Paresh; El-Naggar, Adel K; Santarius, Tom; Collins, V Peter; Grundy, Richard; Taylor, Jack A; Hayes, D Neil; Malkin, David; Foster, Christopher S; Warren, Anne Y; Whitaker, Hayley C; Brewer, Daniel; Eeles, Rosalind; Cooper, Colin; Neal, David; Visakorpi, Tapio; Isaacs, William B; Bova, G Steven; Flanagan, Adrienne M; Futreal, P Andrew; Lynch, Andy G; Chinnery, Patrick F; McDermott, Ultan; Stratton, Michael R; Campbell, Peter J

    2014-01-01

    Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication. PMID:25271376

  18. Initial analysis of non-typical Leber hereditary optic neuropathy (LHON) at onset and late developing demyelinating disease in Italian patients by SSCP and automated DNA sequence analysis

    Energy Technology Data Exchange (ETDEWEB)

    Sartore, M.; Semeraro, A.; Fortina, P. [Univ. of Pennsylvania School of Medicine, Philadelphia, PA (United States)] [and others

    1994-09-01

    LHON is a mitochondrial genetic disease characterized by maternal inheritance and late onset of blindness caused by bilateral retinal degeneration. A number of molecular defects are known affecting expression of seven mitochondrial genes encoding subunits of respiratory chain complex I, III and IV. We screened genomic DNA from Italian patients for seven of the known point mutations in the ND-1, ND-4 and ND-6 subunits of complex I by PCR followed by SSCP and restriction enzyme digestion. Most of the patients had nonfamilial bilateral visual loss with partial or no recovery and normal neurological examination. Fundoscopic examination revealed that none of the patients had features typical of LHON. Nine of 21 patients (43%) showed multifocal CNS demyelination on MRI. Our results show aberrant SSCP patterns for a PCR product from the ND-4 subunit in one affected child and his mother. Sfa NI and Mae III digestions suggested the absence of a previously defined LHON mutation, and automated DNA sequence analysis revealed two A to G neutral sequence polymorphisms in the third position of codons 351 and 353. In addition, PCR products from the same two samples and an unrelated one showed abnormal SSCP patterns for the ND-1 subunit region of complex I due to the presence of a T to C change at nt 4,216 which was demonstrated after Nla III digestion of PCR products and further confirmed by DNA sequence analysis. Our results indicate that additional defects are present in the Italian population, and identification of abnormal SSCP patterns followed by targeted automated DNA sequence analysis is a reasonable strategy for delineation of new LHON mutations.

  19. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

    International Nuclear Information System (INIS)

    Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results

  20. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fortes, F.P. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Kuasne, H. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Marchi, F.A. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Programa Inter-Institucional em Bioinformtica, Instituto de Matemtica e Estatstica, Universidade So Paulo, So Paulo, SP (Brazil); Miranda, P.M. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Rogatto, S.R. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Achatz, M.I. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Oncogentica, A.C. Camargo Cancer Center, So Paulo, SP (Brazil)

    2015-04-28

    Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

  1. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations.

    Science.gov (United States)

    Fortes, F P; Kuasne, H; Marchi, F A; Miranda, P M; Rogatto, S R; Achatz, M I

    2015-07-01

    Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53 mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results. PMID:25945745

  2. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

    Directory of Open Access Journals (Sweden)

    F.P. Fortes

    2015-07-01

    Full Text Available Li-Fraumeni syndrome (LFS is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17 in 30 patients with germline TP53 mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

  3. DNA polymerase ι functions in the generation of tandem mutations during somatic hypermutation of antibody genes.

    Science.gov (United States)

    Maul, Robert W; MacCarthy, Thomas; Frank, Ekaterina G; Donigan, Katherine A; McLenigan, Mary P; Yang, William; Saribasak, Huseyin; Huston, Donald E; Lange, Sabine S; Woodgate, Roger; Gearhart, Patricia J

    2016-08-22

    DNA polymerase ι (Pol ι) is an attractive candidate for somatic hypermutation in antibody genes because of its low fidelity. To identify a role for Pol ι, we analyzed mutations in two strains of mice with deficiencies in the enzyme: 129 mice with negligible expression of truncated Pol ι, and knock-in mice that express full-length Pol ι that is catalytically inactive. Both strains had normal frequencies and spectra of mutations in the variable region, indicating that loss of Pol ι did not change overall mutagenesis. We next examined if Pol ι affected tandem mutations generated by another error-prone polymerase, Pol ζ. The frequency of contiguous mutations was analyzed using a novel computational model to determine if they occur during a single DNA transaction or during two independent events. Analyses of 2,000 mutations from both strains indicated that Pol ι-compromised mice lost the tandem signature, whereas C57BL/6 mice accumulated significant amounts of double mutations. The results support a model where Pol ι occasionally accesses the replication fork to generate a first mutation, and Pol ζ extends the mismatch with a second mutation. PMID:27455952

  4. X-ray induced mutations, DNA and target theory

    International Nuclear Information System (INIS)

    It is stated that X-ray induced, specific locus, germ-line mutation rates vary significantly in eukaryotes. A consideration of the radiobiology of the individual gene by the author leads to three major conclusions. (1) Mutation rates tend to be much lower than radiation theory predicts. (2) Selection and/or repair are major factors that determine the rates. (3) The mouse 7-locus test, which provides a principal data base for the standards of human radiation hygiene, may not provide adequate overall representation of the mutability of the mammalian genome, so more research is needed. (U.K.)

  5. Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

    Science.gov (United States)

    Tang, Sha; Halberg, Michelle C; Floyd, Kristen C; Wang, Jing

    2012-01-01

    Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described. PMID:22215554

  6. Mutation frequency of plasmid DNA and Escherichia coli following long-term space flight on Mir

    International Nuclear Information System (INIS)

    To elucidate the biological influence of space radiation, we studied the effects of long-term space flight on mutation of the bacterial ribosomalprotein L gene (rpsL). We prepared dried samples of plasmid DNA and repair-deficient and wild type cells of Escherichia (E.) coli. After a 40-day space flight on board the Russian space station Mir, the mutation frequencies of the rpsL gene were estimated by transformation of E. coli and by assessment of conversion of rpsL wild type phenotype (SmS) to its mutant phenotype (SmR). Theexperimental findings indicate that mutation frequencies of space samples were not significantlydifferent from those of ground control samples in plasmid DNA and both E. coli strains. It may suggestthat space radiation did not influence mutation frequency. (author)

  7. Automated microfluidic DNA/RNA extraction with both disposable and reusable components

    International Nuclear Information System (INIS)

    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ∼90% for deoxyribonucleic acid (DNA) and ∼54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components

  8. Automated microfluidic DNA/RNA extraction with both disposable and reusable components

    Science.gov (United States)

    Kim, Jungkyu; Johnson, Michael; Hill, Parker; Sonkul, Rahul S.; Kim, Jongwon; Gale, Bruce K.

    2012-01-01

    An automated microfluidic nucleic extraction system was fabricated with a multilayer polydimethylsiloxane (PDMS) structure that consists of sample wells, microvalves, a micropump and a disposable microfluidic silica cartridge. Both the microvalves and micropump structures were fabricated in a single layer and are operated pneumatically using a 100 µm PDMS membrane. To fabricate the disposable microfluidic silica cartridge, two-cavity structures were made in a PDMS replica to fit the stacked silica membranes. A handheld controller for the microvalves and pumps was developed to enable system automation. With purified ribonucleic acid (RNA), whole blood and E. coli samples, the automated microfluidic nucleic acid extraction system was validated with a guanidine-based solid phase extraction procedure. An extraction efficiency of ~90% for deoxyribonucleic acid (DNA) and ~54% for RNA was obtained in 12 min from whole blood and E. coli samples, respectively. In addition, the same quantity and quality of extracted DNA was confirmed by polymerase chain reaction (PCR) amplification. The PCR also presented the appropriate amplification and melting profiles. Automated, programmable fluid control and physical separation of the reusable components and the disposable components significantly decrease the assay time and manufacturing cost and increase the flexibility and compatibility of the system with downstream components.

  9. Hair Dye–DNA Interaction: Plausible Cause of Mutation

    OpenAIRE

    Swati Maiti; Sudarson Sekhar Sinha; Mukesh Singh

    2015-01-01

    Hair dye is one of the most popular cosmetic products which are used more widely and frequently to improve an individual’s appearance. Although the genotoxic effects of dye ingredients are widely reported, hair dye in its usable form is not reported extensively. In this contribution, we report the possible mode of interaction of hair dye with DNA which leads to genotoxicity. The effect of dye DNA interaction was studied on the most popular and globally used hair dye with Calf Thymus DNA and p...

  10. Mitochondrial DNA mutations affect calcium handling in differentiated neurons.

    NARCIS (Netherlands)

    Trevelyan, A.J.; Kirby, D.M.; Smulders-Srinivasan, T.K.; Nooteboom, M.; Acin-Perez, R.; Enriquez, J.A.; Whittington, M.A.; Lightowlers, R.N.; Turnbull, D.M.

    2010-01-01

    Mutations in the mitochondrial genome are associated with a wide range of neurological symptoms, but many aspects of the basic neuronal pathology are not understood. One candidate mechanism, given the well-established role of mitochondria in calcium buffering, is a deficit in neuronal calcium homoeo

  11. A new technique for determining the distribution of N7-methylguanine using an automated DNA sequencer.

    Science.gov (United States)

    Shoukry, S; Anderson, M W; Glickman, B W

    1991-11-01

    We have developed a method to determine rapidly the sequence specificity of DNA alkylation resulting from chemical treatment. The utility of this approach is demonstrated here in a study of the sequence specificity of alkylation by dimethylsulphate (DMS). The method is independent of the sequence chosen and makes use of the polymerase chain reaction (PCR) to generate a fluorescently labelled DNA target. In this study, a 302 bp segment of the Escherichia coli lacI gene was amplified and the product purified by liquid chromatography on a Mono Q column. This DNA was alkylated with DMS and treated with hot piperidine to produce single-strand breaks at sites of N7 alkylation. The distribution of the break points, and hence the position and extent of alkylation, were determined on an Applied Biosystems 370A automated DNA sequencer. PMID:1682064

  12. Automated screening for small organic ligands using DNA-encoded chemical libraries.

    Science.gov (United States)

    Decurtins, Willy; Wichert, Moreno; Franzini, Raphael M; Buller, Fabian; Stravs, Michael A; Zhang, Yixin; Neri, Dario; Scheuermann, Jörg

    2016-04-01

    DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d. PMID:26985574

  13. Aptaligner: Automated Software for Aligning Pseudorandom DNA X-Aptamers from Next-Generation Sequencing Data

    OpenAIRE

    Lu, Emily; Elizondo-Riojas, Miguel-Angel; Chang, Jeffrey T.; Volk, David E.

    2014-01-01

    Next-generation sequencing results from bead-based aptamer libraries have demonstrated that traditional DNA/RNA alignment software is insufficient. This is particularly true for X-aptamers containing specialty bases (W, X, Y, Z, ...) that are identified by special encoding. Thus, we sought an automated program that uses the inherent design scheme of bead-based X-aptamers to create a hypothetical reference library and Markov modeling techniques to provide improved alignments. Aptaligner provid...

  14. Automated serial extraction of DNA and RNA from biobanked tissue specimens

    OpenAIRE

    Mathot, Lucy; Wallin, Monica; Sjöblom, Tobias

    2013-01-01

    Background: With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based te...

  15. Response to: DNA identification by pedigree likelihood ratio accommodating population substructure and mutations

    Directory of Open Access Journals (Sweden)

    Egeland Thore

    2011-03-01

    Full Text Available Abstract Mutation models are important in many areas of genetics including forensics. This letter criticizes the model of the paper 'DNA identification by pedigree likelihood ratio accommodating population substructure and mutations' by Ge et al. (2010. Furthermore, we argue that the paper in some cases misrepresents previously published papers. Please see related letter: http://www.investigativegenetics.com/content/2/1/8.

  16. Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations

    Directory of Open Access Journals (Sweden)

    Leary Jeffry J

    2002-05-01

    Full Text Available Abstract Background The thymidine kinase (tk mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. Methods A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. Results Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture. Conclusions This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a mutations may be modulated by other viral polypeptides cooperating with Pol, and (b the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.

  17. Chemical cleavage reactions of DNA on solid support: application in mutation detection

    Directory of Open Access Journals (Sweden)

    Cotton Richard GH

    2003-05-01

    Full Text Available Abstract Background The conventional solution-phase Chemical Cleavage of Mismatch (CCM method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports. Results DNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO4 (potassium permanganate and hydroxylamine in 3M TEAC (tetraethylammonium chloride solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps. Conclusions The new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.

  18. Identification of DNA polymerase molecules repairing DNA irradiated damage and molecular biological study on modified factors of mutation rate

    International Nuclear Information System (INIS)

    To explain the development mechanism of mutation by radiation, DNA polymerase molecules repairing DNA should be identified. In this study, plasmid was constructed in order to express anti sense DNA of DNA polymerase in the cell and it was introduced into the cell by the calcium phosphate method. Polyclonal antibody of DNA polymerase δ and ε were produced so as to prove no existence of specific polymerase molecules in the cell. When center part of polymerase ε was immunized, antiserum with high antibody titer was obtained. Near terminal C of polymerase δ was immunized, then antiserum was obtained. We discovered very interesting fact that base sequence of polymerase ε published by Syvaoja was not correct. (S.Y.)

  19. Limited clinical relevance of mitochondrial DNA mutation and gene expression analyses in ovarian cancer

    International Nuclear Information System (INIS)

    In recent years, numerous studies have investigated somatic mutations in mitochondrial DNA in various tumours. The observed high mutation rates might reflect mitochondrial deregulation; consequently, mutation analyses could be clinically relevant. The purpose of this study was to determine if mutations in the mitochondrial D-loop region and/or the level of mitochondrial gene expression could influence the clinical course of human ovarian carcinomas. We sequenced a 1320-base-pair DNA fragment of the mitochondrial genome (position 16,000-750) in 54 cancer samples and in 44 corresponding germline control samples. In addition, six transcripts (MT-ATP6, MT-CO1, MT-CYB, MT-ND1, MT-ND6, and MT-RNR1) were quantified in 62 cancer tissues by real-time RT-PCR. Somatic mutations in the D-loop sequence were found in 57% of ovarian cancers. Univariate analysis showed no association between mitochondrial DNA mutation status or mitochondrial gene expression and any of the examined clinicopathologic parameters. A multivariate logistic regression model revealed that the expression of the mitochondrial gene RNR1 might be used as a predictor of tumour sensitivity to chemotherapy. In contrast to many previously published papers, our study indicates rather limited clinical relevance of mitochondrial molecular analyses in ovarian carcinomas. These discrepancies in the clinical utility of mitochondrial molecular tests in ovarian cancer require additional large, well-designed validation studies

  20. Limited clinical relevance of mitochondrial DNA mutation and gene expression analyses in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Rachinger Andrea

    2008-10-01

    Full Text Available Abstract Background In recent years, numerous studies have investigated somatic mutations in mitochondrial DNA in various tumours. The observed high mutation rates might reflect mitochondrial deregulation; consequently, mutation analyses could be clinically relevant. The purpose of this study was to determine if mutations in the mitochondrial D-loop region and/or the level of mitochondrial gene expression could influence the clinical course of human ovarian carcinomas. Methods We sequenced a 1320-base-pair DNA fragment of the mitochondrial genome (position 16,000-750 in 54 cancer samples and in 44 corresponding germline control samples. In addition, six transcripts (MT-ATP6, MT-CO1, MT-CYB, MT-ND1, MT-ND6, and MT-RNR1 were quantified in 62 cancer tissues by real-time RT-PCR. Results Somatic mutations in the D-loop sequence were found in 57% of ovarian cancers. Univariate analysis showed no association between mitochondrial DNA mutation status or mitochondrial gene expression and any of the examined clinicopathologic parameters. A multivariate logistic regression model revealed that the expression of the mitochondrial gene RNR1 might be used as a predictor of tumour sensitivity to chemotherapy. Conclusion In contrast to many previously published papers, our study indicates rather limited clinical relevance of mitochondrial molecular analyses in ovarian carcinomas. These discrepancies in the clinical utility of mitochondrial molecular tests in ovarian cancer require additional large, well-designed validation studies.

  1. Clustering of Caucasian Leber hereditary optic neuropathy patients containing the 11778 or 14484 mutations on an mtDNA lineage

    Energy Technology Data Exchange (ETDEWEB)

    Brown, M.D.; Sun, F.; Wallace, D.C. [Emory Univ. School of Medicine, Atlanta, GA (United States)

    1997-02-01

    Leber hereditary optic neuropathy (LHON) is a type of blindness caused by mtDNA mutations. Three LHON mtDNA mutations at nucleotide positions 3460, 11778, and 14484 are specific for LHON and account for 90% of worldwide cases and are thus designated as {open_quotes}primary{close_quotes} LHON mutations. Fifteen other {open_quotes}secondary{close_quotes} LHON mtDNA mutations have been identified, but their pathogenicity is unclear. mtDNA haplotype and phylogenetic analysis of the primary LHON mutations in North American Caucasian patients and controls has shown that, unlike the 3460 and 11778 mutations, which are distributed throughout the European-derived (Caucasian) mtDNA phylogeny, patients containing the 14484 mutation tended to be associated with European mtDNA haplotype J. To investigate this apparent clustering, we performed {chi}{sup 2}-based statistical analyses to compare the distribution of LHON patients on the Caucasian phylogenetic tree. Our results indicate that, unlike the 3460 and 11778 mutations, the 14484 mutation was not distributed on the phylogeny in proportion to the frequencies of the major Caucasian mtDNA haplogroups found in North America. The 14484 mutation was next shown to occur on the haplogroup J background more frequently that expected, consistent with the observation that {approximately}75% of worldwide 14484-positive LHON patients occur in association with haplogroup J. The 11778 mutation also exhibited a moderate clustering on haplogroup J. These observations were supported by statistical analysis using all available mutation frequencies reported in the literature. This paper thus illustrates the potential importance of genetic background in certain mtDNA-based diseases, speculates on a pathogenic role for a subset of LHON secondary mutations and their interaction with primary mutations, and provides support for a polygenic model for LHON expression in some cases. 18 refs., 3 tabs.

  2. Absence of correlation between serum CRP levels and mitochondrial D-loop DNA mutations in gastro-oesophageal adenocarcinoma

    OpenAIRE

    Tan, Benjamin H L; Richard J. E. Skipworth; Nicholas M Wheelhouse; Fearon, Kenneth C H; Ross, James A.

    2014-01-01

    Introduction: Both inflammation and mitochondrial DNA (mtDNA) mutation are thought to play a role in the many human cancers. The aim of this study was to evaluate the relationship between inflammation and accumulation of mitochondrial DNA (mtDNA) mutations in the D-loop region in carcinogenesis of gastro-oesophageal adenocarcinomas. Materials and Methods: Blood samples of 20 patients with gastro-oesophageal adenocarcinoma were taken for measurement of serum C-reactive protein (CRP) concen...

  3. Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Wessendorf, Petra [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany); Vijg, Jan [Albert Einstein College of Medicine, Michael F. Price Center, 1301 Morris Park Avenue, Bronx, NY 10461 (United States); Nussenzweig, André [Laboratory of Genome Integrity, National Cancer Institute, National Institute of Health, 37 Convent Drive, Room 1106, Bethesda, MD 20892 (United States); Digweed, Martin, E-mail: martin.digweed@charite.de [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany)

    2014-11-15

    Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin.

  4. Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

    International Nuclear Information System (INIS)

    Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin

  5. Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis.

    Science.gov (United States)

    Monnot, Sophie; Samuels, David C; Hesters, Laetitia; Frydman, Nelly; Gigarel, Nadine; Burlet, Philippe; Kerbrat, Violaine; Lamazou, Frédéric; Frydman, René; Benachi, Alexandra; Feingold, Josué; Rotig, Agnes; Munnich, Arnold; Bonnefont, Jean-Paul; Steffann, Julie

    2013-05-01

    Mitochondrial DNA (mtDNA) content is thought to remain stable over the preimplantation period of human embryogenesis that is, therefore, suggested to be entirely dependent on ooplasm mtDNA capital. We have explored the impact of two disease-causing mutations [m.3243A>G myopathy, encephalopathy, lactic acidosis and stroke-like syndrome (MELAS) and m.8344A>G myoclonic epilepsy associated with ragged-red fibers (MERRF)] on mtDNA amounts in human oocytes and day 4-5 preimplantation embryos. The mtDNA amount was stable in MERRF and control materials, whereas gradually increasing from the germinal vesicle of oogenesis to the blastocyst stage of embryogenesis in MELAS cells, MELAS embryos carrying ∼3-fold higher mtDNA amount than control embryos (P = 0.0003). A correlation between mtDNA copy numbers and mutant loads was observed in MELAS embryos (R(2) = 0.42, P < 0.0013), suggestive of a compensation for the respiratory chain defect resulting from high mutation levels. These results suggest that mtDNA can replicate in early embryos and emphasize the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans. PMID:23390135

  6. Sensitivity of Ag:DNA fluorescence to single base mutations of hairpin strands

    Science.gov (United States)

    Gwinn, Elisabeth; Hassanzadeh, Rameen; O'Neill, Patrick; Fygenson, Deborah

    2010-03-01

    DNA strands can stabilize fluorescent silver clusters composed of just a few atoms [1]. The small size of these photon emitters and their formation in single-stranded DNA [2] give Ag:DNA emitters promise for use in optically-active, self-assembled DNA nanostructures. Exploiting this promise requires an understanding of how fluorophore color relates to the sequence and conformation of the host DNA strand. Here we examine the optical properties of Ag:DNA solutions for a family of DNA hairpins that differ by single base mutations in the hairpin loop. Specific mutations result in spectral redistribution of the fluorescence and large changes in brightness, pointing to geometric control as a means to select specific emitter species.[4pt] [1] J.T. Petty, J. Zheng, N.V. Hud and R.M. Dickson, ``DNA-templated Ag nanocluster formation,'' J. Am. Chem. Soc, 126, 5207 (2004).[0pt] [2] E.G. Gwinn, P. O'Neill, A. Guerrero, D. Bouwmeester and D.K. Fygenson, ``Sequence-dependent fluorescence from DNA-hosted silver nanoclusters,'' Advanced Materials 20, 279 (2008).

  7. Somatic mitochondrial DNA mutations in cancer escape purifying selection and high pathogenicity mutations lead to the oncocytic phenotype: pathogenicity analysis of reported somatic mtDNA mutations in tumors

    International Nuclear Information System (INIS)

    The presence of somatic mitochondrial DNA (mtDNA) mutations in cancer cells has been interpreted in controversial ways, ranging from random neutral accumulation of mutations, to positive selection for high pathogenicity, or conversely to purifying selection against high pathogenicity variants as occurs at the population level. Here we evaluated the predicted pathogenicity of somatic mtDNA mutations described in cancer and compare these to the distribution of variations observed in the global human population and all possible protein variations that could occur in human mtDNA. We focus on oncocytic tumors, which are clearly associated with mitochondrial dysfunction. The protein variant pathogenicity was predicted using two computational methods, MutPred and SNPs&GO. The pathogenicity score of the somatic mtDNA variants were significantly higher in oncocytic tumors compared to non-oncocytic tumors. Variations in subunits of Complex I of the electron transfer chain were significantly more common in tumors with the oncocytic phenotype, while variations in Complex V subunits were significantly more common in non-oncocytic tumors. Our results show that the somatic mtDNA mutations reported over all tumors are indistinguishable from a random selection from the set of all possible amino acid variations, and have therefore escaped the effects of purifying selection that act strongly at the population level. We show that the pathogenicity of somatic mtDNA mutations is a determining factor for the oncocytic phenotype. The opposite associations of the Complex I and Complex V variants with the oncocytic and non-oncocytic tumors implies that low mitochondrial membrane potential may play an important role in determining the oncocytic phenotype

  8. Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

    Science.gov (United States)

    Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

    2012-09-01

    Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. PMID:22310206

  9. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    OpenAIRE

    Zhongwei Wu; Zuhong Lu; Quanjun Liu; Lingwei Wu

    2010-01-01

    In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplifica...

  10. DNA transposons and the role of recombination in mutation accumulation in Daphnia pulex

    OpenAIRE

    Schaack, Sarah; Choi, Eunjin; Lynch, Michael; Pritham, Ellen J

    2010-01-01

    Background We identify DNA transposons from the completed draft genome sequence of Daphnia pulex, a cyclically parthenogenetic, aquatic microcrustacean of the class Branchiopoda. In addition, we experimentally quantify the abundance of six DNA transposon families in mutation-accumulation lines in which sex is either promoted or prohibited in order to better understand the role of recombination in transposon proliferation. Results We identified 55 families belonging to 10 of the known superfam...

  11. Mutations in the D-loop region and increased copy number of mitochondrial DNA in human laryngeal squamous cell carcinoma.

    Science.gov (United States)

    Guo, Wei; Yang, Denghua; Xu, Hongbo; Zhang, Yang; Huang, Junwei; Yang, Zheng; Chen, Xiaohong; Huang, Zhigang

    2013-01-01

    The effects of D-loop mutations and the mtDNA copy number alterations in LSCC are poorly understood. Herein, we investigated the features and roles of somatic mutations of the D-loop region and copy number alterations in mtDNA of LSCC. Using direct sequencing and real-time quantitative PCR, we examined D-loop mutations and mtDNA copy number in LSCC tissues, paracancerous normal tissues and peripheral vein blood samples from 40 LSCC patients. A student's t test, ANOVA test and χ(2) test were used to analyze association among mutations, mtDNA copy number alterations with clinicopathologic parameters. The results revealed that 21 tumors (52.5 %) had somatic mtDNA D-loop mutations with a total of 34 mutations. Among them, 28 (82.4 %) and 6 (17.6 %) were located in HVII and HVI, respectively. D-loop mutations correlated with tumor differentiation and p53 mutation (P D-loop mutation was significantly higher than that of the negative cases (P D-loop in LSCC is an unstable region with a high frequency of somatic mutation and polymorphisms. Together with the increase in mtDNA copy number, these factors may play a role in carcinogenesis of the larynx. PMID:23114912

  12. Dyes as bifunctional markers of DNA hybridization on surfaces and mutation detection.

    Science.gov (United States)

    García-Mendiola, Tania; Cerro, María Ramos; López-Moreno, José María; Pariente, Félix; Lorenzo, Encarnación

    2016-10-01

    The interaction of small molecules with DNA has found diagnostic and therapeutic applications. In this work, we propose the use of two different dyes, in particular Azure A and Safranine, as bifunctional markers of on-surface DNA hybridization and potent tools for screening of specific gene mutations directly in real DNA PCR amplicons extracted from blood cells. By combining spectroscopic and electrochemical methods we demonstrate that both dyes can interact with single and double stranded DNA to a different extent, allowing reliable hybridization detection. From these data, we have also elucidated the nature of the interaction. We conclude that the binding mode is fundamentally intercalative with an electrostatic component. The dye fluorescence allows their use as nucleic acid stains for the detection of on-surfaces DNA hybridization. Its redox activity is exploited in the development of selective electrochemical DNA biosensors. PMID:27317997

  13. Effect of cold atmospheric pressure He-plasma jet on DNA change and mutation

    Science.gov (United States)

    Yaopromsiri, C.; Yu, L. D.; Sarapirom, S.; Thopan, P.; Boonyawan, D.

    2015-12-01

    Cold atmospheric pressure plasma jet (CAPPJ) effect on DNA change was studied for assessment of its safety. The experiment utilized a home-developed CAPPJ using 100% helium to directly treat naked DNA plasmid pGFP (plasmid green fluorescent protein). A traversal electric field was applied to separate the plasma components and both dry and wet sample conditions were adopted to investigate various factor roles in changing DNA. Plasma species were measured by using optical emission spectroscopy. DNA topological form change was analyzed by gel electrophoresis. The plasma jet treated DNA was transferred into bacterial Escherichia coli cells for observing mutation. The results show that the He-CAPPJ could break DNA strands due to actions from charge, radicals and neutrals and potentially cause genetic modification of living cells.

  14. A novel class of mutations that affect DNA replication in E. coli

    DEFF Research Database (Denmark)

    Nordman, Jared; Skovgaard, Ole; Wright, Andrew

    2007-01-01

    Over-initiation of DNA replication in cells containing the cold-sensitive dnaA(cos) allele has been shown to lead to extensive DNA damage, potentially due to head-to-tail replication fork collisions that ultimately lead to replication fork collapse, growth stasis and/or cell death. Based on the...... assumption that suppressors of the cold-sensitive phenotype of the cos mutant should include mutations that affect the efficiency and/or regulation of DNA replication, we subjected a dnaA(cos) mutant strain to transposon mutagenesis and selected mutant derivatives that could form colonies at 30°C. Four...... suppressors of the dnaA(cos)-mediated cold sensitivity were identified and further characterized. Based on origin to terminus ratios, chromosome content per cell, measured by flow cytometry, and sensitivity to the replication fork inhibitor hydroxyurea, the suppressors fell into two distinct categories: those...

  15. DNA microarray analysis of fim mutations in Escherichia coli

    DEFF Research Database (Denmark)

    Schembri, Mark; Ussery, David; Workman, Christopher; Hasman, Henrik; Klemm, Per

    2002-01-01

    we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type I fimbriae. Changes in fim...

  16. Screening for mtDNA diabetes mutations in Pima Indians with NIDDM

    Energy Technology Data Exchange (ETDEWEB)

    Sepehrnia, B.; Prezant, T.R.; Rotter, J.I. [UCLA School of Medicine, Los Angeles, CA (United States)] [and others

    1995-03-27

    More than half of the Pima Indians over age 35 years have non-insulin-dependent (type II) diabetes mellitus (NIDDM). Extensive data indicate the importance of maternal diabetes in determining their risk for diabetes. Generally, the risk of having NIDDM is higher in patients with affected mothers than affected fathers. This has been attributed to intrauterine factors, but recently mitochondrial inheritance has been raised as an alternative hypothesis. In other populations, several families and individuals with diabetes due to a mitochondrial DNA point mutation at nucleotide 3243 in the tRNA{sup leu(UUR)} gene have been described, as has one family with a 10.4 kb mitochondrial DNA duplication/deletion. We tested whether these specific mitochondrial gene mutations could explain a portion of the excess maternal transmission seen in the Pima Indians. Mitochondrial DNA obtained from blood lymphocytes of 148 Pima Indians with NIDDM was screened both for the point mutation at nt 3243, and the 10.4 kb duplication/deletion. Neither of these mutations was detected, and although a small proportion of the excess maternal transmission in Pima Indians could still be due to yet undescribed mitochondrial mutations or imprinted nuclear genes, our data support the role of the intrauterine environment in this population. 32 refs, 21 figs.

  17. A New Approach To the Diagnosis of Point Mutations in Native DNA Using Graphene Oxide

    Science.gov (United States)

    Kuznetsov, A.A.; Maksimova, N.R.; Kaimonov, V.S.; Alexandrov, G.N.; Smagulova, S.A.

    2016-01-01

    Development of new methods for the diagnosis of point mutations is a pressing issue. We have developed a new approach to the design of graphene oxide-based test systems for the diagnosis of point mutations in native DNA. This new approach is based on the use of graphene oxide for the adsorption and quenching of fluorescently labeled primers in a post-amplification PCR mixture followed by detection of fluorescently labeled PCR products. It is possible to detect fluorescently labelled amplicons in the presence of an excess of primers in a PCR product solution due to the different affinities of single-stranded and double-stranded DNA molecules to graphene oxide, as well as the ability of graphene oxide to act as a quencher of the fluorophores adsorbed on its surface. The new approach was tested by designing a graphene oxide-based test system for the DNA diagnosis of the point mutation associated with the development of the 3M syndrome in Yakuts. The developed approach enables one to design graphene oxide-based test systems suitable for the diagnosis of any point mutations in native DNA. PMID:27437142

  18. A New Approach To the Diagnosis of Point Mutations in Native DNA Using Graphene Oxide.

    Science.gov (United States)

    Kuznetsov, A A; Maksimova, N R; Kaimonov, V S; Alexandrov, G N; Smagulova, S A

    2016-01-01

    Development of new methods for the diagnosis of point mutations is a pressing issue. We have developed a new approach to the design of graphene oxide-based test systems for the diagnosis of point mutations in native DNA. This new approach is based on the use of graphene oxide for the adsorption and quenching of fluorescently labeled primers in a post-amplification PCR mixture followed by detection of fluorescently labeled PCR products. It is possible to detect fluorescently labelled amplicons in the presence of an excess of primers in a PCR product solution due to the different affinities of single-stranded and double-stranded DNA molecules to graphene oxide, as well as the ability of graphene oxide to act as a quencher of the fluorophores adsorbed on its surface. The new approach was tested by designing a graphene oxide-based test system for the DNA diagnosis of the point mutation associated with the development of the 3M syndrome in Yakuts. The developed approach enables one to design graphene oxide-based test systems suitable for the diagnosis of any point mutations in native DNA. PMID:27437142

  19. Somatic mtDNA mutation spectra in the aging human putamen.

    Directory of Open Access Journals (Sweden)

    Siôn L Williams

    Full Text Available The accumulation of heteroplasmic mitochondrial DNA (mtDNA deletions and single nucleotide variants (SNVs is a well-accepted facet of the biology of aging, yet comprehensive mutation spectra have not been described. To address this, we have used next generation sequencing of mtDNA-enriched libraries (Mito-Seq to investigate mtDNA mutation spectra of putamen from young and aged donors. Frequencies of the "common" deletion and other "major arc" deletions were significantly increased in the aged cohort with the fold increase in the frequency of the common deletion exceeding that of major arc deletions. SNVs also increased with age with the highest rate of accumulation in the non-coding control region which contains elements necessary for translation and replication. Examination of predicted amino acid changes revealed a skew towards pathogenic SNVs in the coding region driven by mutation bias. Levels of the pathogenic m.3243A>G tRNA mutation were also found to increase with age. Novel multimeric tandem duplications that resemble murine control region multimers and yeast ρ(- mtDNAs, were identified in both young and aged specimens. Clonal ∼50 bp deletions in the control region were found at high frequencies in aged specimens. Our results reveal the complex manner in which the mitochondrial genome alters with age and provides a foundation for studies of other tissues and disease states.

  20. Genome sequencing of pediatric medulloblastoma links catastrophic DNA rearrangements with TP53 mutations

    DEFF Research Database (Denmark)

    Rausch, Tobias; Jones, David T W; Zapatka, Marc;

    2012-01-01

    a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals a...

  1. Soluble normal and mutated DNA sequences from single-copy genes in human blood.

    Science.gov (United States)

    Sorenson, G D; Pribish, D M; Valone, F H; Memoli, V A; Bzik, D J; Yao, S L

    1994-01-01

    Healthy individuals have soluble (extracellular) DNA in their blood, and increased amounts are present in cancer patients. Here we report the detection of specific sequences of the cystic fibrosis and K-ras genes in plasma DNA from normal donors by amplification with the polymerase chain reaction. In addition, mutated K-ras sequences are identified by polymerase chain reaction utilizing allele-specific primers in the plasma or serum from three patients with pancreatic carcinoma that contain mutated K-ras genes. The mutations are confirmed by direct sequencing. These results indicate that sequences of single-copy genes can be identified in normal plasma and that the sequences of mutated oncogenes can be detected and identified with allele-specific amplification by polymerase chain reaction in plasma or serum from patients with malignant tumors containing identical mutated genes. Mutated oncogenes in plasma and serum may represent tumor markers that could be useful for diagnosis, determining response to treatment, and predicting prognosis. PMID:8118388

  2. Detection of somatic mutations by high-resolution DNA melting (HRM analysis in multiple cancers.

    Directory of Open Access Journals (Sweden)

    Jesus Gonzalez-Bosquet

    Full Text Available Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each. HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

  3. 4-Aminobiphenyl-DNA adducts and p53 mutations in bladder cancer.

    Science.gov (United States)

    Martone, T; Airoldi, L; Magagnotti, C; Coda, R; Randone, D; Malaveille, C; Avanzi, G; Merletti, F; Hautefeuille, A; Vineis, P

    1998-02-01

    Epidemiologic studies have suggested that smokers of air-cured tobacco (rich in arylamines) are at higher risk of bladder cancer than smokers of flue-cured tobacco. The risk has been shown to be modulated by the N-acetyltransferase genotype. We analyzed the biopsies of 45 patients with bladder cancer. p53 mutations were sought by direct sequencing, and 4-aminobiphenyl-DNA adducts were measured by negative ion gas chromatography-mass spectrometry. 4-Aminobiphenyl-DNA adducts were higher in smokers of air-cured tobacco and in current smokers, but no relationship with the number of cigarettes smoked was found. Adducts were higher in more advanced histologic grades of tumors. No pattern was evident for p53 mutations. Seven of 9 mutations occurred in grade 3 tumors. No association was found between 4-ABP adducts and GSTM1 or NAT2 genetic polymorphisms. PMID:9466649

  4. Mutations in BALB mitochondrial DNA induce CCL20 up-regulation promoting tumorigenic phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Sligh, James [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Janda, Jaroslav [University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Jandova, Jana, E-mail: jjandova@email.arizona.edu [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States)

    2014-11-15

    Highlights: • Alterations in mitochondrial DNA are commonly found in various human cancers. • Mutations in BALB mitochondrial DNA induce up-regulation of chemokine CCL20. • Increased growth and motility of mtBALB cells is associated with CCL20 levels. • mtDNA changes in BALB induce in vivo tumor growth through CCL20 up-regulation. • Mutations in mitochondrial DNA play important roles in keratinocyte neoplasia. - Abstract: mtDNA mutations are common in human cancers and are thought to contribute to the process of neoplasia. We examined the role of mtDNA mutations in skin cancer by generating fibroblast cybrids harboring a mutation in the gene encoding the mitochondrial tRNA for arginine. This somatic mutation (9821insA) was previously reported in UV-induced hyperkeratotic skin tumors in hairless mice and confers specific tumorigenic phenotypes to mutant cybrids. Microarray analysis revealed and RT-PCR along with Western blot analysis confirmed the up-regulation of CCL20 and its receptor CCR6 in mtBALB haplotype containing the mt-Tr 9821insA allele compared to wild type mtB6 haplotype. Based on reported role of CCL20 in cancer progression we examined whether the hyper-proliferation and enhanced motility of mtBALB haplotype would be associated with CCL20 levels. Treatment of both genotypes with recombinant CCL20 (rmCCL20) resulted in enhanced growth and motility of mtB6 cybrids. Furthermore, the acquired somatic alteration increased the in vivo tumor growth of mtBALB cybrids through the up-regulation of CCL20 since neutralizing antibody significantly decreased in vivo tumor growth of these cells; and tumors from anti-CCL20 treated mice injected with mtBALB cybrids showed significantly decreased CCL20 levels. When rmCCL20 or mtBALB cybrids were used as chemotactic stimuli, mtB6 cybrids showed increased motility while anti-CCL20 antibody decreased the migration and in vivo tumor growth of mtBALB cybrids. Moreover, the inhibitors of MAPK signaling and NF

  5. Mutations in BALB mitochondrial DNA induce CCL20 up-regulation promoting tumorigenic phenotypes

    International Nuclear Information System (INIS)

    Highlights: • Alterations in mitochondrial DNA are commonly found in various human cancers. • Mutations in BALB mitochondrial DNA induce up-regulation of chemokine CCL20. • Increased growth and motility of mtBALB cells is associated with CCL20 levels. • mtDNA changes in BALB induce in vivo tumor growth through CCL20 up-regulation. • Mutations in mitochondrial DNA play important roles in keratinocyte neoplasia. - Abstract: mtDNA mutations are common in human cancers and are thought to contribute to the process of neoplasia. We examined the role of mtDNA mutations in skin cancer by generating fibroblast cybrids harboring a mutation in the gene encoding the mitochondrial tRNA for arginine. This somatic mutation (9821insA) was previously reported in UV-induced hyperkeratotic skin tumors in hairless mice and confers specific tumorigenic phenotypes to mutant cybrids. Microarray analysis revealed and RT-PCR along with Western blot analysis confirmed the up-regulation of CCL20 and its receptor CCR6 in mtBALB haplotype containing the mt-Tr 9821insA allele compared to wild type mtB6 haplotype. Based on reported role of CCL20 in cancer progression we examined whether the hyper-proliferation and enhanced motility of mtBALB haplotype would be associated with CCL20 levels. Treatment of both genotypes with recombinant CCL20 (rmCCL20) resulted in enhanced growth and motility of mtB6 cybrids. Furthermore, the acquired somatic alteration increased the in vivo tumor growth of mtBALB cybrids through the up-regulation of CCL20 since neutralizing antibody significantly decreased in vivo tumor growth of these cells; and tumors from anti-CCL20 treated mice injected with mtBALB cybrids showed significantly decreased CCL20 levels. When rmCCL20 or mtBALB cybrids were used as chemotactic stimuli, mtB6 cybrids showed increased motility while anti-CCL20 antibody decreased the migration and in vivo tumor growth of mtBALB cybrids. Moreover, the inhibitors of MAPK signaling and NF

  6. First paraben substituted cyclotetraphosphazene compounds and DNA interaction analysis with a new automated biosensor.

    Science.gov (United States)

    Çiftçi, Gönül Yenilmez; Şenkuytu, Elif; İncir, Saadet Elif; Yuksel, Fatma; Ölçer, Zehra; Yıldırım, Tuba; Kılıç, Adem; Uludağ, Yıldız

    2016-06-15

    Cancer, as one of the leading causes of death in the world, is caused by malignant cell division and growth that depends on rapid DNA replication. To develop anti-cancer drugs this feature of cancer could be exploited by utilizing DNA-damaging molecules. To achieve this, the paraben substituted cyclotetraphosphazene compounds have been synthesized for the first time and their effect on DNA (genotoxicity) has been investigated. The conventional genotoxicity testing methods are laborious, take time and are expensive. Biosensor based assays provide an alternative to investigate this drug/compound DNA interactions. Here for the first time, a new, easy and rapid screening method has been used to investigate the DNA damage, which is based on an automated biosensor device that relies on the real-time electrochemical profiling (REP™) technology. Using both the biosensor based screening method and the in vitro biological assay, the compounds 9 and 11 (propyl and benzyl substituted cyclotetraphosphazene compounds, respectively), have resulted in higher DNA damage than the others with 65% and 80% activity reduction, respectively. PMID:26852202

  7. Development of an Automated Microfluidic System for DNA Collection, Amplification, and Detection of Pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Hagan, Bethany S.; Bruckner-Lea, Cynthia J.

    2002-12-01

    This project was focused on developing and testing automated routines for a microfluidic Pathogen Detection System. The basic pathogen detection routine has three primary components; cell concentration, DNA amplification, and detection. In cell concentration, magnetic beads are held in a flow cell by an electromagnet. Sample liquid is passed through the flow cell and bacterial cells attach to the beads. These beads are then released into a small volume of fluid and delivered to the peltier device for cell lysis and DNA amplification. The cells are lysed during initial heating in the peltier device, and the released DNA is amplified using polymerase chain reaction (PCR) or strand displacement amplification (SDA). Once amplified, the DNA is then delivered to a laser induced fluorescence detection unit in which the sample is detected. These three components create a flexible platform that can be used for pathogen detection in liquid and sediment samples. Future developments of the system will include on-line DNA detection during DNA amplification and improved capture and release methods for the magnetic beads during cell concentration.

  8. A fluoroquinolone resistance associated mutation in gyrA affects DNA supercoiling in Campylobacter jejuni

    Directory of Open Access Journals (Sweden)

    Jing eHan

    2012-03-01

    Full Text Available The prevalence of fluoroquinolone (FQ-resistant Campylobacter has become a concern for public health. To facilitate the control of FQ-resistant Campylobacter, it is necessary to understand the impact of FQ resistance on the fitness of Campylobacter in its natural hosts as understanding fitness will help to determine and predict the persistence of FQ-resistant Campylobacter. Previously it was shown that acquisition of resistance to FQ antimicrobials enhanced the in vivo fitness of FQ-resistant Campylobacter. In this study, we confirmed the role of the Thr-86-Ile mutation in GyrA in modulating Campylobacter fitness by reverting the mutation to the wild-type allele, which resulted in the loss of the fitness advantage. Additionally, we determined if the resistance-conferring GyrA mutations alter the enzymatic function of the DNA gyrase. Recombinant wild-type gyrase and mutant gyrases with three different types of mutations (Thr-86-Ile, Thr-86-Lys, and Asp-90-Asn, which are associated with FQ resistance in Campylobacter, were generated in E. coli and compared for their supercoiling activities using an in vitro assay. The mutant gyrase with the Thr-86-Ile change showed a greatly reduced supercoiling activity compared with the wild-type gyrase, while other mutant gyrases did not show an altered supercoiling. Furthermore, we measured DNA supercoiling within Campylobacter cells using a reporter plasmid. Consistent with the results from the in vitro supercoiling assay, the FQ-resistant mutant carrying the Thr-86-Ile change in GyrA showed much less DNA supercoiling than the wild-type strain and the mutant strains carrying other mutations. Together, these results indicate that the Thr-86-Ile mutation, which is predominant in clinical FQ-resistant Campylobacter, modulates DNA supercoiling homeostasis in FQ-resistant Campylobacter.

  9. Rapid DNA analysis for automated processing and interpretation of low DNA content samples

    OpenAIRE

    Turingan, Rosemary S.; Vasantgadkar, Sameer; Palombo, Luke; Hogan, Catherine; Jiang, Hua; Tan, Eugene; Selden, Richard F.

    2016-01-01

    Background Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or f...

  10. Branch migration displacement assay with automated heuristic analysis for discrete DNA length measurement using DNA microarrays

    OpenAIRE

    Pourmand, Nader; Caramuta, Stefano; Villablanca, Andrea; Mori, Silvia; Karhanek, Miloslav; Wang, Shan X.; Ronald W Davis

    2007-01-01

    The analysis of short tandem repeats (STRs) plays an important role in forensic science, human identification, genetic mapping, and disease diagnostics. Traditional STR analysis utilizes gel- or column-based approaches to analyze DNA repeats. Individual STR alleles are separated and distinguished according to fragment length; thus the assay is generally hampered by its low multiplex capacity. However, use of DNA microarray would employ a simple hybridization and detection for field forensics ...

  11. Automated Line Tracking of lambda-DNA for Single-Molecule Imaging

    CERN Document Server

    Guan, Juan; Granick, Steve

    2011-01-01

    We describe a straightforward, automated line tracking method to visualize within optical resolution the contour of linear macromolecules as they rearrange shape as a function of time by Brownian diffusion and under external fields such as electrophoresis. Three sequential stages of analysis underpin this method: first, "feature finding" to discriminate signal from noise; second, "line tracking" to approximate those shapes as lines; third, "temporal consistency check" to discriminate reasonable from unreasonable fitted conformations in the time domain. The automated nature of this data analysis makes it straightforward to accumulate vast quantities of data while excluding the unreliable parts of it. We implement the analysis on fluorescence images of lambda-DNA molecules in agarose gel to demonstrate its capability to produce large datasets for subsequent statistical analysis.

  12. Detection of HIV cDNA Point Mutations with Rolling-Circle Amplification Arrays

    Directory of Open Access Journals (Sweden)

    Zhongwei Wu

    2010-01-01

    Full Text Available In this paper we describe an isothermal rolling-circle amplification (RCA protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP.The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.

  13. Role of mitochondrial DNA mutations in brain tumors: A mini-review.

    Science.gov (United States)

    Mohamed Yusoff, Abdul Aziz

    2015-01-01

    Brain tumor is molecularly a heterogeneous group of diseases, and genetic factors seem to play a crucial role in its genesis. Even though multiple alterations in the nuclear-encoded genes such as tumor suppressor and oncogenes are believed to play a key role in brain tumorigenesis, the involvement of the mitochondrial genome to this event remains controversial to date. Mitochondrial DNA (mtDNA) has been suspected to be associated with the carcinogenesis because of its high sensitivity to mutations and inefficient repair mechanisms in comparison to nuclear DNA. Thus, defects in mtDNA could also lead to the development of brain tumor. By virtue of their clonal nature and high copy number, mtDNA mutations may provide a new effective molecular biomarker for the cancer detection. It has been suggested that establishing mtDNA defective pattern might be useful in cancer diagnostics and detection, the prognosis of cancer outcome, and/or the response to certain treatments. This mini-review gives a brief overview on the several aspects of mtDNA, with a particular focus on its role in tumorigenesis and progression of brain tumor. Understanding the role of mitochondria and brain tumor development could potentially translate into therapeutic strategies for patients with these tumors. PMID:26458578

  14. Role of mitochondrial DNA mutations in brain tumors: A mini-review

    Directory of Open Access Journals (Sweden)

    Abdul Aziz Mohamed Yusoff

    2015-01-01

    Full Text Available Brain tumor is molecularly a heterogeneous group of diseases, and genetic factors seem to play a crucial role in its genesis. Even though multiple alterations in the nuclear-encoded genes such as tumor suppressor and oncogenes are believed to play a key role in brain tumorigenesis, the involvement of the mitochondrial genome to this event remains controversial to date. Mitochondrial DNA (mtDNA has been suspected to be associated with the carcinogenesis because of its high sensitivity to mutations and inefficient repair mechanisms in comparison to nuclear DNA. Thus, defects in mtDNA could also lead to the development of brain tumor. By virtue of their clonal nature and high copy number, mtDNA mutations may provide a new effective molecular biomarker for the cancer detection. It has been suggested that establishing mtDNA defective pattern might be useful in cancer diagnostics and detection, the prognosis of cancer outcome, and/or the response to certain treatments. This mini-review gives a brief overview on the several aspects of mtDNA, with a particular focus on its role in tumorigenesis and progression of brain tumor. Understanding the role of mitochondria and brain tumor development could potentially translate into therapeutic strategies for patients with these tumors.

  15. Relationship Between Mitochondrial DNA Mutations and Aging. Estimation of Age-at-death.

    Science.gov (United States)

    Zapico, Sara C; Ubelaker, Douglas H

    2016-04-01

    Some studies have pointed to the relationship between mitochondrial DNA (mtDNA) mutations and age in different tissues, which are potentially interesting in aging research and in forensic identification because they could help to improve the estimation of age-at-death. The present study aims to evaluate the mutations in mtDNA from dentin and pulp and their relation with age. Healthy erupted third molars were extracted from individuals from two Spanish populations, aged 20-70. When analyzing the amplification of hypervariable region 2 of the mtDNA by real-time polymerase chain reaction, a negative strong linear correlation was found between the mtDNA amplification and age in dentin from both populations. In contrast, a significant correlation between mtDNA amplification and age in pulp was not discovered, probably due to the majority of the mitochondria are placed in dentin. A difference in mtDNA damage between these two populations was also detected, indicating the role of ancestry as a component. The findings from this research enrich the current studies related to aging and mitochondrial damage and provide a new quantitative tool for estimating the age-at-death that, in combination with traditional age markers, could improve identification accuracy in forensic cases. PMID:26286606

  16. Rapid photometric detection of thymine residues partially flipped out of double helix as a method for direct scanning of point mutations and apurinic DNA sites.

    Science.gov (United States)

    Logvina, N A; Yakubovskaya, M G; Dolinnaya, N G

    2011-02-01

    A spectroscopic assay for detection of extrahelical thymine residues in DNA heteroduplexes under their modification by potassium permanganate has been developed. The assay is based on increase in absorbance at 420 nm due to accumulation of thymidine oxidation intermediates and soluble manganese dioxide. The analysis was carried out using a set of 19-bp DNA duplexes containing unpaired thymidines opposite tetrahydrofuranyl derivatives mimicking a widespread DNA damage (apurinic (AP) sites) and a library of 50-bp DNA duplexes containing all types of base mismatches in different surroundings. The relation between the selectivity of unpaired T oxidation and the thermal stability of DNA double helix was investigated. The method described here was shown to discriminate between DNA duplexes with one or two AP sites and to reveal thymine-containing mismatches and all noncanonical base pairs in AT-surroundings. Comparative results of CCM analysis and the rapid photometric assay for mismatch detection are demonstrated for the first time in the same model system. The chemical reactivity of target thymines was shown to correlate with local disturbance of double helix at the mismatch site. As the spectroscopic assay does not require the DNA cleavage reaction and gel electrophoresis, it can be easily automated and used for primary screening of somatic mutations. PMID:21568858

  17. Structural Insight into Processive Human Mitochondrial DNA Synthesis and Disease-Related Polymerase Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young-Sam; Kennedy, W. Dexter; Yin, Y. Whitney; (Texas)

    2010-09-07

    Human mitochondrial DNA polymerase (Pol {gamma}) is the sole replicase in mitochondria. Pol {gamma} is vulnerable to nonselective antiretroviral drugs and is increasingly associated with mutations found in patients with mitochondriopathies. We determined crystal structures of the human heterotrimeric Pol {gamma} holoenzyme and, separately, a variant of its processivity factor, Pol {gamma}B. The holoenzyme structure reveals an unexpected assembly of the mitochondrial DNA replicase where the catalytic subunit Pol {gamma}A interacts with its processivity factor primarily via a domain that is absent in all other DNA polymerases. This domain provides a structural module for supporting both the intrinsic processivity of the catalytic subunit alone and the enhanced processivity of holoenzyme. The Pol {gamma} structure also provides a context for interpreting the phenotypes of disease-related mutations in the polymerase and establishes a foundation for understanding the molecular basis of toxicity of anti-retroviral drugs targeting HIV reverse transcriptase.

  18. Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

    Directory of Open Access Journals (Sweden)

    Monica K Akre

    Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

  19. DNA microarray analysis of fim mutations in Escherichia coli

    DEFF Research Database (Denmark)

    Schembri, Mark; Ussery, David; Workman, Christopher;

    2002-01-01

    Bacterial adhesion is often mediated by complex polymeric surface structures referred to as fimbriae. Type I fimbriae of Escherichia coli represent the archetypical and best characterised fimbrial system. These adhesive organelles mediate binding to D-mannose and are directly associated with...... we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type I fimbriae. Changes in fim...

  20. Automated and Manual Methods of DNA Extraction for Aspergillus fumigatus and Rhizopus oryzae Analyzed by Quantitative Real-Time PCR▿

    OpenAIRE

    Francesconi, Andrea; Kasai, Miki; Harrington, Susan M.; Beveridge, Mara G; Petraitiene, Ruta; Petraitis, Vidmantas; Schaufele, Robert L.; Walsh, Thomas J.

    2008-01-01

    Quantitative real-time PCR (qPCR) may improve the detection of fungal pathogens. Extraction of DNA from fungal pathogens is fundamental to optimization of qPCR; however, the loss of fungal DNA during the extraction process is a major limitation to molecular diagnostic tools for pathogenic fungi. We therefore studied representative automated and manual extraction methods for Aspergillus fumigatus and Rhizopus oryzae. Both were analyzed by qPCR for their ability to extract DNA from propagules a...

  1. Transcription Restores DNA Repair to Heterochromatin, Determining Regional Mutation Rates in Cancer Genomes

    Directory of Open Access Journals (Sweden)

    Christina L. Zheng

    2014-11-01

    Full Text Available Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs arising in an XPC−/− background. XPC−/− cells lack global genome nucleotide excision repair (GG-NER, thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.

  2. [Clinical features and DGUOK mutations of an infant with mitochondrial DNA depletion syndrome].

    Science.gov (United States)

    Deng, Mei; Lin, Wei-Xia; Guo, Li; Zhang, Zhan-Hui; Song, Yuan-Zong

    2016-06-01

    The aim of this study was to investigate the clinical features and DGUOK gene mutations of an infant with mitochondrial DNA depletion syndrome (MDS). The patient (more than 7 months old) manifested as hepatosplenomegaly, abnormal liver function, nystagmus and psychomotor retardation. Genetic DNA was extracted from peripheral blood samples of the patient and her parents. Targeted Exome Sequencing was performed to explore the genetic causes. Sanger sequencing was carried out to confirm the detected mutations. The sequencing results showed that the patient was a compound heterozygote for c.679G>A and c.817delT in the DGUOK gene. The former was a reportedly pathogenic missense mutation of maternal origin, while the latter, a frameshift mutation from the father, has not been described yet. The findings in this study expand the mutation spectrum of DGUOK gene, and provide molecular evidence for the etiologic diagnosis of the patient as well as for the genetic counseling and prenatal diagnosis in the family. PMID:27324545

  3. Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNA.

    Science.gov (United States)

    Seeger, Christoph; Sohn, Ji A

    2016-08-01

    Hepatitis B virus (HBV) causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV. Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA. In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15,000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB. PMID:27203444

  4. EGFR-activating mutations, DNA copy number abundance of ErbB family, and prognosis in lung adenocarcinoma

    Science.gov (United States)

    Chen, Hsuan-Yu; Liu, Chia-Hsin; Chang, Ya-Hsuan; Yu, Sung-Liang; Ho, Bing-Ching; Hsu, Chung-Ping; Yang, Tsung-Ying; Chen, Kun-Chieh; Hsu, Kuo-Hsuan; Tseng, Jeng-Sen; Hsia, Jiun-Yi; Chuang, Cheng-Yen; Chang, Chi-Sheng; Li, Yu-Cheng; Li, Ker-Chau; Chang, Gee-Chen; Yang, Pan-Chyr

    2016-01-01

    In this study, EGFR-activating mutation status and DNA copy number abundances of members of ErbB family were measured in 261 lung adenocarcinomas. The associations between DNA copy number abundances of ErbB family, EGFR-activating mutation status, and prognosis were explored. Results showed that DNA copy number abundances of EGFR, ERBB2, ERBB3, and ERBB4 had associations with overall survival in lung adenocarcinoma with EGFR-activating mutations. In the stratification analysis, only ERBB2 showed significant discrepancy in patients carrying wild type EGFR and other members of ErbB family in patients carrying EGFR-activating mutation. This indicated that CNAs of ErbB family had effect modifications of EGFR-activating mutation status. Findings of this study demonstrate potential molecular guidance of patient management of lung adenocarcinoma with or without EGFR-activating mutations. PMID:26824984

  5. Gamma radiation-induced heritable mutations at repetitive DNA loci in out-bred mice

    International Nuclear Information System (INIS)

    Recent studies have shown that expanded-simple-tandem-repeat (ESTR) DNA loci are efficient genetic markers for detecting radiation-induced germ line mutations in mice. Dose responses following irradiation, however, have only been characterized in a small number of inbred mouse strains, and no studies have applied Esters to examine potential modifiers of radiation risk, such as adaptive response. We gamma-irradiated groups of male out-bred Swiss-Webster mice with single acute doses of 0.5 and 1.0 Gy, and compared germ line mutation rates at ESTR loci to a sham-irradiated control. To test for evidence of adaptive response we treated a third group with a total dose of 1.1 Gy that was fractionated into a 0.1 Gy adapting dose, followed by a challenge dose of 1.0 Gy 24 h later. Paternal mutation rates were significantly elevated above the control in the 0.5 Gy (2.8-fold) and 1.0 Gy (3.0-fold) groups, but were similar to each other despite the difference in radiation dose. The doubling dose for paternal mutation induction was 0.26 Gy (95% CI = 0.14-0.51 Gy). Males adapted with a 0.1 Gy dose prior to a 1.0 Gy challenge dose had mutation rates that were not significantly elevated above the control, and were 43% reduced compared to those receiving single doses. We conclude that pre-meiotic male germ cells in out-bred Swiss-Webster mice are sensitive to ESTR mutations induced by acute doses of ionizing radiation, but mutation induction may become saturated at a lower dose than in some strains of inbred mice. Reduced mutation rates in the adapted group provide intriguing evidence for suppression of ESTR mutations in the male germline through adaptive response. Repetitive DNA markers may be useful tools for exploration of biological factors affecting the probability of heritable mutations caused by low-dose ionizing radiation exposure. The biological significance of ESTR mutations in terms of radiation risk assessment, however, is still undetermined

  6. Human aging and somatic point mutations in mtDNA: a comparative study of generational differences (grandparents and grandchildren

    Directory of Open Access Journals (Sweden)

    Anderson Nonato do Rosário Marinho

    2011-01-01

    Full Text Available The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years and their 62 grandchildren (mean age: 15 ± 4.1 years, the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers and in a heteroplasmic state in an additional 13 grandmothers; no mutations were detected in the remaining 15 grandmothers. However, cloning followed by DNA sequencing in 12 grandmothers confirmed homoplasia in only one of the three mutations previously considered to be homoplasmic and did not confirm heteroplasmy in three out of nine grandmothers found to be heteroplasmic by direct sequencing. Thus, of 12 grandmothers in whom mtDNA was analyzed by cloning, eight were heteroplasmic for mutations not detected in their grandchildren. In this study, the use of genetically related subjects allowed us to demonstrate the occurrence of age-related (> 60 years old mutations (homoplasia and heteroplasmy. It is possible that both of these situations (homoplasia and heteroplasmy were a long-term consequence of mitochondrial oxidative phosphorylation that can lead to the accumulation of mtDNA mutations throughout life.

  7. Hepatitis B virus basal core promoter mutations show lower replication fitness associated with cccDNA acetylation status.

    Science.gov (United States)

    Koumbi, Lemonica; Pollicino, Teresa; Raimondo, Giovanni; Stampoulis, Dimitrios; Khakoo, Salim; Karayiannis, Peter

    2016-07-15

    In chronic hepatitis B virus (HBV) infection, variants with mutations in the basal core promoter (BCP) and precore region predominate and associate with more severe disease forms. Studies on their effect on viral replication remain controversial. Increasing evidence shows that epigenetic modifications of cccDNA regulate HBV replication and disease outcome. Here we determined the transcription and viral replication efficiency of well-defined BCP and precore mutations and their effect on cccDNA epigenetic control. HBV monomers bearing BCP mutations A1762T/G1764A and A1762T/G1764A/C1766T, and precore mutations G1896A, G1899A and G1896A/G1899A, were transfected into HepG2 cells using a plasmid-free approach. Viral RNA transcripts were detected by Northern blot hybridization and RT PCR, DNA replicative intermediates by Southern blotting and RT PCR, and viral release was measured by ELISA. Acetylation of cccDNA-bound histones was assessed by Chromatin ImmunoPrecipitation (ChIP) assay and methylation of cccDNA by bisulfite sequencing. BCP mutations resulted in low viral release, mRNA transcription and pgRNA/cccDNA ratios that paralleled the acetylation of cccDNA-bound H4 histone and inversely correlated with the HDAC1 recruitment onto cccDNA. Independently of the mutations, cccDNA was a target for methylation, accompanied by the upregulation of DNMT1 expression and DNMT1 recruitment onto cccDNA. Our results suggest that BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones while precore mutations do not have a significant effect on viral replication. These data provide evidence that epigenetic factors contribute to the regulation of HBV viral replication. PMID:27132039

  8. Mutations in the two ribosomal RNA genes in mitochondrial DNA among Finnish children with hearing impairment

    OpenAIRE

    Häkli, Sanna; Luotonen, Mirja; Sorri, Martti; Majamaa, Kari

    2015-01-01

    Background Mutations in the two MT-RNR genes in mitochondrial DNA can cause hearing impairment that presents with variable severity and age of onset. In order to study the prevalence of mutations in MT-RNR1 and MT-RNR2 genes among Finnish children, we studied a ten-year cohort of hearing impaired children born in Northern Finland. Methods We studied children, who had been born in Northern Finland in 1993–2002 and who had been ascertained to have hearing impairment by 31 December 2007. Samples...

  9. Suppression of thermosensitive initiation of DNA replication in a dnaR mutant of Escherichia coli by a rifampin resistance mutation in the rpoB gene.

    OpenAIRE

    Sakakibara, Y

    1995-01-01

    The thermosensitivity of the Escherichia coli dnaR130 mutant in initiation of DNA replication was suppressed by a spontaneous rifampin resistance mutation in rpoB, the gene for the beta subunit of RNA polymerase. Among the dnaR-suppressing rpoB alleles obtained was rpoB22, which was able to suppress the thermosensitivity of the dnaA46 or dnaA167 mutant, but not that of the dnaA5 mutant, in initiation of replication. Some dnaA-suppressing rpoB alleles obtained from rifampin-resistant derivativ...

  10. Absence of correlation between serum CRP levels and mitochondrial D-loop DNA mutations in gastro-oesophageal adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Benjamin H. L. Tan

    2014-01-01

    Full Text Available Introduction: Both inflammation and mitochondrial DNA (mtDNA mutation are thought to play a role in the many human cancers. The aim of this study was to evaluate the relationship between inflammation and accumulation of mitochondrial DNA (mtDNA mutations in the D-loop region in carcinogenesis of gastro-oesophageal adenocarcinomas. Materials and Methods: Blood samples of 20 patients with gastro-oesophageal adenocarcinoma were taken for measurement of serum C-reactive protein (CRP concentration. Direct sequencing of mtDNA in the D-loop region was done in the 20 adenocarcinoma samples and their corresponding surrounding non-cancerous tissue. Sequences were compared with existing mtDNA databases to identify mutations. Results: mtDNA mutations in the D-loop region occur commonly with almost identical frequency in both non-cancerous tissue (3.0 ΁ 1.6 and adenocarcinoma (3.1 ΁ 1.9 (P = 0.916, paired t-test. CRP levels are not predictive of the number of D-loop mutations in both adenocarcinoma (β: -0.131; 95% CI: -2.354-1.364; P = 0.583 and non-cancerous tissue samples (β: 0.130; 95% CI: -1.125-1.933; P = 0.586. Five new mutations were identified that were not recorded previously in mtDNA databases. Conclusion: D-loop mtDNA mutations are common in both gastro-oesophageal adenocarcinoma and surrounding non-cancerous tissue. However, the accumulation of such mutations appears to occur independent of systemic inflammation. The frequency of D-loop mutations is likely not useful as a marker for carcinogenesis in gastro-oesophageal adenocarcinoma.

  11. A high sensitivity, high throughput, automated single-cell gel electrophoresis ('Comet') DNA damage assay

    International Nuclear Information System (INIS)

    A fully automated microscopy machine vision image capture and analysis system for the collection of data from slides of 'comets' has been developed. The novel image processing algorithms employed in delineating the 'comet head' from the 'comet tail' allow us to determine accurately very low levels of damage. In conjunction with calibrated and automated image capture methods, we are able to eliminate operator subjectivity and analyse large numbers of cells (>2500) in a short time (<1 hour). The image processing algorithm is designed to handle particularly difficult nuclei containing a high degree of structure, due to DNA clumping. We also present techniques used to extend the assay's dynamic range by removing interfering background fluorescence and to define a region of interest. If subtle biological variations are to be quantified (e.g. cell cycle dependant damage), then the use of large cell populations is dictated. Under those circumstances, the use of a fully automated system is particularly advantageous providing that the manner in which data is extracted does not introduce any inadvertent bias. In practice, it is essential that the image processing steps are geared towards the correct recognition of an acceptable cell nucleus, i.e. comet 'head'. We acknowledge the financial support of CRUK, Programme Grant C133/A1812 - SP 2195-01/02 and the US Department of Energy Low Dose Radiation Research Program grant DE-FG07-99ER62878

  12. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients

    OpenAIRE

    Olmedillas López, Susana; García-Olmo, Dolores C; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-01-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy”) by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorect...

  13. Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection

    OpenAIRE

    Frey Daniel; Edmondson Sonia; Crone Stephanie; Sauter Marion; Wagen Sandro; Kuchen Melanie; Olieric Natacha; Ostermeier Christian; Steinmetz Michel O; Jaussi Rolf

    2010-01-01

    Abstract Background Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products. Results Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong express...

  14. A Key Evolutionary Mutation Enhances DNA Binding of the FOXP2 Forkhead Domain.

    Science.gov (United States)

    Morris, Gavin; Fanucchi, Sylvia

    2016-04-01

    Forkhead box (FOX) transcription factors share a conserved forkhead DNA binding domain (FHD) and are key role players in the development of many eukaryotic species. Their involvement in various congenital disorders and cancers makes them clinically relevant targets for novel therapeutic strategies. Among them, the FOXP subfamily of multidomain transcriptional repressors is unique in its ability to form DNA binding homo and heterodimers. The truncated FOXP2 FHD, in the absence of the leucine zipper, exists in equilibrium between monomeric and domain-swapped dimeric states in vitro. As a consequence, determining the DNA binding properties of the FOXP2 FHD becomes inherently difficult. In this work, two FOXP2 FHD hinge loop mutants have been generated to successfully prevent both the formation (A539P) and the dissociation (F541C) of the homodimers. This allows for the separation of the two species for downstream DNA binding studies. Comparison of DNA binding of the different species using electrophoretic mobility shift assay, fluorescence anisotropy and isothermal titration calorimetry indicates that the wild-type FOXP2 FHD binds DNA as a monomer. However, comparison of the DNA-binding energetics of the monomer and wild-type FHD, reveals that there is a difference in the mechanism of binding between the two species. We conclude that the naturally occurring reverse mutation (P539A) seen in the FOXP subfamily increases DNA binding affinity and may increase the potential for nonspecific binding compared to other FOX family members. PMID:26950495

  15. Human aging and somatic point mutations in mtDNA: A comparative study of generational differences (grandparents and grandchildren)

    OpenAIRE

    Anderson Nonato do Rosário Marinho; Milene Raiol de Moraes; Sidney Santos; Ândrea Ribeiro-dos- Santos

    2011-01-01

    The accumulation of somatic mutations in mtDNA is correlated with aging. In this work, we sought to identify somatic mutations in the HVS-1 region (D-loop) of mtDNA that might be associated with aging. For this, we compared 31 grandmothers (mean age: 63 ± 2.3 years) and their 62 grandchildren (mean age: 15 ± 4.1 years), the offspring of their daughters. Direct DNA sequencing showed that mutations absent in the grandchildren were detected in a presumably homoplasmic state in three grandmothers...

  16. A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

    Science.gov (United States)

    Thierry, Alain R

    2016-01-01

    Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics

  17. Identification of unique repeated patterns, location of mutation in DNA finger printing using artificial intelligence technique.

    Science.gov (United States)

    Mukunthan, B; Nagaveni, N

    2014-01-01

    In genetic engineering, conventional techniques and algorithms employed by forensic scientists to assist in identification of individuals on the basis of their respective DNA profiles involves more complex computational steps and mathematical formulae, also the identification of location of mutation in a genomic sequence in laboratories is still an exigent task. This novel approach provides ability to solve the problems that do not have an algorithmic solution and the available solutions are also too complex to be found. The perfect blend made of bioinformatics and neural networks technique results in efficient DNA pattern analysis algorithm with utmost prediction accuracy. PMID:24589835

  18. Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Miotke, Laura; Maity, Arindam; Ji, Hanlee; Brewer, Jonathan; Astakhova, Kira

    2015-01-01

    BACKGROUND: Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of...... methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence...... microscopy and nucleic acid analogues have been proposed so far. METHODS AND RESULTS: Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and...

  19. Alternative mechanisms of telomere lengthening: Permissive mutations, DNA repair proteins and tumorigenic progression

    Energy Technology Data Exchange (ETDEWEB)

    Gocha, April Renee Sandy; Harris, Julia [Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna, E-mail: joanna.groden@osumc.edu [Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

    2013-03-15

    Highlights: ► Neoplastic cells maintain telomeres by telomerase or ALT. ► Genetic mutations in p53, ATRX, DAXX or H3F3A may activate ALT. ► Many DNA repair proteins are involved in ALT. ► Tumor progression is favored by telomerase expression. - Abstract: Telomeres protect chromosome termini to maintain genomic stability and regulate cellular lifespan. Maintenance of telomere length is required for neoplastic cells after the acquisition of mutations that deregulate cell cycle control and increase cellular proliferation, and can occur through expression of the enzyme telomerase or in a telomerase-independent manner termed alternative lengthening of telomeres (ALT). The precise mechanisms that govern the activation of ALT or telomerase in tumor cells are unknown, although cellular origin may favor one or the other mechanisms. ALT pathways are incompletely understood to date; however, recent publications have increasingly broadened our understanding of how ALT is activated, how it proceeds, and how it influences tumor growth. Specific mutational events influence ALT activation, as mutations in genes that suppress recombination and/or alterations in the regulation of telomerase expression are associated with ALT. Once engaged, ALT uses DNA repair proteins to maintain telomeres in the absence of telomerase; experiments that manipulate the expression of specific proteins in cells using ALT are illuminating some of its mechanisms. Furthermore, ALT may influence tumor growth, as experimental and clinical data suggest that telomerase expression may favor tumor progression. This review summarizes recent findings in mammalian cells and models, as well as clinical data, that identify the genetic mutations permissive to ALT, the DNA repair proteins involved in ALT mechanisms and the importance of telomere maintenance mechanisms for tumor progression. A comprehensive understanding of the mechanisms that permit tumor cell immortalization will be important for identifying

  20. Mitochondrial DNA (mt DNA) A3243G mutation associated with an annular perimacular retinal atrophy

    OpenAIRE

    Adjadj E.

    2010-01-01

    Résumé:Background:La mutation 3243 de 1'ADN mitochondrial est associee avec le syndrome l\\/HDD (surdite, diabète transmis par la mère) et le syndrome MELAS (Myopathie, Encéphalopathie, acidose Lactique et attaques cérébrales). Elle est aussi associe à des troubles cardiaques, digestifs, endo- et exocrines. Nous rapportons deux cas de maculopathie associée à cette mutation.Histoire et symptomes: pCas l: il s'agit d'une femme de 60 ans soufrant d'un diabète et d'une surdité sans plainte visuell...

  1. CSF studies facilitate DNA diagnosis in familial Alzheimer's disease due to a presenilin-1 mutation.

    OpenAIRE

    Bot, S.T. de; Kremer, H. P. H.; Dooijes, D.; Verbeek, M. M.

    2009-01-01

    In sporadic Alzheimer's disease (AD), cerebrospinal fluid (CSF) analysis is becoming increasingly relevant to establish an early diagnosis. We present a case of familial AD due to a presenilin-1 mutation in which CSF studies suggested appropriate DNA diagnostics. A 38 year old Dutch man presented with dementia, spastic paraparesis, and frontal executive function impairments, mimicking familial Creutzfeldt Jakob disease and frontotemporal dementia. CSF studies, revealing increased total tau an...

  2. Mutation patterns of mitochondrial H- and L-strand DNA in closely related Cyprinid fishes.

    OpenAIRE

    Bielawski, Joseph P.; John R. Gold

    2002-01-01

    Mitochondrial genome replication is asymmetric. Replication starts from the origin of heavy (H)-strand replication, displacing the parental H-strand as it proceeds along the molecule. The H-strand remains single stranded until light (L)-strand replication is initiated from a second origin of replication. It has been suggested that single-stranded H-strand DNA is more sensitive to mutational damage, giving rise to substitutional rate differences between the two strands and among genes in mamma...

  3. MtDNA analysis reveals enriched pathogenic mutations in Tibetan highlanders

    Science.gov (United States)

    Kang, Longli; Zheng, Hong-Xiang; Zhang, Menghan; Yan, Shi; Li, Lei; Liu, Lijun; Liu, Kai; Hu, Kang; Chen, Feng; Ma, Lifeng; Qin, Zhendong; Wang, Yi; Wang, Xiaofeng; Jin, Li

    2016-01-01

    Tibetan highlanders, including Tibetans, Monpas, Lhobas, Dengs and Sherpas, are considered highly adaptive to severe hypoxic environments. Mitochondrial DNA (mtDNA) might be important in hypoxia adaptation given its role in coding core subunits of oxidative phosphorylation. In this study, we employed 549 complete highlander mtDNA sequences (including 432 random samples) to obtain a comprehensive view of highlander mtDNA profile. In the phylogeny of a total of 36,914 sequences, we identified 21 major haplogroups representing founding events of highlanders, most of which were coalesced in 10 kya. Through founder analysis, we proposed a three-phase model of colonizing the plateau, i.e., pre-LGM Time (30 kya, 4.68%), post-LGM Paleolithic Time (16.8 kya, 29.31%) and Neolithic Time (after 8 kya, 66.01% in total). We observed that pathogenic mutations occurred far more frequently in 22 highlander-specific lineages (five lineages carrying two pathogenic mutations and six carrying one) than in the 6,857 haplogroups of all the 36,914 sequences (P = 4.87 × 10−8). Furthermore, the number of possible pathogenic mutations carried by highlanders (in average 3.18 ± 1.27) were significantly higher than that in controls (2.82 ± 1.40) (P = 1.89 × 10−4). Considering that function-altering and pathogenic mutations are enriched in highlanders, we therefore hypothesize that they may have played a role in hypoxia adaptation. PMID:27498855

  4. Obtaining insurance after DNA diagnostics: a survey among hypertrophic cardiomyopathy mutation carriers

    OpenAIRE

    Christiaans, Imke; Kok, Tjitske M; van Langen, Irene M.; Birnie, Erwin; Bonsel, Gouke J.; Wilde, Arthur A. M.; Smets, Ellen M. A.

    2009-01-01

    Hypertrophic cardiomyopathy (HCM) is a common hereditary heart disease associated with increased mortality. Disclosure of DNA test results may have social implications such as low access to insurance. In the Netherlands, insurance companies are restricted in the use of genetic information of their clients by the Medical Examination Act. A cross-sectional survey was used to assess the frequency and type of problems encountered by HCM mutation carriers applying for insurance, and associations w...

  5. DNA crosslinking, sister-chromatid exchange and specific-locus mutations.

    Science.gov (United States)

    Carrano, A V; Thompson, L H; Stetka, D G; Minkler, J L; Mazrimas, J A; Fong, S

    1979-11-01

    Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds. PMID:522865

  6. DNA crosslinking, sister-chromatid exchange and specific-locus mutations

    Energy Technology Data Exchange (ETDEWEB)

    Carrano, A.V.; Thompson, L.H.; Stetka, D.G.; Minkler, J.L.; Mazrimas, J.A.; Fong, S.

    1979-01-01

    Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.

  7. Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

    Directory of Open Access Journals (Sweden)

    Elena Ordoñez

    2013-01-01

    Full Text Available Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL, the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

  8. Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

    OpenAIRE

    Knopf, K W; Kaufman, E R; Crumpacker, C

    1981-01-01

    The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purifie...

  9. Mitochondrial DNA mutations provoke dominant inhibition of mitochondrial inner membrane fusion.

    Directory of Open Access Journals (Sweden)

    Cécile Sauvanet

    Full Text Available Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA. We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP or to maternally inherited Leigh Syndrome (MILS in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from

  10. Triplet repeat DNA structures and human genetic disease: dynamic mutations from dynamic DNA

    Indian Academy of Sciences (India)

    Richard R Sinden; Vladimir N Potaman; Elena A Oussatcheva; Christopher E Pearson; Yuri L Lyubchenko; Luda S Shlyakhtenko

    2002-02-01

    Fourteen genetic neurodegenerative diseases and three fragile sites have been associated with the expansion of (CTG)n•(CAG)n, (CGG)n•(CCG)n, or (GAA)n•(TTC)n repeat tracts. Different models have been proposed for the expansion of triplet repeats, most of which presume the formation of alternative DNA structures in repeat tracts. One of the most likely structures, slipped strand DNA, may stably and reproducibly form within triplet repeat sequences. The propensity to form slipped strand DNA is proportional to the length and homogeneity of the repeat tract. The remarkable stability of slipped strand DNA may, in part, be due to loop-loop interactions facilitated by the sequence complementarity of the loops and the dynamic structure of three-way junctions formed at the loop-outs.

  11. MtDNA mutations are a common cause of severe disease phenotypes in children with Leigh syndrome.

    Science.gov (United States)

    Naess, Karin; Freyer, Christoph; Bruhn, Helene; Wibom, Rolf; Malm, Gunilla; Nennesmo, Inger; von Döbeln, Ulrika; Larsson, Nils-Göran

    2009-05-01

    Leigh syndrome is a common clinical manifestation in children with mitochondrial disease and other types of inborn errors of metabolism. We characterised clinical symptoms, prognosis, respiratory chain function and performed extensive genetic analysis of 25 Swedish children suffering from Leigh syndrome with the aim to obtain insights into the molecular pathophysiology and to provide a rationale for genetic counselling. We reviewed the clinical history of all patients and used muscle biopsies in order to perform molecular, biochemical and genetic investigations, including sequencing the entire mitochondrial DNA (mtDNA), the mitochondrial DNA polymerase (POLGA) gene and the surfeit locus protein 1 (SURF1) gene. Respiratory chain enzyme activity measurements identified five patients with isolated complex I deficiency and five with combined enzyme deficiencies. No patient presented with isolated complex IV deficiency. Seven patients had a decreased ATP production rate. Extensive sequence analysis identified eight patients with pathogenic mtDNA mutations and one patient with mutations in POLGA. Mutations of mtDNA are a common cause of LS and mtDNA analysis should always be included in the diagnosis of LS patients, whereas SURF1 mutations are not a common cause of LS in Sweden. Unexpectedly, age of onset, clinical symptoms and prognosis did not reveal any clear differences in LS patients with mtDNA or nuclear DNA mutations. PMID:19103152

  12. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease.

    Directory of Open Access Journals (Sweden)

    Timothy M Butler

    Full Text Available The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.

  13. Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

    Science.gov (United States)

    Butler, Timothy M.; Johnson-Camacho, Katherine; Peto, Myron; Wang, Nicholas J.; Macey, Tara A.; Korkola, James E.; Koppie, Theresa M.; Corless, Christopher L.; Gray, Joe W.; Spellman, Paul T.

    2015-01-01

    The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient’s resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor. PMID:26317216

  14. Actionable mutations in plasma cell-free DNA in patients with advanced cancers referred for experimental targeted therapies

    OpenAIRE

    Janku, Filip; Angenendt, Philipp; Tsimberidou, Apostolia M.; Fu, Siqing; Naing, Aung; Falchook, Gerald S.; David S Hong; Holley, Veronica R.; Cabrilo, Goran; Jennifer J Wheler; Piha-Paul, Sarina A.; Zinner, Ralph G.; Bedikian, Agop Y.; Overman, Michael J.; Kee, Bryan K.

    2015-01-01

    Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable source of biologic material for mutation analysis. Plasma samples from 157 patients with advanced cancers who progressed on systemic therapy were tested for 21 mutations in BRAF, EGFR, KRAS, and PIK3CA using the BEAMing method and results were compared to mutation analysis of archival tumor tissue from a CLIA-certified laboratory obtained as standard of care from diagnostic or therapeutic procedures. Results were ...

  15. Actionable mutations in plasma cell-free DNA in patients with advanced cancers referred for experimental targeted therapies

    Science.gov (United States)

    Janku, Filip; Angenendt, Philipp; Tsimberidou, Apostolia M.; Fu, Siqing; Naing, Aung; Falchook, Gerald S.; Hong, David S.; Holley, Veronica R.; Cabrilo, Goran; Wheler, Jennifer J.; Piha-Paul, Sarina A.; Zinner, Ralph G.; Bedikian, Agop Y.; Overman, Michael J.; Kee, Bryan K.; Kim, Kevin B.; Kopetz, E. Scott; Luthra, Rajyalakshmi; Diehl, Frank; Meric-Bernstam, Funda; Kurzrock, Razelle

    2015-01-01

    Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable source of biologic material for mutation analysis. Plasma samples from 157 patients with advanced cancers who progressed on systemic therapy were tested for 21 mutations in BRAF, EGFR, KRAS, and PIK3CA using the BEAMing method and results were compared to mutation analysis of archival tumor tissue from a CLIA-certified laboratory obtained as standard of care from diagnostic or therapeutic procedures. Results were concordant for archival tissue and plasma cfDNA in 91% cases for BRAF mutations (kappa = 0.75, 95% confidence interval [CI] 0.63 – 0.88), in 99% cases for EGFR mutations (kappa = 0.90, 95% CI 0.71– 1.00), in 83% cases for KRAS mutations (kappa = 0.67, 95% CI 0.54 – 0.80) and in 91% cases for PIK3CA mutations (kappa = 0.65, 95% CI 0.46 – 0.85). Patients (n = 41) with > 1% of KRAS mutant cfDNA had a shorter median survival compared to 20 patients with 1% of mutant cfDNA (BRAF, EGFR, KRAS, or PIK3CA) had a shorter median survival compared to 33 patients with DNA (5.5 vs. 9.8 months, p = 0.001), which was confirmed in multivariable analysis. PMID:25980577

  16. Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Tan, H.

    1999-03-31

    The purpose of this research is to develop a multiplexed sample processing system in conjunction with multiplexed capillary electrophoresis for high-throughput DNA sequencing. The concept from DNA template to called bases was first demonstrated with a manually operated single capillary system. Later, an automated microfluidic system with 8 channels based on the same principle was successfully constructed. The instrument automatically processes 8 templates through reaction, purification, denaturation, pre-concentration, injection, separation and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were implemented to manage flow direction and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an 8-capillary array in a hot air thermal cycler. Subsequently, the sequencing ladders are directly loaded into a corresponding size-exclusion chromatographic column operated at {approximately} 60 C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at {approximately} 70 C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. DNA sequencing data from this system allow base calling up to 460 bases with accuracy of 98%.

  17. The prognostic value of KRAS mutated plasma DNA in advanced non-small cell lung cancer

    DEFF Research Database (Denmark)

    Nygaard, Anneli Dowler; Garm Spindler, Karen-Lise; Pallisgaard, Niels;

    2013-01-01

    BACKGROUND: Lung cancer is one of the most common malignant diseases worldwide and associated with considerable morbidity and mortality. New agents targeting the epidermal growth factor system are emerging, but only a subgroup of the patients will benefit from the therapy. Cell free DNA (cf......DNA) in the blood allows for tumour specific analyses, including KRAS-mutations, and the aim of the study was to investigate the possible prognostic value of plasma mutated KRAS (pmKRAS) in patients with non-small cell lung cancer (NSCLC). MATERIAL AND METHODS: Patients with newly diagnosed, advanced NSCLC eligible......-KRAS mutation had a significantly shorter OS and PFS compared to the wild type (WT) patients (median OS 4.8 months versus 9.5 months, HR 1.87, 95% CI 1.23-2.84, p=0.0002 and median PFS 3.0 months versus 5.6 months, HR 1.60, 95% CI 1.09-2.37, p=0.0043). A multivariate Cox regression analysis confirmed...

  18. Platelet hexosaminidase a enzyme assay effectively detects carriers missed by targeted DNA mutation analysis.

    Science.gov (United States)

    Nakagawa, Sachiko; Zhan, Jie; Sun, Wei; Ferreira, Jose Carlos; Keiles, Steven; Hambuch, Tina; Kammesheidt, Anja; Mark, Brian L; Schneider, Adele; Gross, Susan; Schreiber-Agus, Nicole

    2012-01-01

    Biochemical testing of hexosaminidase A (HexA) enzyme activity has been available for decades and has the ability to detect almost all Tay-Sachs disease (TSD) carriers, irrespective of ethnic background. This is increasingly important, as the gene pool of those who identify as Ashkenazi Jewish is diversifying. Here we describe the analysis of a cohort of 4,325 individuals arising from large carrier screening programs and tested by the serum and/or platelet HexA enzyme assays and by targeted DNA mutation analysis. Our results continue to support the platelet assay as a highly effective method for TSD carrier screening, with a low inconclusive rate and the ability to detect possible disease-causing mutation carriers that would have been missed by targeted DNA mutation analysis. Sequence analysis performed on one such platelet assay carrier, who had one non-Ashkenazi Jewish parent, identified the amino acid change Thr259Ala (A775G). Based on crystallographic modeling, this change is predicted to be deleterious, as threonine 259 is positioned proximal to the HexA alpha subunit active site and helps to stabilize key residues therein. Accordingly, if individuals are screened for TSD in broad-based programs by targeted molecular testing alone, they must be made aware that there is a more sensitive and inexpensive test available that can identify additional carriers. Alternatively, the enzyme assays can be offered as a first tier test, especially when screening individuals of mixed or non-Jewish ancestry. PMID:23430931

  19. A BRCA1-mutation associated DNA methylation signature in blood cells predicts sporadic breast cancer incidence and survival.

    OpenAIRE

    Anjum, S; Fourkala, E O; Zikan, M.; Wong, A.; Gentry-Maharaj, A.; Jones, A.; HARDY, R.; Cibula, D.; Kuh, D.; Jacobs, I. J.; Teschendorff, A.E.; Menon, U; Widschwendter, M

    2014-01-01

    Background BRCA1 mutation carriers have an 85% risk of developing breast cancer but the risk of developing non-hereditary breast cancer is difficult to assess. Our objective is to test whether a DNA methylation (DNAme) signature derived from BRCA1 mutation carriers is able to predict non-hereditary breast cancer. Methods In a case/control setting (72 BRCA1 mutation carriers and 72 BRCA1/2 wild type controls) blood cell DNA samples were profiled on the Illumina 27 k methylation array. Using th...

  20. Prognostic role of PIK3CA mutations of cell‐free DNA in early‐stage triple negative breast cancer

    OpenAIRE

    Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto‐Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

    2015-01-01

    PIK3CA is an oncogene that encodes the p110α component of phosphatidylinositol 3‐kinase (PI3K); it is the second most frequently mutated gene following the TP53 gene. In the clinical setting, PIK3CA mutations may have favorable prognostic value for hormone receptor‐positive breast cancer patients and, during the past few years, PIK3CA mutations of cell‐free DNA (cfDNA) have attracted attention as a potential noninvasive biomarker of cancer. However, there are few reports on the clinical impli...

  1. Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

    OpenAIRE

    Elena Ordoñez; Laura Rueda; M. Paz Cañadas; Carme Fuster; Vincenzo Cirigliano

    2013-01-01

    Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 15...

  2. Performance verification of the Maxwell 16 Instrument and DNA IQ Reference Sample Kit for automated DNA extraction of known reference samples.

    Science.gov (United States)

    Krnajski, Z; Geering, S; Steadman, S

    2007-12-01

    Advances in automation have been made for a number of processes conducted in the forensic DNA laboratory. However, because most robotic systems are designed for high-throughput laboratories batching large numbers of samples, smaller laboratories are left with a limited number of cost-effective options for employing automation. The Maxwell 16 Instrument and DNA IQ Reference Sample Kit marketed by Promega are designed for rapid, automated purification of DNA extracts from sample sets consisting of sixteen or fewer samples. Because the system is based on DNA capture by paramagnetic particles with maximum binding capacity, it is designed to generate extracts with yield consistency. The studies herein enabled evaluation of STR profile concordance, consistency of yield, and cross-contamination performance for the Maxwell 16 Instrument. Results indicate that the system performs suitably for streamlining the process of extracting known reference samples generally used for forensic DNA analysis and has many advantages in a small or moderate-sized laboratory environment. PMID:25869266

  3. Mechanisms of mtDNA segregation and mitochondrial signalling in cells with the pathogenic A3243G mutation

    NARCIS (Netherlands)

    Jahangir Tafrechi, Roshan Sakineh

    2008-01-01

    Using newly developed single cell A3243G mutation load assays a novel mechanism of mtDNA segregation was identified in which the multi-copy mtDNA nucleoid takes a central position. Furthermore, likely due to low level changes in gene expression, no genes or gene sets could be identified with gene wi

  4. Clonal expansion of early to mid-life mitochondrial DNA point mutations drives mitochondrial dysfunction during human ageing.

    NARCIS (Netherlands)

    Greaves, L.C.; Nooteboom, M.; Elson, J.L.; Tuppen, H.A.; Taylor, G.A.; Commane, D.M.; Arasaradnam, R.P.; Khrapko, K.; Taylor, R.W.; Kirkwood, T.B.; Mathers, J.C.; Turnbull, D.M.

    2014-01-01

    Age-related decline in the integrity of mitochondria is an important contributor to the human ageing process. In a number of ageing stem cell populations, this decline in mitochondrial function is due to clonal expansion of individual mitochondrial DNA (mtDNA) point mutations within single cells. Ho

  5. SOLAR RADIATION AND INDUCTION OF DNA DAMAGE, MUTATIONS AND SKIN CANCERS.

    Energy Technology Data Exchange (ETDEWEB)

    SETLOW,R.B.

    2007-05-10

    An understanding of the effects of sunlight on human skin begins with the effects on DNA and extends to cells, animals and humans. The major DNA photoproducts arising from UVB (280-320 nm) exposures are cyclobutane pyrimidine dimers. If unrepaired, they may kill or mutate cells and result in basal and squamous cell carcinomas. Although UVA (320-400 nm) and visible wavelengths are poorly absorbed by DNA, the existing data indicate clearly that exposures to these wavelengths are responsible, in an animal model, for {approx}95 % of the incidence of cutaneous malignant melanoma (CMM). Six lines of evidence, to be discussed in detail, support the photosensitizing role of melanin in the induction of this cancer. They are: (1) Melanomas induced in backcross hybrids of small tropical fish of the genus Xiphophorus, exposed to wavelengths from 302-547 nm, indicate that {approx}95% of the cancers induced by exposure to sunlight would arise from UVA + visible wavelengths; (2) The action spectrum for inducing melanin-photosensitized oxidant production is very similar to the spectrum for inducing melanoma; (3) Albino whites and blacks, although very sensitive to sunburn and the sunlight induction of non-CMM, have very low incidences of CMM; (4) The incidence of CMM as a function of latitude is very similar to that of UVA, but not UVB; (5) Use of UVA-exposing sun-tanning parlors by the young increases the incidence rate of CMM and (6) Major mutations observed in CMM are not UVB-induced.

  6. A triple-helix forming oligonucleotide targeting genomic DNA fails to induce mutation.

    Science.gov (United States)

    Reshat, Reshat; Priestley, Catherine C; Gooderham, Nigel J

    2012-11-01

    Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity. PMID:22914677

  7. Analysis of morphology, DNA and isozyme of leaf mutation in Brassica napus L

    International Nuclear Information System (INIS)

    This paper aims to study the rule of irradiating effects, provide the effective way of analyzing mutant, and discuss the production application of mutant. By irradiating the 040B of Brassica napus L with . 0Co γ- ray, an obvious leaf mutation (ML) with large leaf area was found. The ML which has been inherited stably after three generations was compared with wide-type (CK) on the morphologic, DNA and isozymic levels. Results showed that S 4 and S17 from RAPD were two molecular markers which can express good polymorphism and have close relationships with leaf mutation sites. And in the analysis of EST and POD between ML and CK, the polymorphisms also proved that many discrepancies exist between ML and CK on the protein level. In addition, the research results in question can be applied to the breeding and genetic research of Brassica napus L

  8. Mismatch oxidation assay: detection of DNA mutations using a standard UV/Vis microplate reader.

    Science.gov (United States)

    Tabone, Tania; Sallmann, Georgina; Cotton, Richard G H

    2009-01-01

    Simple, low-cost mutation detection assays that are suitable for low-throughput analysis are essential for diagnostic applications where the causative mutation may be different in every family. The mismatch oxidation assay is a simple optical absorbance assay to detect nucleotide substitutions, insertions, and deletions in heteroduplex DNA. The method relies on detecting the oxidative modification products of mismatched thymine and cytosine bases by potassium permanganate as it is reduced to manganese dioxide. This approach, unlike other methods commonly used to detect sequence variants, does not require costly labeled probes or primers, toxic chemicals, or a time-consuming electrophoretic separation step. The oxidation rate, and hence the presence of a sequence variant, is detected by measuring the formation of the potassium permanganate reduction product (hypomanganate diester), which absorbs at the 420-nm visible wavelength, using a standard UV/vis microplate reader. PMID:19768598

  9. CENTRIFUGAL LABTUBE FOR FULLY AUTOMATED DNA EXTRACTION & LAMP AMPLIFICATION BASED ON AN INTEGRATED, LOW-COST HEATING SYSTEM

    OpenAIRE

    Hoehl, Melanie Margarete; Weibert, Michael; Paust, Nils; Zengerle, Roland; Slocum, Alexander H.; Steigert, Juergen

    2013-01-01

    In this paper, we introduce a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA-extraction platform (LabTube). We demonstrate fully automated, fully closed extraction of as little as 100 DNA copies of verotoxin-producing (VTEC) Escherichia coli lysate in water, milk and apple juice in a standard laboratory centrifuge, followed by subsequent automatic LAMP amplification with an overall time-to-result of 1.5hrs. The ...

  10. Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line.

    Science.gov (United States)

    Meng, Ran; Zhou, Jin; Sui, Meng; Li, ZhiYong; Feng, GuoSheng; Yang, BaoFeng

    2010-01-01

    This study aimed to investigate the effects of arsenic trioxide (As(2)O(3)) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 micromol/L As(2)O(3) in vitro, and the primary APL cells were treated with 2.0 micromol/L As(2)O(3) in vitro and 0.16 mg kg(-1) d(-1) As(2)O(3) in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As(2)O(3) use, but the mutation spots were remarkably increased after As(2)O(3) treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r (NB4-As2O3)=0.973818, and r (APL-As2O3)=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As(2)O(3) aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As(2)O(3) in APL treatment. PMID:20596959

  11. Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair, chromosomal alterations, and mutations.

    Science.gov (United States)

    Natarajan, A T; van Zeeland, A A; Zwanenburg, T S

    1983-01-01

    The influence of inhibitors of poly(ADP-ribose) polymerase such as 3-aminobenzamide (3AB) and benzamide (B) on the spontaneously occurring as well as mutagen induced chromosomal aberrations, sister chromatid exchanges (SCEs) and point mutations has been studied. In addition, we have measured the influence of 3AB on DNA repair following treatment with physical and chemical mutagens. Post treatment of X-irradiated mammalian cells with 3AB increases the frequencies of induced chromosomal aberrations by a factor of 2 to 3. Both acentric fragments and exchanges increase indicating that the presence of 3AB slows down the repair of DNA strand breaks (probably DNA double strand breaks), thus making breaks available for interaction with each other to give rise to exchanges. 3AB, when present in the medium containing bromodeoxyuridine(BrdUrd) during two cell cycles, increases the frequencies of SCEs in Chinese hamster ovary cells (CHO) in a concentration dependent manner leading to about a 10-fold increase at 10 mM concentration. Most 3AB induced SCEs occur during the second cell cycle, in which DNA containing bromouridine (BU) is used as template for replication. BU containing DNA appears to be prone to errors during replication. The extent of increase in the frequencies of SCEs by 3AB is correlated with the amount of BU incorporated in the DNA of the cells. The frequencies of spontaneously occurring DNA single strand breaks in cells grown in BrdUrd containing medium are higher than in the cells grown in normal medium and this increase depends on the amount of BU incorporated in the DNA of these cells. We have studied the extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines, including those derived from patients suffering from genetic diseases such as ataxia telangiectasia (A-T), Fanconi's anemia (FA), and Huntington's chorea. None of these syndromes showed any increased response when compared to normal cells. 3AB, however, increased the

  12. Loss-of-function mutations in MGME1 impair mtDNA replication and cause multi-systemic mitochondrial disease

    OpenAIRE

    Kornblum, Cornelia; Nicholls, Thomas J.; Haack, Tobias B.; Schöler, Susanne; Peeva, Viktoriya; Danhauser, Katharina; Hallmann, Kerstin; Zsurka, Gábor; Rorbach, Joanna; Iuso, Arcangela; Wieland, Thomas; Sciacco, Monica; Ronchi, Dario; Comi, Giacomo P; Moggio, Maurizio

    2013-01-01

    Known disease mechanisms in mitochondrial DNA (mtDNA) maintenance disorders alter either the mitochondrial replication machinery (POLG1, POLG22 and C10orf23) or the biosynthesis pathways of deoxyribonucleoside 5′-triphosphates for mtDNA synthesis4–11. However, in many of these disorders, the underlying genetic defect has not yet been discovered. Here, we identified homozygous nonsense and missense mutations in the orphan gene C20orf72 in three families with a mitochondrial syndrome characteri...

  13. Robust DNA Damage Response and Elevated Reactive Oxygen Species in TINF2-Mutated Dyskeratosis Congenita Cells.

    Science.gov (United States)

    Pereboeva, Larisa; Hubbard, Meredith; Goldman, Frederick D; Westin, Erik R

    2016-01-01

    Dyskeratosis Congenita (DC) is an inherited multisystem premature aging disorder with characteristic skin and mucosal findings as well as a predisposition to cancer and bone marrow failure. DC arises due to gene mutations associated with the telomerase complex or telomere maintenance, resulting in critically shortened telomeres. The pathogenesis of DC, as well as several congenital bone marrow failure (BMF) syndromes, converges on the DNA damage response (DDR) pathway and subsequent elevation of reactive oxygen species (ROS). Historically, DC patients have had poor outcomes following bone marrow transplantation (BMT), perhaps as a consequence of an underlying DNA hypersensitivity to cytotoxic agents. Previously, we demonstrated an activated DDR and increased ROS, augmented by chemotherapy and radiation, in somatic cells isolated from DC patients with a mutation in the RNA component of telomerase, TERC. The current study was undertaken to determine whether previous findings related to ROS and DDR in TERC patients' cells could be extended to other DC mutations. Of particular interest was whether an antioxidant approach could counter increased ROS and decrease DC pathologies. To test this, we examined lymphocytes from DC patients from different DC mutations (TERT, TINF2, and TERC) for the presence of an active DDR and increased ROS. All DC mutations led to increased steady-state p53 (2-fold to 10-fold) and ROS (1.5-fold to 2-fold). Upon exposure to ionizing radiation (XRT), DC cells increased in both DDR and ROS to a significant degree. Exposing DC cells to hydrogen peroxide also revealed that DC cells maintain a significant oxidant burden compared to controls (1.5-fold to 3-fold). DC cell culture supplemented with N-acetylcysteine, or alternatively grown in low oxygen, afforded significant proliferative benefits (proliferation: maximum 2-fold increase; NAC: 5-fold p53 decrease; low oxygen: maximum 3.5-fold p53 decrease). Together, our data supports a mechanism

  14. Robust DNA Damage Response and Elevated Reactive Oxygen Species in TINF2-Mutated Dyskeratosis Congenita Cells.

    Directory of Open Access Journals (Sweden)

    Larisa Pereboeva

    Full Text Available Dyskeratosis Congenita (DC is an inherited multisystem premature aging disorder with characteristic skin and mucosal findings as well as a predisposition to cancer and bone marrow failure. DC arises due to gene mutations associated with the telomerase complex or telomere maintenance, resulting in critically shortened telomeres. The pathogenesis of DC, as well as several congenital bone marrow failure (BMF syndromes, converges on the DNA damage response (DDR pathway and subsequent elevation of reactive oxygen species (ROS. Historically, DC patients have had poor outcomes following bone marrow transplantation (BMT, perhaps as a consequence of an underlying DNA hypersensitivity to cytotoxic agents. Previously, we demonstrated an activated DDR and increased ROS, augmented by chemotherapy and radiation, in somatic cells isolated from DC patients with a mutation in the RNA component of telomerase, TERC. The current study was undertaken to determine whether previous findings related to ROS and DDR in TERC patients' cells could be extended to other DC mutations. Of particular interest was whether an antioxidant approach could counter increased ROS and decrease DC pathologies. To test this, we examined lymphocytes from DC patients from different DC mutations (TERT, TINF2, and TERC for the presence of an active DDR and increased ROS. All DC mutations led to increased steady-state p53 (2-fold to 10-fold and ROS (1.5-fold to 2-fold. Upon exposure to ionizing radiation (XRT, DC cells increased in both DDR and ROS to a significant degree. Exposing DC cells to hydrogen peroxide also revealed that DC cells maintain a significant oxidant burden compared to controls (1.5-fold to 3-fold. DC cell culture supplemented with N-acetylcysteine, or alternatively grown in low oxygen, afforded significant proliferative benefits (proliferation: maximum 2-fold increase; NAC: 5-fold p53 decrease; low oxygen: maximum 3.5-fold p53 decrease. Together, our data supports a

  15. An automated image cytometry system for monitoring DNA ploidy and other cell features of radiotherapy and chemotherapy patients

    International Nuclear Information System (INIS)

    DNA content and distribution in cell nuclei were studied in samples of fine-needle aspiration (FNA) from 27 locally advanced breast and head and neck cancers in two going randomized trials that compared accelerated fractionation to standard fractionation radiation in locally advanced breast cancer and head and neck cancer. Two image cytometry methods were compared: a new, fully automated DNA image cytometry system (AIC) and a conventional image cytometry (CIC) system with manual selection, focusing, and segmentation of cells. The results of both techniques were compared on the basis of DNA histogram parameters including DNA index (DI), mean DNA values (MDV), and Auer's DNA histogram patterns. An excellent correlation was achieved between the two imaging techniques in terms of DI (r=0.985, p<0.001) and MDV (r=0.951, p<0.001) as well as between Auer's histogram patterns, where both methods agreed completely. It was concluded in these analyses that the two image cytometry methods were equivalent. However, the AIC offered an advantage by scanning samples in a fully automated way, which represented significant time saving for cytopathologists working with the system, as well as a larger number of cells used in the automated analysis. With the automated image cytometer, 500 relevant cells were collected and analyzed in about 10 minutes, where with the interactive (manual) method, it took typically an hour to collect and analyze only about 250 cells. Seventeen samples were sufficient for flow analysis. Image cytometry and flow cytometry showed good agreement in DI determination; however, three cases reported as diploid by flow cytometry were found to be aneuploid by image cytometry techniques. (author)

  16. Modulation of radiation-induced mutation frequencies and DNA synthesis by deoxycytidine in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    The goal of this project is to elucidate and model the impact of perturbations in cellular metabolism on the cellular response to DNA damage. Previously reported experiments established roles for DNA synthesis and the inhibition of DNA synthesis in the expression of radiation-induced mutations. This year, these experiments were continued and the study was expanded. Two different Chinese hamster ovary (CHO) cell lines were examined. Experimental measurements were obtained for cells in one of two growth media: (1) medium A, which consisted of Ham's F-12 containing 10% fetal calf serum; or (2) medium B, which consisted of medium A containing 2 mm deoxycytidine (CdR). The presence of CdR differentially affected the nature of DNA replication, the inhibition of replication by radiation, and the corresponding mutation frequency responses. Results suggest that the effect of CdR on mutation-induction is not locus-specific. Moreover, the phenomenal reduction in the mutation response in the presence of CdR is also seen following exposure to ionizing radiation, which implies that the effect of CdR is not mutagen-specific. Time-dependent changes in profiles of pulse-labeled DNA on alkaline sucrose gradients were employed to measure and compare nascent DNA chain growth processes in the two media. Results suggest that a more rapid DNA chain elongation and/or replicon mutation rate occurs in cells in medium B. In contrast, the sedimentation of one pulse-labeled CHO cell line DNA is always greater when cells are grown in medium A. UV radiation appears to inhibit postirradiation replicon initiation events and results in reduced mutation frequencies when cells are grown in the medium that promotes the more rapid rate of polymerization

  17. Control region mutations and the 'common deletion' are frequent in the mitochondrial DNA of patients with esophageal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Tang Ze-Zong

    2004-07-01

    Full Text Available Abstract Background North central China has some of the highest rates of esophageal squamous cell carcinoma in the world with cumulative mortality surpassing 20%. Mitochondrial DNA (mtDNA accumulates more mutations than nuclear DNA and because of its high abundance has been proposed as a early detection device for subjects with cancer at various sites. We wished to examine the prevalence of mtDNA mutation and polymorphism in subjects from this high risk area of China. Methods We used DNA samples isolated from tumors, adjacent normal esophageal tissue, and blood from 21 esophageal squamous cell carcinoma cases and DNA isolated from blood from 23 healthy persons. We completely sequenced the control region (D-Loop from each of these samples and used a PCR assay to assess the presence of the 4977 bp common deletion. Results Direct DNA sequencing revealed that 7/21 (33%, 95% CI = 17–55% tumor samples had mutations in the control region, with clustering evident in the hyper-variable segment 1 (HSV1 and the homopolymeric stretch surrounding position 309. The number of mutations per subject ranged from 1 to 16 and there were a number of instances of heteroplasmy. We detected the 4977 bp 'common deletion' in 92% of the tumor and adjacent normal esophageal tissue samples examined, whereas no evidence of the common deletion was found in corresponding peripheral blood samples. Conclusions Control region mutations were insufficiently common to warrant attempts to develop mtDNA mutation screening as a clinical test for ESCC. The common deletion was highly prevalent in the esophageal tissue of cancer cases but absent from peripheral blood. The potential utility of the common deletion in an early detection system will be pursued in further studies.

  18. DNA Hydroxymethylation Profiling Reveals that WT1 Mutations Result in Loss of TET2 Function in Acute Myeloid Leukemia

    Directory of Open Access Journals (Sweden)

    Raajit Rampal

    2014-12-01

    Full Text Available Somatic mutations in IDH1/IDH2 and TET2 result in impaired TET2-mediated conversion of 5-methylcytosine (5mC to 5-hydroxymethylcytosine (5hmC. The observation that WT1 inactivating mutations anticorrelate with TET2/IDH1/IDH2 mutations in acute myeloid leukemia (AML led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant AML patients have reduced 5hmC levels similar to TET2/IDH1/IDH2 mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5hmC, and WT1 silencing reduced 5hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/IDH2 and WT1 mutations define an AML subtype defined by dysregulated DNA hydroxymethylation.

  19. Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs.

    Science.gov (United States)

    Kang, Eunju; Wang, Xinjian; Tippner-Hedges, Rebecca; Ma, Hong; Folmes, Clifford D L; Gutierrez, Nuria Marti; Lee, Yeonmi; Van Dyken, Crystal; Ahmed, Riffat; Li, Ying; Koski, Amy; Hayama, Tomonari; Luo, Shiyu; Harding, Cary O; Amato, Paula; Jensen, Jeffrey; Battaglia, David; Lee, David; Wu, Diana; Terzic, Andre; Wolf, Don P; Huang, Taosheng; Mitalipov, Shoukhrat

    2016-05-01

    The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24-72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications. PMID:27151456

  20. Poor correlations in the levels of pathogenic mitochondrial DNA mutations in polar bodies versus oocytes and blastomeres in humans.

    Science.gov (United States)

    Gigarel, Nadine; Hesters, Laetitia; Samuels, David C; Monnot, Sophie; Burlet, Philippe; Kerbrat, Violaine; Lamazou, Frédéric; Benachi, Alexandra; Frydman, René; Feingold, Josué; Rotig, Agnes; Munnich, Arnold; Bonnefont, Jean-Paul; Frydman, Nelly; Steffann, Julie

    2011-04-01

    Because the mtDNA amount remains stable in the early embryo until uterine implantation, early human development is completely dependent on the mtDNA pool of the mature oocyte. Both quantitative and qualitative mtDNA defects therefore may negatively impact oocyte competence or early embryonic development. However, nothing is known about segregation of mutant and wild-type mtDNA molecules during human meiosis. To investigate this point, we compared the mutant levels in 51 first polar bodies (PBs) and their counterpart (oocytes, blastomeres, or whole embryos), at risk of having (1) the "MELAS" m.3243A>G mutation in MT-TL1 (n = 30), (2) the "MERRF" m.8344A>G mutation in MT-TK (n = 15), and (3) the m.9185T>G mutation located in MT-ATP6 (n = 6). Seven out of 51 of the PBs were mutation free and had homoplasmic wild-type counterparts. In the heteroplasmic PBs, measurement of the mutant load was a rough estimate of the counterpart mutation level (R(2) = 0.52), and high mutant-load differentials between the two populations were occasionally observed (ranging from -34% to +34%). The mutant-load differentials between the PB and its counterpart were higher in highly mutated PBs, suggestive of a selection process acting against highly mutated cells during gametogenesis or early embryonic development. Finally, individual discrepancies in mutant loads between PBs and their counterparts make PB-based preconception diagnosis unreliable for the prevention of mtDNA disorder transmission. Such differences were not observed in animal models, and they emphasize the need to conduct thorough studies on mtDNA segregation in humans. PMID:21473984

  1. Diamondoid-functionalized gold nanogaps as sensors for natural, mutated, and epigenetically modified DNA nucleotides

    Science.gov (United States)

    Sivaraman, Ganesh; Amorim, Rodrigo G.; Scheicher, Ralph H.; Fyta, Maria

    2016-05-01

    Modified tiny hydrogen-terminated diamond structures, known as diamondoids, show a high efficiency in sensing DNA molecules. These diamond cages, as recently proposed, could offer functionalization possibilities for gold junction electrodes. In this investigation, we report on diamondoid-functionalized electrodes, showing that such a device would have a high potential in sensing and sequencing DNA. The smallest diamondoid including an amine modification was chosen for the functionalization. Here, we report on the quantum tunneling signals across diamondoid-functionalized Au(111) electrodes. Our work is based on quantum-transport calculations and predicts the expected signals arising from different DNA units within the break junctions. Different gating voltages are proposed in order to tune the sensitivity of the functionalized electrodes with respect to specific nucleotides. The relation of this sensitivity to the coupling or decoupling of the electrodes is discussed. Our results also shed light on the sensing capability of such a device in distinguishing the DNA nucleotides, in their natural and mutated forms.

  2. Distinct nuclear gene expression profiles in cells with mtDNA depletion and homoplasmic A3243G mutation

    Energy Technology Data Exchange (ETDEWEB)

    Jahangir Tafrechi, Roshan S. [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Svensson, Peter J. [Department of Toxicogenetics, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Department of Oncology, Radiology and Clinical Immunology, University Hospital, 75185 Uppsala (Sweden); Janssen, George M.C. [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Szuhai, Karoly [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Maassen, J. Antonie [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands); Raap, Anton K. [Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9503, 2300 RA Leiden (Netherlands)]. E-mail: A.K.Raap@lumc.nl

    2005-10-15

    The pathobiochemical pathways determining the wide variability in phenotypic expression of mitochondrial DNA (mtDNA) mutations are not well understood. Most pathogenic mtDNA mutations induce a general defect in mitochondrial respiration and thereby ATP synthesis. Yet phenotypic expression of the different mtDNA mutations shows large variations that are difficult to reconcile with ATP depletion as sole pathogenic factor, implying that additional mechanisms contribute to the phenotype. Here, we use DNA microarrays to identify changes in nuclear gene expression resulting from the presence of the A3243G diabetogenic mutation and from a depletion of mtDNA ({rho}{sup 0} cells). We find that cells respond mildly to these mitochondrial states with both general and specific changes in nuclear gene expression. This observation indicates that cells can sense the status of mtDNA. A number of genes show divergence in expression in {rho}{sup 0} cells compared to cells with the A3243G mutation, such as genes involved in oxidative phosphorylation. As a common response in A3243G and {rho}{sup 0} cells, mRNA levels for extracellular matrix genes are up-regulated, while the mRNA levels of genes involved in ubiquitin-mediated protein degradation and in ribosomal protein synthesis is down-regulated. This reduced expression is reflected at the level of cytosolic protein synthesis in both A3243G and {rho}{sup 0} cells. Our finding that mitochondrial dysfunction caused by different mutations affects nuclear gene expression in partially distinct ways suggests that multiple pathways link mitochondrial function to nuclear gene expression and contribute to the development of the different phenotypes in mitochondrial disease.

  3. Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria

    International Nuclear Information System (INIS)

    Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmides were detected by hybridization to 32P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G x C → C x G or G x C → T x A transversions or single nucleotide deletions. The authors conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria

  4. PCR-SSCP-DNA sequencing method in detecting PTEN gene mutation and its significance in human gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Chuan-Yong Guo; Xuan-Fu Xu; Jian-Ye Wu; Shu-Fang Liu

    2008-01-01

    AIM: To discuss the possible effect of PTEN gene mutations on occurrence and development of gastric cancer.METHODS: Fifty-three gastric cancer specimens were selected to probe PTEN gene mutations in genome of gastric cancer and paracancerous tissues using PCR-SSCP-DNA sequencing method based on microdissection and to observe the protein expression by immunohistochemistry technique.RESULTS: PCR-SSCP-DNA sequencing indicated that 4 kinds of mutation sites were found in 5 of 53 gastric cancer specimens.One kind of mutation was found in exons.AA-TCC mutation was located at 40bp upstream of 3' lateral exert 7 (115946 AA-TCC).Such mutations led to terminator formation in the 297th codon of the PTEN gene.The other 3 kinds of mutation were found in introns,including a G-C point mutation at 91 bp upstream of 5' lateral exon 5(90896 G-C),a T-G point mutation at 24 bp upstream of 5' lateral exon 5 (90963 T-G),and a single base A mutation at 7 bp upstream of 5' lateral exon 5 (90980 A del).The PTEN protein expression in gastric cancer and paracancerous tissues detected using immunohistochemistry technique indicated that the total positive rate of PTEN protein expression was 66% in gastric cancer tissue,which was significantly lower than that (100%) in paracancerous tissues (P<0.005).CONCLUSION: PTEN gene mutation and expression may play an important role in the occurrence and development of gastric cancer.(C)2008 The WJG Press.All rights reserved.

  5. p53 Mutation analysis in breast tumors by a DNA microarray method.

    Science.gov (United States)

    Tennis, Meredith; Krishnan, Shiva; Bonner, Matthew; Ambrosone, Christine B; Vena, John E; Moysich, Kirsten; Swede, Helen; McCann, Susan; Hall, Per; Shields, Peter G; Freudenheim, Jo L

    2006-01-01

    The p53 gene acts as a regulator of cell growth and DNA repair in normal cells; inactivation of the gene seems to lead to cancer. It is the most commonly mutated gene in human cancers, and a high-throughput sequencing method is needed for cancer etiology studies using large sample sets. In our population-based case-control study of breast cancer, the p53 gene was amplified by PCR for 392 subjects from seven hospitals in Western New York using the Affymetrix GeneChip technology. One hundred thirty-eight (35%) of the breast tumors had p53 mutations, of which 88% were located in exons 5 to 8. New hotspots were identified at codons 179, 195, 196, 213, 217, 249, 254, 278, 281, and 298, and previously reported hotspots were found at codons 175, 248, and 273. Manual sequencing for exons 5 to 9 of the p53 gene was done for 139 tumors to validate the Affymetrix assay. The two methods had 100% concordance for mutations detectable by the Affymetrix assay. We also successfully assayed paraffin-embedded breast and lung tumors from as early as 1958 and employed a nested PCR strategy to improve weak PCR amplification. To have statistical power, the investigation of gene environment interactions and cancer requires a large number of tumor analyses, which are frequently only available from archived tissue from multiple sources. We have shown the utility of the Affymetrix GeneChip method under these challenging conditions and provided new data for the mutational spectra of breast cancer in a population-based study. PMID:16434591

  6. The Use of EGFR Exon 19 and 21 Unlabeled DNA Probes to Screen for Activating Mutations in Non–Small Cell Lung Cancer

    OpenAIRE

    Willmore-Payne, Carlynn; Holden, Joseph A.; Wittwer, Carl T.; Layfield, Lester J.

    2008-01-01

    Activating mutations in epidermal growth factor receptor-1 (EGFR) are found in 10–15% of Caucasian patients with non–small cell lung carcinoma (NSCLC). Approximately 90% of the mutations are deletions of several amino acids in exon 19 or point mutations in exon 21. Some studies suggest that these mutations identify patients that might benefit from targeted EGFR inhibitor therapy. DNA melting analysis of polymerase chain reaction products can screen for these mutations to identify this patient...

  7. Mitochondrial DNA mutations--candidate biomarkers for breast cancer diagnosis in Bangladesh

    Directory of Open Access Journals (Sweden)

    Rowshan Ara Begum

    2012-09-01

    Full Text Available Breast cancer is a major health problem that affects more than 24% of women in Bangladesh. Further- more, among low-income countries including Bangladesh, individuals have a high risk for developing breast cancer. This study aimed to identify candidate mitochondrial DNA (mtDNA biomarkers for breast cancer diagnosis in Bangladeshi women to be used as a preventive approach. We screened the blood samples from 24 breast cancer patients and 20 healthy controls to detect polymorphisms in the D-loop and the ND3- and ND4-coding regions of mtDNA by direct sequencing. Among 14 distinct mutations, 10 polymorphisms were found in the D-loop, 3 were found in the ND3-coding region, and 1 was found in the ND4-coding region. The frequency of two novel polymorphisms in the D-loop, one at position 16290 (T-ins and the other at position 16293 (A-del, was higher in breast cancer patients than in control subjects (position 16290: odds ratio = 6.011, 95% confidence interval = 1.2482 to 28.8411, P = 0.002; position 16293: odds ratio = 5.6028, 95% confidence interval = 1.4357 to 21.8925, P = 0.010. We also observed one novel mutation in the ND3-coding region at position 10316 (A > G in 69% of breast cancer patients but not in control subjects. The study suggests that two novel polymorphisms in the D-loop may be candidate biomarkers for breast cancer diagnosis in Bangladeshi women.

  8. Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations

    OpenAIRE

    Douglas C. Wallace; Stugard, Carol; Murdock, Deborah; Schurr, Theodore; Brown, Michael D.

    1997-01-01

    Nuclear-localized mtDNA pseudogenes might explain a recent report describing a heteroplasmic mtDNA molecule containing five linked missense mutations dispersed over the contiguous mtDNA CO1 and CO2 genes in Alzheimer’s disease (AD) patients. To test this hypothesis, we have used the PCR primers utilized in the original report to amplify CO1 and CO2 sequences from two independent ρ° (mtDNA-less) cell lines. CO1 and CO2 sequences amplified from both of the ρ° cells, ...

  9. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients.

    Science.gov (United States)

    Olmedillas López, Susana; García-Olmo, Dolores C; García-Arranz, Mariano; Guadalajara, Héctor; Pastor, Carlos; García-Olmo, Damián

    2016-01-01

    KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR) therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma ("liquid biopsy") by droplet digital PCR (ddPCR) has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066). Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286). Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002). In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker. PMID:27043547

  10. KRAS G12V Mutation Detection by Droplet Digital PCR in Circulating Cell-Free DNA of Colorectal Cancer Patients

    Directory of Open Access Journals (Sweden)

    Susana Olmedillas López

    2016-04-01

    Full Text Available KRAS mutations are responsible for resistance to anti-epidermal growth factor receptor (EGFR therapy in colorectal cancer patients. These mutations sometimes appear once treatment has started. Detection of KRAS mutations in circulating cell-free DNA in plasma (“liquid biopsy” by droplet digital PCR (ddPCR has emerged as a very sensitive and promising alternative to serial biopsies for disease monitoring. In this study, KRAS G12V mutation was analyzed by ddPCR in plasma DNA from 10 colorectal cancer patients and compared to six healthy donors. The percentage of KRAS G12V mutation relative to wild-type sequences in tumor-derived DNA was also determined. KRAS G12V mutation circulating in plasma was detected in 9 of 10 colorectal cancer patients whose tumors were also mutated. Colorectal cancer patients had 35.62 copies of mutated KRAS/mL plasma, whereas in healthy controls only residual copies were found (0.62 copies/mL, p = 0.0066. Interestingly, patients with metastatic disease showed a significantly higher number of mutant copies than M0 patients (126.25 versus 9.37 copies/mL, p = 0.0286. Wild-type KRAS was also significantly elevated in colorectal cancer patients compared to healthy controls (7718.8 versus 481.25 copies/mL, p = 0.0002. In conclusion, KRAS G12V mutation is detectable in plasma of colorectal cancer patients by ddPCR and could be used as a non-invasive biomarker.

  11. Mutation of mtDNA ND1 Gene in 20 Type 2 Diabetes Mellitus Patients of Gorontalonese and Javanese Ethnicity

    Directory of Open Access Journals (Sweden)

    AMIEN RAMADHAN ISHAK

    2014-12-01

    Full Text Available Mitochondrial gene mutation plays a role in the development of type two diabetes mellitus (T2DM. A point mutation in the mitochondrial gene Nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND1 gene mainly reported as the most common mutation related to T2DM. However, several studies have identified another SNP (single-nucleotide polymorphisms in the RNA region of mtDNA from patients from specific ethnic populations in Indonesia. Building on those findings, this study aimed to use PCR and DNA sequencing technology to identify nucleotides in RNA and ND1 fragment from 20 Gorontalonese and 20 Javanese T2DM patients, that may trigger T2DM expression. The results showed successful amplification of RNA along 294 bp for all samples. From these samples, we found two types of point mutation in Javanese patients in the G3316A and T3200C points of the rRNA and ND1 gene. In samples taken from Gorontalonese patients, no mutation were found in the RNA or ND1 region. We conclude that T2DM was triggered differently in our two populations. While genetic mutation is implicated for the 20 Javanese patients, T2DM pathogenesis in the Gorontalonese patients must be traced to other genetic, environmental, or behavioral factors.

  12. Long-term bezafibrate treatment improves skin and spleen phenotypes of the mtDNA mutator mouse.

    Directory of Open Access Journals (Sweden)

    Lloye M Dillon

    Full Text Available Pharmacological agents, such as bezafibrate, that activate peroxisome proliferator-activated receptors (PPARs and PPAR γ coactivator-1α (PGC-1α pathways have been shown to improve mitochondrial function and energy metabolism. The mitochondrial DNA (mtDNA mutator mouse is a mouse model of aging that harbors a proofreading-deficient mtDNA polymerase γ. These mice develop many features of premature aging including hair loss, anemia, osteoporosis, sarcopenia and decreased lifespan. They also have increased mtDNA mutations and marked mitochondrial dysfunction. We found that mutator mice treated with bezafibrate for 8-months had delayed hair loss and improved skin and spleen aging-like phenotypes. Although we observed an increase in markers of fatty acid oxidation in these tissues, we did not detect a generalized increase in mitochondrial markers. On the other hand, there were no improvements in muscle function or lifespan of the mutator mouse, which we attributed to the rodent-specific hepatomegaly associated with fibrate treatment. These results showed that despite its secondary effects in rodent's liver, bezafibrate was able to improve some of the aging phenotypes in the mutator mouse. Because the associated hepatomegaly is not observed in primates, long-term bezafibrate treatment in humans could have beneficial effects on tissues undergoing chronic bioenergetic-related degeneration.

  13. LOW-COST BACTERIAL DETECTION SYSTEM FOR FOOD SAFETY BASED ON AUTOMATED DNA EXTRACTION, AMPLIFICATION AND READOUT

    OpenAIRE

    Hoehl, Melanie Margarete; Bocholt, Eva Schulte; Karippai, Nobu; Zengerle, Roland; Steigert, Juergen; Slocum, Alexander H.

    2013-01-01

    To ensure food, medical and environmental safety and quality, rapid, low-cost and easy-to-use detection methods are desirable. Here, the LabSystem is introduced for integrated, automated DNA purification and amplification. It consists of a disposable, centrifugally-driven DNA purification platform (LabTube) and the subsequent amplification in a low-cost UV/vis-reader (LabReader). In this paper, food safety was chosen as the first sample application with pathogenic verotoxin-producing (VTEC) E...

  14. A novel approach for rapid detection of X-ray irradiation induced mtDNA D310 mutation

    International Nuclear Information System (INIS)

    Human breast cancer cell line MCF-7 was irradiated with 4 Gy X-ray, collected at 0, 2, 4, 8, 16, 24, 48, 72, 144 h after irradiation,respectively. Whole genome DNA including mtDNA were extracted at each time point, and amplified by polymerase chain reaction (PCR). Then the PCR product was subjected to BSAXI digestion, all of digestion product then underwent a brief electrophoresis. Results showed D310 mutation can be induced by 4 Gy X-ray irradiation and D310 mutation can overwhelm the normal phenotype 144 h after irradiation. (authors)

  15. A nonsense mutation in the DNA repair factor Hebo causes mild bone marrow failure and microcephaly.

    Science.gov (United States)

    Zhang, Shu; Pondarre, Corinne; Pennarun, Gaelle; Labussiere-Wallet, Helene; Vera, Gabriella; France, Benoit; Chansel, Marie; Rouvet, Isabelle; Revy, Patrick; Lopez, Bernard; Soulier, Jean; Bertrand, Pascale; Callebaut, Isabelle; de Villartay, Jean-Pierre

    2016-05-30

    Inherited bone marrow failure syndromes are human conditions in which one or several cell lineages of the hemopoietic system are affected. They are present at birth or may develop progressively. They are sometimes accompanied by other developmental anomalies. Three main molecular causes have been recognized to result in bone marrow failure syndromes: (1) defects in the Fanconi anemia (FA)/BRCA DNA repair pathway, (2) defects in telomere maintenance, and (3) abnormal ribosome biogenesis. We analyzed a patient with mild bone marrow failure and microcephaly who did not present with the typical FA phenotype. Cells from this patient showed increased sensitivity to ionizing radiations and phleomycin, attesting to a probable DNA double strand break (dsb) repair defect. Linkage analysis and whole exome sequencing revealed a homozygous nonsense mutation in the ERCC6L2 gene. We identified a new ERCC6L2 alternative transcript encoding the DNA repair factor Hebo, which is critical for complementation of the patient's DNAdsb repair defect. Sequence analysis revealed three structured regions within Hebo: a TUDOR domain, an adenosine triphosphatase domain, and a new domain, HEBO, specifically present in Hebo direct orthologues. Hebo is ubiquitously expressed, localized in the nucleus, and rapidly recruited to DNAdsb's in an NBS1-dependent manner. PMID:27185855

  16. Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples

    International Nuclear Information System (INIS)

    MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50-200 ng of PCR product and 1-3 μg of Thermus thermophilus his6-MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET, hMSH2, hMLH1, BRCA1, BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations

  17. Methods for detection of ataxia telangiectasia mutations

    Science.gov (United States)

    Gatti, Richard A.

    2005-10-04

    The present invention is directed to a method of screening large, complex, polyexonic eukaryotic genes such as the ATM gene for mutations and polymorphisms by an improved version of single strand conformation polymorphism (SSCP) electrophoresis that allows electrophoresis of two or three amplified segments in a single lane. The present invention also is directed to new mutations and polymorphisms in the ATM gene that are useful in performing more accurate screening of human DNA samples for mutations and in distinguishing mutations from polymorphisms, thereby improving the efficiency of automated screening methods.

  18. The SRS2 suppressor of rad6 mutations of Saccharomyces cerevisiae acts by channeling DNA lesions into the RAD52 DNA repair pathway

    International Nuclear Information System (INIS)

    rad6 mutants of Saccharomyces cerevisiae are defective in the repair of damaged DNA, DNA damage induced mutagenesis, and sporulation. In order to identify genes that can substitute for RAD6 function, the authors have isolated genomic suppressors of the UV sensitivity of rad6 deletion (rad6Δ) mutations and show that they also suppress the γ-ray sensitivity but not the UV mutagenesis or sporulation defects of rad6. The suppressors show semidominance for suppression of UV sensitivity and dominance for suppression of γ-ray sensitivity. The six suppressor mutations they isolated are all alleles of the same locus and are also allelic to a previously described suppressor of the rad6-1 nonsense mutation, SRS2. They show that suppression of rad6Δ is dependent on the RAD52 recombinational repair pathway since suppression is not observed in the rad6Δ SRS2 strain containing an additional mutation in either the RAD51, RAD52, RAD54, RAD55 or RAD57 genes. Possible mechanisms by which SRS2 may channel unrepaired DNA lesions into the RAD52 DNA repair pathway are discussed

  19. Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice

    Energy Technology Data Exchange (ETDEWEB)

    Swayne, Breanne G.; Kawata, Alice [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Behan, Nathalie A. [Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Williams, Andrew; Wade, Mike G. [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); MacFarlane, Amanda J. [Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada); Yauk, Carole L., E-mail: carole.yauk@hc-sc.ga.ca [Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9 (Canada)

    2012-09-01

    To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations. The majority of those studied are classical mutagens like radiation and anti-cancer drugs. Very little is known about the dietary variables influencing germline mutation rates. Folate is essential for DNA synthesis and methylation and can impact chromatin structure. We therefore determined the effects of folic acid-deficient (0 mg/kg), control (2 mg/kg) and supplemented (6 mg/kg) diets in early development and during lactation or post-weaning on mutation rates and chromatin quality in sperm of adult male Balb/c mice. The sperm chromatin structure assay and mutation frequencies at expanded simple tandem repeats (ESTRs) were used to evaluate germline DNA integrity. Treatment of a subset of mice fed the control diet with the mutagen ethylnitrosourea (ENU) at 8 weeks of age was included as a positive control. ENU treated mice exhibited decreased cauda sperm counts, increased DNA fragmentation and increased ESTR mutation frequencies relative to non-ENU treated mice fed the control diet. Male mice weaned to the folic acid deficient diet had decreased cauda sperm numbers, increased DNA fragmentation index, and increased ESTR mutation frequency. Folic acid deficiency in early development did not lead to changes in sperm counts or chromatin integrity in adult mice. Folic acid supplementation in early development or post-weaning did not affect germ cell measures. Therefore, adequate folic acid intake in adulthood is important for preventing chromatin damage and mutation in the male germline. Folic acid supplementation at the level achieved in this study does not improve nor is it detrimental to male germline chromatin integrity.

  20. Investigating the effects of dietary folic acid on sperm count, DNA damage and mutation in Balb/c mice

    International Nuclear Information System (INIS)

    To date, fewer than 50 mutagens have been studied for their ability to cause heritable mutations. The majority of those studied are classical mutagens like radiation and anti-cancer drugs. Very little is known about the dietary variables influencing germline mutation rates. Folate is essential for DNA synthesis and methylation and can impact chromatin structure. We therefore determined the effects of folic acid-deficient (0 mg/kg), control (2 mg/kg) and supplemented (6 mg/kg) diets in early development and during lactation or post-weaning on mutation rates and chromatin quality in sperm of adult male Balb/c mice. The sperm chromatin structure assay and mutation frequencies at expanded simple tandem repeats (ESTRs) were used to evaluate germline DNA integrity. Treatment of a subset of mice fed the control diet with the mutagen ethylnitrosourea (ENU) at 8 weeks of age was included as a positive control. ENU treated mice exhibited decreased cauda sperm counts, increased DNA fragmentation and increased ESTR mutation frequencies relative to non-ENU treated mice fed the control diet. Male mice weaned to the folic acid deficient diet had decreased cauda sperm numbers, increased DNA fragmentation index, and increased ESTR mutation frequency. Folic acid deficiency in early development did not lead to changes in sperm counts or chromatin integrity in adult mice. Folic acid supplementation in early development or post-weaning did not affect germ cell measures. Therefore, adequate folic acid intake in adulthood is important for preventing chromatin damage and mutation in the male germline. Folic acid supplementation at the level achieved in this study does not improve nor is it detrimental to male germline chromatin integrity.

  1. Mitochondrial DNA mutation m.10680G > A is associated with Leber hereditary optic neuropathy in Chinese patients

    Directory of Open Access Journals (Sweden)

    Zhang A-Mei

    2012-03-01

    Full Text Available Abstract Background Leber hereditary optic neuropathy (LHON is a mitochondrial disorder with gender biased and incomplete penetrance. The majority of LHON patients are caused by one of the three primary mutations (m.3460G > A, m.11778G > A and m.14484T > C. Rare pathogenic mutations have been occasionally reported in LHON patients. Methods We screened mutation m.10680G > A in the MT-ND4L gene in 774 Chinese patients with clinical features of LHON but lacked the three primary mutations by using allele specific PCR (AS-PCR. Patients with m.10680G > A were further determined entire mtDNA genome sequence. Results The optimal AS-PCR could detect as low as 10% heteroplasmy of mutation m.10680G > A. Two patients (Le1263 and Le1330 were identified to harbor m.10680G > A. Analysis of the complete mtDNA sequences of the probands suggested that they belonged to haplogroups B4a1 and D6a1. There was no other potentially pathogenic mutation, except for a few private yet reported variants in the MT-ND1 and MT-ND5 genes, in the two lineages. A search in reported mtDNA genome data set (n = 9277; excluding Chinese LHON patients identified no individual with m.10680G > A. Frequency of m.10680G > A in Chinese LHON patients analyzed in this study and our previous studies (3/784 was significantly higher than that of the general populations (0/9277 (P = 0.0005. Conclusion Taken together, we speculated that m.10680G > A may be a rare pathogenic mutation for LHON in Chinese. This mutation should be included in future clinical diagnosis.

  2. Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM located in the C-terminal domain of fission yeast DNA polymerase δ subunit Cdc27

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    Warbrick Emma

    2004-12-01

    Full Text Available Abstract Background DNA polymerases α and δ play essential roles in the replication of chromosomal DNA in eukaryotic cells. DNA polymerase α (Pol α-primase is required to prime synthesis of the leading strand and each Okazaki fragment on the lagging strand, whereas DNA polymerase δ (Pol δ is required for the elongation stages of replication, a function it appears capable of performing on both leading and lagging strands, at least in the absence of DNA polymerase ε (Pol ε. Results Here it is shown that the catalytic subunit of Pol α, Pol1, interacts with Cdc27, one of three non-catalytic subunits of fission yeast Pol δ, both in vivo and in vitro. Pol1 interacts with the C-terminal domain of Cdc27, at a site distinct from the previously identified binding sites for Cdc1 and PCNA. Comparative protein sequence analysis identifies a protein sequence motif, called the DNA polymerase interaction motif (DPIM, in Cdc27 orthologues from a wide variety of eukaryotic species, including mammals. Mutational analysis shows that the DPIM in fission yeast Cdc27 is not required for effective DNA replication, repair or checkpoint function. Conclusions The absence of any detectable phenotypic consequences arising from mutation of the DPIM suggests that despite its evolutionary conservation, the interaction between the two polymerases mediated by this motif is a non-essential one.

  3. Dlx5 Homeodomain:DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome.

    Science.gov (United States)

    Proudfoot, Andrew; Axelrod, Herbert L; Geralt, Michael; Fletterick, Robert J; Yumoto, Fumiaki; Deacon, Ashley M; Elsliger, Marc-André; Wilson, Ian A; Wüthrich, Kurt; Serrano, Pedro

    2016-03-27

    The Dlx5 homeodomain is a transcription factor related to the Drosophila distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including split hand and foot malformation 1 in humans. Our characterization of a Dlx5 homeodomain:(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A Q186H mutation linked to split hand and foot malformation 1 likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and they likely destabilize the formation of functional complexes. PMID:26829219

  4. Frequent mutations in EGFR, KRAS and TP53 genes in human lung cancer tumors detected by ion torrent DNA sequencing.

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    Xin Cai

    Full Text Available Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations.

  5. EGFR Mutations Detection in Non-small Cell Lung Cancer Tissues by Real-time PCR and DNA Sequencing

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    Qinghua ZHOU

    2009-12-01

    Full Text Available Background and objective Small molecule tyrosine kinase inhibitors (TKIs, such as gefitinib and erlotinib that target the kinase domain of epidermal growth factor receptor (EGFR are making successful progression for advanced non-small cell lung cancer patients treatment. The growing evidences revealed that EGFR exon 19 and 21 mutation status in NSCLC patients was correlated with the outcome for EGFR-TKI treatment. In this study, two methods of Real-time PCR and DNA sequencing were compared to detected EGFR exon 19 and 21 mutations. Methods EGFR exon19 mutation del-E746-A750 and exon 21mutation L858R were detected by Real-time PCR and DNA sequencing in 103 NSCLC patients. Chi-square test was used to analyze the consistance. Results There was no significant difference between the two methods. However, Real-time PCR was more convenient and sensitive compared to DNA sequencing. Conclusion Real-time PCR was more suitable for clinical testing than DNA sequencing.

  6. GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy.

    Science.gov (United States)

    Kuschal, Christiane; Botta, Elena; Orioli, Donata; Digiovanna, John J; Seneca, Sara; Keymolen, Kathelijn; Tamura, Deborah; Heller, Elizabeth; Khan, Sikandar G; Caligiuri, Giuseppina; Lanzafame, Manuela; Nardo, Tiziana; Ricotti, Roberta; Peverali, Fiorenzo A; Stephens, Robert; Zhao, Yongmei; Lehmann, Alan R; Baranello, Laura; Levens, David; Kraemer, Kenneth H; Stefanini, Miria

    2016-04-01

    The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP. PMID:26996949

  7. Roles of DNA mutation in the coding region and DNA methylation in the 5' flanking region of BRCA1 in canine mammary tumors.

    Science.gov (United States)

    Qiu, Hengbin; Lin, Deigui

    2016-07-01

    The Breast cancer 1, early onset gene (BRCA1) is known to be significantly associated with human familial breast cancer and is identified to play an important role in canine mammary tumors. Here, genetic variations in the coding region and DNA methylation in the 5' flanking region of BRCA1 in canine mammary tumor samples, 15 each of benign and malignant against 10 normal canine mammary tissue samples, were analyzed using the direct sequencing method. The results indicated two point mutations each in the coding region of canine BRCA1 in one benign mammary tumor sample (4702G >T and 4765G >T) and in one malignant canine mammary tumor sample (3619A >G and 4006G >A). No mutations were detected in the normal canine mammary tissue samples. The 4702G >T mutation was found to terminate further translation. The physical effect of the 4765G >T mutation was found to be the repalacement of the glutamate residue with glutamine. The physical effect of the 3619A >G mutation was found to be the replacement of the threonine residue with alanine, and that of mutation 4006G >A was the replacement of the valine residue with isoleucine in the BRCA1 protein. Bisulfite sequencing detected methylated CpG sites in one canine malignant mammary tumor sample. In conclusion, the present study elucidated the mutational status of the BRCA1 coding region and methylation status of the 5' flanking region of BRCA1 in canine mammary tumors. PMID:26888582

  8. Leber Hereditary Optic Neuropathy: Do Folate Pathway Gene Alterations Influence the Expression of Mitochondrial DNA Mutation?

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    A Aleyasin

    2010-09-01

    Full Text Available "nBackground: Leber hereditary optic neuropathy (LHON is an inherited form of bilateral optic atrophy leading to the loss of central vision.  The primary cause of vision loss is mutation in the mitochondrial DNA (mtDNA, however, unknown secon­dary genetic and/or epigenetic risk factors are suggested to influence its neuropathology.  In this study folate gene polymor­phisms were examined as a possible LHON secondary genetic risk factor in Iranian patients."nMethods: Common polymorphisms in the MTHFR (C677T and A1298C and MTRR (A66G genes were tested in 21 LHON patients and 150 normal controls."nResults:  Strong associations were observed between the LHON syndrome and C677T (P= 0.00 and A66G (P= 0.00 polymor­phisms.  However, no significant association was found between A1298C (P =0.69 and the LHON syndrome."nConclusion: This is the first study that shows MTHFR C677T and MTRR A66G polymorphisms play a role in the etiology of the LHON syndrome.  This finding may help in the better understanding of mechanisms involved in neural degeneration and vision loss by LHON and hence the better treatment of patients.

  9. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    Science.gov (United States)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  10. Mutational analysis of Mycobacterium UvrD1 identifies functional groups required for ATP hydrolysis, DNA unwinding, and chemomechanical coupling

    OpenAIRE

    Sinha, Krishna Murari; Glickman, Michael S.; Shuman, Stewart

    2009-01-01

    Mycobacterial UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3’ to 5’ DNA helicase. The UvrD1 motor domain resembles that of the prototypal superfamily I helicases UvrD and PcrA. Here we performed a mutational analysis of UvrD1 guided by the crystal structure of a DNA-bound E. coli UvrD-ADP-MgF3 transition state mimetic. Alanine scanning and conservative substitutions identified five amino acids essential for both ATP hydrolysis and duplex unwinding, including those implicated in phosphoh...

  11. Temporal changes of EGFR mutations and T790M levels in tumour and plasma DNA following AZD9291 treatment.

    Science.gov (United States)

    Chia, Puey Ling; Do, Hongdo; Morey, Adrienne; Mitchell, Paul; Dobrovic, Alexander; John, Thomas

    2016-08-01

    AZD9291, a T790M specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has demonstrated impressive response rates in tumours harbouring the EGFR T790M resistance mutation. Emergence of resistance to AZD9291 has been shown to occur through several different mechanisms including the development of new mutations (e.g. C797S) in the EGFR tyrosine kinase domain. We studied two patients with paired tumour biopsies and blood samples pre- and post-progression on AZD9291 to explore possible resistance mechanisms. Pre- and Post-AZD9291 tumour biopsies as well as serial plasma samples were collected from two patients on the AURA clinical study (AZD9291 First Time in Patients Ascending Dose study). Droplet digital PCR (ddPCR) assays were used to quantify T790M, the driver EGFR mutation, and the C797S mutation in genomic DNA from paired tumour biopsies and plasma cell-free DNA. In the first patient, both EGFR T790M and L858R became undetectable in the plasma within 1 month after treatment with AZD9291. However, the T790M and the original L858R mutation re-emerged with radiologically confirmed resistance to AZD9291. In patient two, the levels of T790M were undetectable at the time of radiological resistance to AZD9291 but increasing levels of the original EGFR exon 19 deletion was detected. MET amplification was detected in a biopsy performed on progression. The EGFR C797S mutation was not detected in either patient at the time of relapse. ddPCR of cell free DNA enables real time monitoring of patients on 3rd generation TKIs. As resistance mechanisms are variable, monitoring levels of the initial activating EGFR mutation may facilitate more reliable detection of progression. PMID:27393503

  12. Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

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    Grafodatskaya Daria

    2013-01-01

    Full Text Available Abstract Background A number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment. Results Using a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP. Conclusions We have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain.

  13. POLG1 p.R722H mutation associated with multiple mtDNA deletions and a neurological phenotype

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    Finnilä Saara

    2010-05-01

    Full Text Available Abstract Background The c.2447G>A (p.R722H mutation in the gene POLG1 of the catalytic subunit of human mitochondrial polymerase gamma has been previously found in a few occasions but its pathogenicity has remained uncertain. We set out to ascertain its contribution to neuromuscular disease. Methods Probands from two families with probable mitochondrial disease were examined clinically, muscle and buccal epithelial DNA were analyzed for mtDNA deletions, and the POLG1, POLG2, ANT1 and Twinkle genes were sequenced. Results An adult proband presented with progressive external ophthalmoplegia, sensorineural hearing impairment, diabetes mellitus, dysphagia, a limb myopathy and dementia. Brain MRI showed central and cortical atrophy, and 18F-deoxyglucose PET revealed reduced glucose uptake. Histochemical analysis of muscle disclosed ragged red fibers and cytochrome c oxidase-negative fibers. Electron microscopy showed subsarcolemmal aggregates of morphologically normal mitochondria. Multiple mtDNA deletions were found in the muscle, and sequencing of the POLG1 gene revealed a homozygous c.2447G>A (p.R722H mutation. His two siblings were also homozygous with respect to the p.R722H mutation and presented with dementia and sensorineural hearing impairment. In another family the p.R722H mutation was found as compound heterozygosity with the common p.W748S mutation in two siblings with mental retardation, ptosis, epilepsy and psychiatric symptoms. The estimated carrier frequency of the p.R722H mutation was 1:135 in the Finnish population. No mutations in POLG2, ANT1 and Twinkle genes were found. Analysis of the POLG1 sequence by homology modeling supported the notion that the p.R722H mutation is pathogenic. Conclusions The recessive c.2447G>A (p.R722H mutation in the linker region of the POLG1 gene is pathogenic for multiple mtDNA deletions in muscle and is associated with a late-onset neurological phenotype as a homozygous state. The onset of the disease

  14. Mutagenesis by site-specific arylamine adducts in plasmid DNA: Enhancing replication of the adducted strand alters mutation frequency

    International Nuclear Information System (INIS)

    Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by >7-fold to 2.9% and of the AAF adduct by >12-fold to 0.75%. The AF adduct produced primarily single-base deletions in the absence of uracil but only base substitutions in the uracil-containing constructs. The AAF adduct produced mutations only in the uracil-containing DNA, which included both frame shifts and base substitutions. Mutations produced by both adducts were SOS dependent

  15. Molecular analysis of the human laminin alpha3a chain gene (LAMA3a): a strategy for mutation identification and DNA-based prenatal diagnosis in Herlitz junctional epidermolysis bullosa.

    Science.gov (United States)

    Pulkkinen, L; Cserhalmi-Friedman, P B; Tang, M; Ryan, M C; Uitto, J; Christiano, A M

    1998-09-01

    Mutations in the genes (LAMA3, LAMB3, and LAMC2) encoding the subunit polypeptides of the cutaneous basement membrane zone protein laminin 5 have been reported in different forms of junctional epidermolysis bullosa (JEB), an inherited blistering skin disease. In this study, we present the complete exon-intron organization of the "a" transcript of the laminin alpha3 chain gene, LAMA3a, which is expressed primarily in the skin. We have performed fine-resolution mapping of this gene on chromosome 18q11.2 using a human-hamster radiation hybrid panel. We have also developed a mutation-detection strategy based on the exon-intron structure of LAMA3a. This strategy, based on PCR amplification of genomic sequences, followed by heteroduplex scanning and automated nucleotide sequencing, was used for successful mutation screening in a family with the lethal (Herlitz) type of JEB, and two novel LAMA3 mutations were identified in the proband. The mutations consisted of a single-base pair deletion in LAMA3a exon A11 on the paternal allele, designated 1239delC, and a two-base pair deletion in LAMA3a exon A23 on the maternal allele, designated 2959delGG. This information was also used for DNA-based prenatal testing in a subsequent pregnancy in this family. Collectively, these results attest to our expanding capability to elucidate the genetic basis of various forms of epidermolysis bullosa using molecular techniques. PMID:9759651

  16. The use of EGFR exon 19 and 21 unlabeled DNA probes to screen for activating mutations in non-small cell lung cancer.

    Science.gov (United States)

    Willmore-Payne, Carlynn; Holden, Joseph A; Wittwer, Carl T; Layfield, Lester J

    2008-07-01

    Activating mutations in epidermal growth factor receptor-1 (EGFR) are found in 10-15% of Caucasian patients with non-small cell lung carcinoma (NSCLC). Approximately 90% of the mutations are deletions of several amino acids in exon 19 or point mutations in exon 21. Some studies suggest that these mutations identify patients that might benefit from targeted EGFR inhibitor therapy. DNA melting analysis of polymerase chain reaction products can screen for these mutations to identify this patient population. However, amplicon DNA melting analysis, although easily capable of detecting heterozygous mutations by heterodimer formation, becomes more difficult if mutations are homozygous or if the mutant allele is selectively amplified over wild type. Amplification of EGFR is common in NSCLC and this could compromise mutation detection by amplicon melting analysis. To overcome this potential limitation, we developed unlabeled, single-stranded DNA probes, complimentary to EGFR exon 19 and exon 21 where the common activating mutations occur. The unlabeled probes are incorporated into a standard polymerase chain reaction during the amplification of EGFR exons 19 and 21. The probe melting peak is easily distinguished from the amplicon melting peak, and probe melting is altered if mutations are present. This allows for easy identification of activating mutations even in homozygous or amplified states and is useful in the screening of NSCLC for the common EGFR activating mutations. PMID:19137110

  17. Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Frøslev, Tobias G; Frank-Hansen, Rune;

    2011-01-01

    We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Männedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO...... automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid...... 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the...

  18. Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair, chromosomal alterations, and mutations

    Energy Technology Data Exchange (ETDEWEB)

    Natarajan, A.T.; van Zeeland, A.A.; Zwanenburg, T.S.

    1983-01-01

    The influence of inhibitors of poly(ADP-ribose) polymerase such as 3-aminobenzamide (3AB) and benzamide (B) on the spontaneously occurring as well as mutagen induced chromosomal aberrations, sister chromatid exchanges (SCEs) and point mutations has been studied. In addition, the influence of 3AB on DNA repair was measured following treatment with physical and chemical mutagens. Post treatment of X-irradiated mammalian cells with 3AB increases the frequencies of induced chromosomal aberrations by a factor of 2 to 3. 3AB, when present in the medium containing bromodeoxyuridine(BrdUrd) during two cell cycles, increases the frequencies of SCEs in Chinese hamster ovary cells (CHO) in a concentration dependent manner leading to about a 10-fold increase at 10 mM concentration. The extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines has been studied, including those derived from patients suffering from genetic diseases such as ataxia telangiectasia (A-T), Fanconi's anemia (FA), and Huntington's chorea. None of these syndromes showed any increased response when compared to normal cells. 3AB, however, increased the frequencies of spontaneously occurring chromosomal aberrations in A-T and FA cells. 3AB does not influence the frequencies of SCEs induced by UV or mitomycin C (MMC) in CHO cells. However, it increases the frequencies of SCEs induced by ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). Under the conditions in which 3AB increases the frequencies of spontaneously occurring as well as induced SCEs, it does not increase the frequencies of point mutations in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus. 3AB does not influence the amount of repair replication following dimethylsulphate (DMS) treatment of human fibroblasts, or UV irradiated human lymphocytes.

  19. DNA synthesis time in germinating rice and pattern of diethylsulphate induced mutations in pre-soaked seeds

    International Nuclear Information System (INIS)

    DNA synthesis pattern in germinating rice seeds, pre-soaked in water for varying periods upto 48 hr, was determined by following the pulse incorporation of 3H-thymidine into the TCA-insoluble nucleoprotein. Synthesis of DNA commenced at 24 hr, progressively increased to a first peak at about 38 hr, thereafter showed a 1/3rd drop and subsequently increased to a 2nd and still higher peak at 46 to 48 hr of pre-soaking. Treatments of diethylsulphate (dES) at a low concentration (0.2%-2hr) administered at various progressing stages of DNA synthesis resulted in decrease in seedling height and survival, and increase in mutation frequency at 45 hr. pre-soaking, maximum mutation frequencies of 20, 10 and 2% on M1 plants, M1 spikes and M2 seedling bases, respectively were observed. Higher dES concentration (0.3%-2hr) given at later periods of pre-soaking showed near lethal effects and consequently decreased mutation frequencies. Treatments of sodium fluoride given singly or in combination with dES did not show any substantially different results as compared to those of the respective controls. Mutation spectra observed after dES treatments to germinating seeds, at different pre-soaking periods, were quite dissimilar. Specific mutations of economic importance like semi-dwarf mutants were isolated from the treatment of germinating seeds pre-soaked for 37.5 hr or more when shoot apex cells were undergoing DNA synthesis. (author)

  20. Decreased heat shock protein 27 expression and altered autophagy in human cells harboring A8344G mitochondrial DNA mutation.

    Science.gov (United States)

    Chen, Chin-Yi; Chen, Hsueh-Fu; Gi, Siao-Jhen; Chi, Tang-Hao; Cheng, Che-Kun; Hsu, Chi-Fu; Ma, Yi-Shing; Wei, Yau-Huei; Liu, Chin-Shan; Hsieh, Mingli

    2011-09-01

    Mitochondrial DNA (mtDNA) mutations are responsible for human neuromuscular diseases caused by mitochondrial dysfunction. Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a maternally inherited mitochondrial encephalomyopathy with various syndromes involving both muscular and nervous systems. The most common mutation in MERRF syndrome, A8344G mutation in mtDNA, has been associated with severe defects in protein synthesis. This defect impairs assembly of complexes in electron transport chain and results in decreased respiratory function of mitochondria. In this study, we showed a significant decrease of the heat shock protein 27 (Hsp27) in lymphoblastoid cells derived from a MERRF patient and in cybrid cells harboring MERRF A8344G mutation. However, normal cytoplasmic distributions of Hsp27 and normal heat shock responses were observed in both wild type and mutant cybrids. Furthermore, overexpression of wild type Hsp27 in mutant MERRF cybrids significantly decreased cell death under staurosporine (STS) treatment, suggesting a protective function of Hsp27 in cells harboring the A8344G mutation of mtDNA. Meanwhile, reverse transcriptase PCR showed no difference in the mRNA level between normal and mutant cybrids, indicating that alterations may occur at the protein level. Evidenced by the decreased levels of Hsp27 upon treatment with proteasome inhibitor, starvation and rapamycin and the accumulation of Hsp27 upon lysosomal inhibitor treatment; Hsp27 may be degraded by the autophagic pathway. In addition, the increased formation of LC3-II and autophagosomes was found in MERRF cybrids under the basal condition, indicating a constitutively-activated autophagic pathway. It may explain, at least partially, the faster turnover of Hsp27 in MERRF cybrids. This study provides information for us to understand that Hsp27 is degraded through the autophagic pathway and that Hsp27 may have a protective role in MERRF cells. Regulating Hsp27 and the autophagic pathway

  1. Somatic point mutations in mtDNA control region are influenced by genetic background and associated with healthy aging: a GEHA study

    DEFF Research Database (Denmark)

    Rose, Giuseppina; Romeo, Giuseppe; Dato, Serena;

    2010-01-01

    Tissue specific somatic mutations occurring in the mtDNA control region have been proposed to provide a survival advantage. Data on twins and on relatives of long-lived subjects suggested that the occurrence/accumulation of these mutations may be genetically influenced. To further investigate....... We found a significant correlation of the mtDNA control region heteroplasmy between sibs, confirming a genetic influence on this phenomenon. Furthermore, many subjects showed heteroplasmy due to mutations different from the C150T transition. In these cases heteroplasmy was correlated within sibpairs...... performance and of mortality risk in the elderly. Our study provides new evidence on the relevance of mtDNA somatic mutations in aging and longevity and confirms that the occurrence of specific point mutations in the mtDNA control region may represent a strategy for the age-related remodelling of organismal...

  2. A Novel Homoplasmic Mutation in mtDNA with a Single Evolutionary Origin as a Risk Factor for Cardiomyopathy

    OpenAIRE

    Shin, Wee Soo; Tanaka, Masashi; Suzuki, Jun-ichi; Hemmi, Chieko; Toyo-oka, Teruhiko

    2000-01-01

    To clarify the relationship between variation in mtDNA and the development of cardiomyopathy (CM), the complete sequences of mtDNAs of two brothers with dilated CM were compared with those of 181 patients who had CM and with those of 168 control subjects. Five patients with CM shared a novel homoplasmic point mutation (G12192A tRNAHis), and all of them demonstrated the evolutionarily related D-loop sequence. The results suggest that this novel mutation originated from the same ancestor and th...

  3. Mitochondrial DNA A10398G Mutation is not Associated with Breast Cancer Risk in a Sample of Iraqi Women

    Directory of Open Access Journals (Sweden)

    Rawaa A. Zahid

    2013-05-01

    Full Text Available The aim of this study was to investigate if there is a relationship between mtDNA polymorphism (A10398G and breast cancer in a sample of 59 Iraqi women. Breast cancer is the second most common diagnosed cause of cancer death in the developed countries and accounts for 23% of the total cancers. Different studies reported that breast cancer accounts for 14% of all cancer deaths in females. It is well documented that the different factors such as genetics and environment factors are involved in tumorigenesis. Mutations in the mitochondrial DNA D-loop region and somatic mutations are emerging as early genetic markers of cancer. Identification of such markers for breast cancer would prevent late detection and increase the chance of recovery and survival rate. In breast cancer different mtDNA alterations were reported. The A10398G mutation in NADH Dehyrogenase (ND3 a subunit of complex I of the Oxidative Phosphorylation process (OXPHOS is perhaps one of the most studied mutations with conflicting reports of its association with breast cancer. Genomic DNA was extracted from 21 unrelated women with malignant tumors, 22 women with benign tumors and 16 healthy women blood donors. Subsequently, PCR amplification was performed using specific primers, PCR products were subjected to a suitable restriction enzyme. No genetic variants were identified in mtDNA among malignant tumoral group and controls while 9% of benign tumor cases exhibited the variant. Our finding indicated that A10398G polymorphism cannot be used as a biomarker for breast cancer detection in Iraqi women.

  4. Mutations Affecting Potassium Import Restore the Viability of the Escherichia coli DNA Polymerase III holD Mutant

    Science.gov (United States)

    Durand, Adeline

    2016-01-01

    Mutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo. PMID:27280472

  5. Effect of increased intake of dietary animal fat and fat energy on oxidative damage, mutation frequency, DNA adduct level and DNA repair in rat colon and liver

    DEFF Research Database (Denmark)

    Vogel, Ulla Birgitte; Danesvar, B.; Autrup, H.;

    2003-01-01

    liver, colon, or urine. Thus, lard intake at the expense of other nutrients and a large increase in the fat energy consumption affects the redox state locally in the liver cytosol, but does not induce DNA-damage, systemic oxidative stress or a dose-dependent increase in mutation frequency in rat colon...... was observed. Intake of lard fat resulted in increased ascorbate synthesis and affected markers of oxidative damage to proteins in liver cytosol, but not in plasma. The effect was observed at all lard doses and was not dose-dependent. However, no evidence of increased oxidative DNA damage was found in...

  6. Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise Garm; Pallisgaard, Niels; Vogelius, Ivan Storgaard; Jakobsen, Anders

    2012-01-01

    The present study investigated the levels of circulating cell-free DNA (cfDNA) in plasma from patients with metastatic colorectal cancer (mCRC) in relation to third-line treatment with cetuximab and irinotecan and the quantitative relationship of cfDNA with tumor-specific mutations in plasma....

  7. Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma

    DEFF Research Database (Denmark)

    Asmar, Fazila; Punj, Vasu; Christensen, Jesper Aagaard; Pedersen, Marianne T; Pedersen, Anja; Nielsen, Anders B; Hother, Christoffer; Ralfkiaer, Ulrik; Brown, Peter; Ralfkiaer, Elisabeth; Helin, Kristian; Grønbæk, Kirsten

    2013-01-01

    The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and...... recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7...

  8. Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

    Science.gov (United States)

    Lanman, Richard B.; Mortimer, Stefanie; Zill, Oliver A.; Kim, Kyoung-Mee; Jang, Kee Taek; Kim, Seok-Hyung; Park, Se Hoon; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Eltoukhy, Helmy; Kang, Won Ki; Lee, Woo Yong; Kim, Hee-Cheol; Park, Keunchil; Lee, Jeeyun; Talasaz, AmirAli

    2015-01-01

    Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer. We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy. PMID:26452027

  9. Mutational and Biochemical Analysis of the DNA-entry Nuclease EndA from Streptococcus pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    M Midon; P Schafer; A Pingoud; M Ghosh; A Moon; M Cuneo; R London; G Meiss

    2011-12-31

    EndA is a membrane-attached surface-exposed DNA-entry nuclease previously known to be required for genetic transformation of Streptococcus pneumoniae. More recent studies have shown that the enzyme also plays an important role during the establishment of invasive infections by degrading extracellular chromatin in the form of neutrophil extracellular traps (NETs), enabling streptococci to overcome the innate immune system in mammals. As a virulence factor, EndA has become an interesting target for future drug design. Here we present the first mutational and biochemical analysis of recombinant forms of EndA produced either in a cell-free expression system or in Escherichia coli. We identify His160 and Asn191 to be essential for catalysis and Asn182 to be required for stability of EndA. The role of His160 as the putative general base in the catalytic mechanism is supported by chemical rescue of the H160A variant of EndA with imidazole added in excess. Our study paves the way for the identification and development of protein or low-molecular-weight inhibitors for EndA in future high-throughput screening assays.

  10. Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis

    NARCIS (Netherlands)

    Wu, Y; Hofstra, RMW; Scheffer, H; Uitterlinden, AG; Mullaart, E; Buys, CHCM; Vijg, J

    1996-01-01

    The large number of possible disease causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for mutation

  11. Molecular diagnosis of mucopolysaccharidosis Type II (Hunter syndrome) by automated sequencing and computer-assisted interpretation: Toward mutation mapping of the Iduronate-2-sulfatase gene

    Energy Technology Data Exchange (ETDEWEB)

    Jonsson, J.J.; Aronovich, E.L.; Braun, S.E.; Whitley, C.B. [Univ. of Minnesota Medical School, Minneapolis, MN (United States)

    1995-03-01

    Virtually all mutations causing Hunter syndrome (mucopolysaccharidosis type II) are expected to be new mutations. Therefore, as a means of molecular diagnosis, we developed a rapid method to sequence the entire iduronate-2-sulfatase (IDS) coding region. PCR amplicons representing the IDS cDNA were sequenced with an automatic instrument, and output was analyzed by computer-assisted interpretation of tracings, using Staden programs on a Sun computer. Mutations were found in 10 of 11 patients studied. Unique missense mutations were identified in five patients: H229Y (685{r_arrow}T, severe phenotype); P358R (1073C{r_arrow}G, severe); R468W (1402C{r_arrow}T, mild); P469H (1406C{r_arrow}A, mild); and Y523C (1568A{r_arrow}G, mild). Nonsense mutations were identified in two patients: R172X (514C{r_arrow}T, severe) and Q389X (1165C{r_arrow}T, severe). Two other patients with severe disease had insertions of 1 and 14 bp, in exons 3 and 6, respectively. In another patient with severe disease, the predominant (<95%) IDS message resulted from aberrant splicing, which skipped exon 3. In this last case, consensus sequences for splice sites in exon 3 were intact, but a 395C{r_arrow}G mutation was identified 24 bp upstream from the 3` splice of exon 3. This mutation created a cryptic 5` splice site with a better consensus sequence for 5` splice sites than the natural 5` splice site of intron 3. A minor population of the IDS message was processed by using this cryptic splice site; however, no correctly spliced message was detected in leukocytes from this patient. The mutational topology of the IDS gene is presented. 46 refs., 6 figs., 2 tabs.

  12. Distinctive Drug-resistant Mutation Profiles and Interpretations of HIV-1 Proviral DNA Revealed by Deep Sequencing in Reverse Transcriptase

    Institute of Scientific and Technical Information of China (English)

    YIN Qian Qian; SHAO Yi Ming; MA Li Ying; LI Zhen Peng; ZHAO Hai; PAN Dong; WANG Yan; XU Wei Si; XING Hui; FENGYi; JIANG Shi Bo

    2016-01-01

    ObjectiveTo investigate distinctive features in drug-resistant mutations(DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients. MethodsForty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA. ResultsCompared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher’s exact test,P ConclusionCompared with viral RNA, the distinctive information of DRMsand drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.

  13. SIMILARITY ANALYSIS OF DNA SEQUENCES BASED ON THE CHEMICAL PROPERTIES OF NUCLEOTIDE BASES, FREQUENCY AND POSITION OF GROUP MUTATIONS

    Directory of Open Access Journals (Sweden)

    Fatima KABLI

    2016-01-01

    Full Text Available The DNA sequences similarity analysis approaches have been based on the representation and the frequency of sequences components; however, the position inside sequence is important information for the sequence data. Whereas, insufficient information in sequences representations is important reason that causes poor similarity results. Based on three classifications of the DNA bases according to their chemical properties, the frequencies and average positions of group mutations have been grouped into two twelve-components vectors, the Euclidean distances among introduced vectors applied to compare the coding sequences of the first exon of beta globin gene of 11 species.

  14. A Mitochondrial DNA A8701G Mutation Associated with Maternally Inherited Hypertension and Dilated Cardiomyopathy in a Chinese Pedigree of a Consanguineous Marriage

    Institute of Scientific and Technical Information of China (English)

    Ye Zhu; Xiang Gu; Chao Xu

    2016-01-01

    Background: Cardiovascular diseases, including dilated cardiomyopathy (DCM) and hypertension, are the leading cause of death worldwide.The role of mitochondrial DNA (mtDNA) in the pathogenesis of these diseases has not been completely clarified.In this study, we evaluate whether A8701G mutation is associated with maternally inherited hypertension and DCM in a Chinese pedigree of a consanguineous marriage.Methods: Fourteen subjects in a three-generation Han Chinese family with hypertension and DCM, in which consanguineous marriage was present in the parental generation, were interviewed.We divided all the family members into case (7 maternal members) and control group (7 nonmaternal members) for comparison.Clinical evaluations and sequence analysis ofmtDNA were obtained from all participants.Frequency differences between maternal and nonmaternal members were tested to locate the disease-associated mutations.Results: The majority of the family members presented with a maternal inheritance of hypertension and DCM.Sequence analysis of mtDNA in this pedigree identified eight mtDNA mutations.Among the mutations identified, there was only one significant mutation: A8701G (P =0.005), which is a homoplasmic mitochondrial missense mutation in all the matrilineal relatives.There was no clear evidence for any synergistic effects between A8701G and other mutations.Conclusions: A8701G mutation may act as an inherited risk factor for the matrilineal transmission of hypertension and DCM in conj unction with genetic disorders caused by consanguineous marriage.

  15. Significance of the pathogenic mutation T372R in the Yin Yang 1 protein interaction with DNA--thermodynamic studies.

    Science.gov (United States)

    Nieborak, Anna; Górecki, Andrzej

    2016-03-01

    This work focuses on the pathogenic missense mutation in YY1 protein correlated with insulinomas. Based on in vitro studies, we demonstrate that the mutation does not affect the secondary structure of either zinc fingers or the N-terminal fragment (NTF) of the protein. Apart from a slight increase in the protein's compactness, no changes in the tertiary structure were observed. The introduced mutation significantly alters DNA-binding properties, both the affinity and enthalpy-entropy contribution of the process, which are highly dependent on the recognized sequence. Obtained results indicate concerted rather than a modular mode of sequence recognition by YY1 with the significant impact of a disordered NTF. PMID:26910132

  16. Preparatory methods for DNA hydrolysis, cytochemistry, immunocytochemistry and ploidy analysis. Their application to automated and routine diagnostic cytopathology.

    Science.gov (United States)

    Husain, O A; Watts, K C

    1987-06-01

    A review is presented of some methods used to prepare cytologic specimens for analytical and/or automated studies, with the steps of the procedures detailed in appendices. The preparation of the cell monolayers required for optimal automated cell image analysis and classification, e.g., by the Cytoscan 110, is discussed, as is the preparation of poly-L-lysine-coated slides used in the production of monolayered specimens. These monolayers, which can be prepared from a variety of specimens, are also useful for cytochemical and immunocytochemical studies and DNA ploidy analysis. For DNA analysis, a modified gallocyanin chrome alum staining procedure is described as a stoichiometric alternative to the time-consuming Feulgen reaction. The hydrolysis technique required by the latter method is also detailed. The freeze-fracturing technique for the enhancement of monoclonal antibody immunocytochemical staining of detectable antigens is described, along with an indirect immunoalkaline phosphatase staining method. The use of enzyme cytochemical reactions for glucose 6 phosphate dehydrogenase and lysosomal naphthylamidase is also presented. PMID:3620061

  17. High-fat diet and FGF21 cooperatively promote aerobic thermogenesis in mtDNA mutator mice

    OpenAIRE

    Wall, Christopher E.; Whyte, Jamie; Suh, Jae M.; Fan, Weiwei; Collins, Brett; Liddle, Christopher; Yu, Ruth T; Atkins, Annette R.; Naviaux, Jane C.; Li, Kefeng; Bright, Andrew Taylor; Alaynick, William A; Downes, Michael; Naviaux, Robert K.; Evans, Ronald M.

    2015-01-01

    Fibroblast growth factor 21 (FGF21), a hormone that mediates an adaptive response to starvation, is also a long-standing marker of mitochondrial disease. In this article, we describe the metabolic benefits induced by mild mitochondrial stress via FGF21 induction in polymerase gamma mtDNA mutator (POLG) mice, a model of mitochondrial disease and premature aging. When challenged with a high-fat diet (HFD), these mice resist diet-induced obesity and its underlying associated disease states. In a...

  18. Nitric Oxide Induces Early Viral Transcription Coincident with Increased DNA Damage and Mutation Rates in Human Papillomavirus–Infected Cells

    OpenAIRE

    Wei, Lanlan; Gravitt, Patti E.; Song, Hebin; Maldonado, Anastacia M.; Ozbun, Michelle A.

    2009-01-01

    High-risk human papillomavirus (HPV) infections are necessary but insufficient causes of cervical cancers. Other risk factors for cervical cancer (e.g., pregnancy, smoking, infections causing inflammation) can lead to high and sustained nitric oxide (NO) concentrations in the cervix, and high NO levels are related to carcinogenesis through DNA damage and mutation. However, the effects of NO exposure in HPV-infected cells have not been investigated. In this study, we used the NO donor DETA-NO ...

  19. A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

    International Nuclear Information System (INIS)

    Highlights: • We studied the combined role of MutY and Fpg DNA glycosylases in M. smegmatis. • Loss of MutY showed increased sensitivity to oxidative damage. • Loss of MutY together with the Fpg glycosylases showed increased mutation rates. • Our data indicate interplay between these enzymes to control mutagenesis. - Abstract: Hydroxyl radical (·OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen (102) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to ·OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei homologues did

  20. A combinatorial role for MutY and Fpg DNA glycosylases in mutation avoidance in Mycobacterium smegmatis

    Energy Technology Data Exchange (ETDEWEB)

    Hassim, Farzanah; Papadopoulos, Andrea O.; Kana, Bavesh D.; Gordhan, Bhavna G., E-mail: bhavna.gordhan@nhls.ac.za

    2015-09-15

    Highlights: • We studied the combined role of MutY and Fpg DNA glycosylases in M. smegmatis. • Loss of MutY showed increased sensitivity to oxidative damage. • Loss of MutY together with the Fpg glycosylases showed increased mutation rates. • Our data indicate interplay between these enzymes to control mutagenesis. - Abstract: Hydroxyl radical (·OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen ({sup 1}0{sub 2}) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to ·OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei

  1. Detection of Onchocerca volvulus in Latin American black flies for pool screening PCR using high-throughput automated DNA isolation for transmission surveillance.

    Science.gov (United States)

    Rodríguez-Pérez, Mario A; Gopal, Hemavathi; Adeleke, Monsuru Adebayo; De Luna-Santillana, Erick Jesús; Gurrola-Reyes, J Natividad; Guo, Xianwu

    2013-11-01

    The posttreatment entomological surveillance (ES) of onchocerciasis in Latin America requires quite large numbers of flies to be examined for parasite infection to prove that the control strategies have worked and that the infection is on the path of elimination. Here, we report a high-throughput automated DNA isolation of Onchocerca volvulus for PCR using a major Latin American black fly vector of onchocerciasis. The sensitivity and relative effectiveness of silica-coated paramagnetic beads was evaluated in comparison with phenol chloroform (PC) method which is known as the gold standard of DNA extraction for ES in Latin America. The automated method was optimized in the laboratory and validated in the field to detect parasite DNA in Simulium ochraceum sensu lato flies in comparison with PC. The optimization of the automated method showed that it is sensitive to detect O. volvulus with a pool size of 100 flies as compared with PC which utilizes 50 flies pool size. The validation of the automated method in comparison with PC in an endemic community showed that 5/67 and 3/134 heads pools were positive for the two methods, respectively. There was no statistical variation (P < 0.05) in the estimation of transmission indices generated by automated method when compared with PC method. The fact that the automated method is sensitive to pool size up to 100 confers advantage over PC method and can, therefore, be employed in large-scale ES of onchocerciasis transmission in endemic areas of Latin America. PMID:24030195

  2. Mutational Analysis of Bacterial NAD+-dependent DNA Ligase:Role of Motif Ⅳ in Ligation Catalysis

    Institute of Scientific and Technical Information of China (English)

    Hong FENG

    2007-01-01

    The bacterial DNA ligase as a multiple domain protein is involved in DNA replication, repair and recombination. Its catalysis of ligation can be divided into three steps. To delineate the roles of amino acid residues in motif Ⅳ in ligation catalysis, site-directed mutants were constructed in a bacterial NAD+-dependent DNA ligase from Thermus sp. TAK16D. It was shown that four conserved residues (D286, G287, V289 and K291) in motif Ⅳ had significant roles on the overall ligation. Under single turnover conditions, the observed apparent rates of D286E, G287A, V289I and K291R mutants were clearly reduced compared with that of WT ligase on both match and mismatch nicked substrates. The effects of D286E mutation on overall ligation may not only be ascribed to the third step. The G287A mutation has a major effect on the second step. The effects of V289I and K291R mutation on overall ligation are not on the third step, perhaps other aspects, such as conformation change of ligase protein in ligation catalysis, are involved. Moreover, the amino acid substitutions of above four residues were more sensitive on mismatch nicked substrate, indicating an enhanced ligation fidelity.

  3. Mitochondrial DNA A1555G mutation screening using a testing kit method and its significance in preventing aminoglycoside-related hearing loss

    Institute of Scientific and Technical Information of China (English)

    LIU Xin; YANG Weiyan; HAN Dongyi; JIN Zhengce; GUAN Minxin; DAI Pu; HUANG Deliang; YUAN Huijun; LI Weiming; YU Fei; ZHANG Xin; KANG Dongyang; CAO Juyang

    2006-01-01

    To report a new screening method for mitochondrial DNA 1555A→G mutation and the results of genotype analysis in 19 maternal inherited deafness pedigrees. Method Five hundred and forty-six non-syndromic neuro-sensory hearing loss patients were tested for 1555A→G mutation using a new compact testing kit, which allows clear distinction between wild type and 1555 A→G mutated mtDNAs. Results Nineteen subjects among the 546 patients (3.48%) were found to carry mtDNA A1555G mutation. The results were confirmed by sequencing in an ABI 3100 Avant sequencer. Conclusions Maternal inherited deafness families are a frequently seen in outpatient group. The detection ofmtDNA 1555 A→G mutation with a low cost, ready to use detection kit is needed and suitable in China for large scale screening and preventive testing before usage of aminoglycoside antibiotics.

  4. D-loop somatic mutations and ∼5 kb "common" deletion in mitochondrial DNA: important molecular markers to distinguish oral precancer and cancer.

    Science.gov (United States)

    Datta, Sayantan; Chattopadhyay, Esita; Ray, Jay Gopal; Majumder, Mousumi; Roy, Puspita Das; Roy, Bidyut

    2015-04-01

    Apart from genomic DNA, mutations at mitochondrial DNA (mtDNA) have been hypothesized to play vital roles in cancer development. In this study, ∼5 kb deletion and D-loop mutations in mtDNA and alteration in mtDNA content were investigated in buccal smears from 104 healthy controls and 74 leukoplakia and 117 cancer tissue samples using Taqman-based quantitative assay and re-sequencing. The ∼5 kb deletion in mtDNA was significantly less (9.8 and 10.5 folds, P mutations in D-loop, investigated in 54 controls, 50 leukoplakias and 56 cancer patients, were found to be significantly more in cancer tissues, but not in leukoplakia tissues, compared to control (Z-score = 5.4). MtDNA contents were observed to be significantly more in leukoplakia (2.1 folds, P = 0.004) and cancer (1.6 folds, P = 0.03) tissues compared to control tissues. So, D-loop somatic mutations and ∼5 kb deletion patterns could be used as distinguishing markers between precancer and cancer tissues. This observation further suggests that somatic mutations in D-loop may facilitate carcinogenesis and cancer cells with less ∼5 kb deletion, i.e., intact mtDNA, may become resistant to apoptosis. PMID:25527154

  5. Automated property optimization via ab initio O(N) elongation method: Application to (hyper-)polarizability in DNA

    Science.gov (United States)

    Orimoto, Yuuichi; Aoki, Yuriko

    2016-07-01

    An automated property optimization method was developed based on the ab initio O(N) elongation (ELG) method and applied to the optimization of nonlinear optical (NLO) properties in DNA as a first test. The ELG method mimics a polymerization reaction on a computer, and the reaction terminal of a starting cluster is attacked by monomers sequentially to elongate the electronic structure of the system by solving in each step a limited space including the terminal (localized molecular orbitals at the terminal) and monomer. The ELG-finite field (ELG-FF) method for calculating (hyper-)polarizabilities was used as the engine program of the optimization method, and it was found to show linear scaling efficiency while maintaining high computational accuracy for a random sequenced DNA model. Furthermore, the self-consistent field convergence was significantly improved by using the ELG-FF method compared with a conventional method, and it can lead to more feasible NLO property values in the FF treatment. The automated optimization method successfully chose an appropriate base pair from four base pairs (A, T, G, and C) for each elongation step according to an evaluation function. From test optimizations for the first order hyper-polarizability (β) in DNA, a substantial difference was observed depending on optimization conditions between "choose-maximum" (choose a base pair giving the maximum β for each step) and "choose-minimum" (choose a base pair giving the minimum β). In contrast, there was an ambiguous difference between these conditions for optimizing the second order hyper-polarizability (γ) because of the small absolute value of γ and the limitation of numerical differential calculations in the FF method. It can be concluded that the ab initio level property optimization method introduced here can be an effective step towards an advanced computer aided material design method as long as the numerical limitation of the FF method is taken into account.

  6. Automated property optimization via ab initio O(N) elongation method: Application to (hyper-)polarizability in DNA.

    Science.gov (United States)

    Orimoto, Yuuichi; Aoki, Yuriko

    2016-07-14

    An automated property optimization method was developed based on the ab initio O(N) elongation (ELG) method and applied to the optimization of nonlinear optical (NLO) properties in DNA as a first test. The ELG method mimics a polymerization reaction on a computer, and the reaction terminal of a starting cluster is attacked by monomers sequentially to elongate the electronic structure of the system by solving in each step a limited space including the terminal (localized molecular orbitals at the terminal) and monomer. The ELG-finite field (ELG-FF) method for calculating (hyper-)polarizabilities was used as the engine program of the optimization method, and it was found to show linear scaling efficiency while maintaining high computational accuracy for a random sequenced DNA model. Furthermore, the self-consistent field convergence was significantly improved by using the ELG-FF method compared with a conventional method, and it can lead to more feasible NLO property values in the FF treatment. The automated optimization method successfully chose an appropriate base pair from four base pairs (A, T, G, and C) for each elongation step according to an evaluation function. From test optimizations for the first order hyper-polarizability (β) in DNA, a substantial difference was observed depending on optimization conditions between "choose-maximum" (choose a base pair giving the maximum β for each step) and "choose-minimum" (choose a base pair giving the minimum β). In contrast, there was an ambiguous difference between these conditions for optimizing the second order hyper-polarizability (γ) because of the small absolute value of γ and the limitation of numerical differential calculations in the FF method. It can be concluded that the ab initio level property optimization method introduced here can be an effective step towards an advanced computer aided material design method as long as the numerical limitation of the FF method is taken into account. PMID:27421397

  7. Identification of DNA polymerase molecules repairing DNA irradiated damage and molecular biological study on modified factors of mutation rate

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Koichi; Inoue, Shuji [National Inst. of Healthand Nutrition, Tokyo (Japan)

    1999-02-01

    DNA repairing polymerase has not been identified in human culture cells because the specificities of enzyme inhibitors used in previous studies were not so high. In this study, anti-sense oligonucleotides were transfected into human fibroblast cells by electroporation and several clones selected by geneticin treatment were found to express the RNA of the incorporated DNA. However, the expression was not significant and its reproducibility was poor. Then, a study on repairing mechanism was made using XP30 RO and XP 115 LO cells which are variant cells of xeroderma pigmentosum, a human hereditary disease aiming to identify the DNA polymerase related to the disease. There were abnormalities in DNA polymerase subunit {delta} or {epsilon} which consists DNA replication complex. Thus, it was suggested that the DNA replication of these mutant cells might terminate at the site containing such abnormality. (M.N.)

  8. Identification of DNA polymerase molecules repairing DNA irradiated damage and molecular biological study on modified factors of mutation rate

    International Nuclear Information System (INIS)

    DNA repairing polymerase has not been identified in human culture cells because the specificities of enzyme inhibitors used in previous studies were not so high. In this study, anti-sense oligonucleotides were transfected into human fibroblast cells by electroporation and several clones selected by geneticin treatment were found to express the RNA of the incorporated DNA. However, the expression was not significant and its reproducibility was poor. Then, a study on repairing mechanism was made using XP30 RO and XP 115 LO cells which are variant cells of xeroderma pigmentosum, a human hereditary disease aiming to identify the DNA polymerase related to the disease. There were abnormalities in DNA polymerase subunit δ or ε which consists DNA replication complex. Thus, it was suggested that the DNA replication of these mutant cells might terminate at the site containing such abnormality. (M.N.)

  9. Genotypic Characterization of UL23 Thymidine Kinase and UL30 DNA Polymerase of Clinical Isolates of Herpes Simplex Virus: Natural Polymorphism and Mutations Associated with Resistance to Antivirals▿

    OpenAIRE

    Burrel, Sonia; Deback, Claire; Agut, Henri; Boutolleau, David

    2010-01-01

    The molecular mechanisms of herpes simplex virus (HSV) resistance to antiviral drugs interfering with viral DNA synthesis reported so far rely on the presence of mutations within UL23 (thymidine kinase [TK]) and UL30 (DNA polymerase) genes. The interpretation of genotypic antiviral resistance assay results requires the clear distinction between resistance mutations and natural interstrain sequence variations. The objectives of this work were to describe extensively the natural polymorphism of...

  10. Identification of a New Motif in Family B DNA Polymerases by Mutational Analyses of the Bacteriophage T4 DNA Polymerase

    OpenAIRE

    Li, Vincent; Hogg, Matthew; Reha-Krantz, Linda J.

    2010-01-01

    Structure-based protein sequence alignments of family B DNA polymerases revealed a conserved motif that is formed from interacting residues between loops from the N-terminal and palm domains and between the N-terminal loop and a conserved proline residue. The importance of the motif for function of the bacteriophage T4 DNA polymerase was revealed by suppressor analysis. T4 DNA polymerases that form weak replicating complexes cannot replicate DNA when the dGTP pool is reduced. The conditional ...

  11. Detection of DNA Aneuploidy in Exfoliated Airway Epithelia Cells of Sputum Specimens by the Automated Image Cytometry and Its Clinical Value in the Identification of Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    杨健; 周宜开

    2004-01-01

    To evaluate the value of detecton of DNA aneuploidy in exfoliated airway epithelia cells of sputum specimens by the automated image cytometry for the identification of lung cancer, 100patients were divided into patient group (50 patients with lung cancer)and control group (30 patients with tuberculosis and 20 healthy people). Sputum was obtained for the quantitative analysis of DNA content of exfoliated airway epithelial cells with the automated image cytometry, together with the examinations of brush cytology and conventional sputum cytology. Our results showed that DNA aneuploidy (DI>2.5 or 5c) was found in 20 out of 50 sputum samples of lung cancer, 1 out of 30 sputum samples from tuberculosis patients, and none of 20 sputum samples from healthy people. The positive rates of conventional sputum cytology and brush cytology were 16 % and 32 %,which was lower than that of DNA aneuploidy detection by the automated image cytometry (P<0.01 ,P>0.05). Our study showed that automated image cytometry, which uses DNA aneuploidy as a marker for tumor, can detect the malignant cells in sputum samples of lung cancer and it is a sensitive and specific method serving as a complement for the diagnosis of lung cancer.

  12. Molecular nature of X-ray-induced mutations compared with that of spontaneous ones in human c-hprt gene integrated into mammalian chromosomal DNA

    International Nuclear Information System (INIS)

    X-ray-induced mutations were analysed at molecular levels in comparison with spontaneous mutations. Altered sequences were determined tentatively of 30 independent X-ray-induced mutations in a cDNA of the human hprt gene which was integrated into mammalian chromosome as a part of a shuttle vector. Mutations consisted of base substitutions (37 %), frameshifts (27 %), deletions (27 %) and others (10 %). All these mutational events were distributed randomly over the gene without there being hot spots. The spectrum and distribution of X-ray-induced mutations resembled those of spontaneous mutations. Among base substitutions, transversions were predominant and base substitution mutations occurred more at A:T sites than at G:C sites, which is also the case in spontaneous mutations. Most of the frameshift and deletion mutations induced by X-rays, as well as those spontaneously arising, were characterized by the existence of short direct repeats of several identical bases in a row at the sites of the mutations. A slippage misalignment mechanism in replication well accounts for the generation of these classes of mutations. Judging from the data accumulated so far, it can be concluded that X-ray-induced mutations at molecular levels are similar to those spontaneously occurring. (author)

  13. Comparative synchronous fluorescence spectrophotometry and 32P-postlabeling analysis of PAH-DNA adducts in human lung and the relationship to TP53 mutations

    DEFF Research Database (Denmark)

    Andreassen, Åshild; Kure, Elin H.; Nielsen, Per Sabro; Autrup, Herman; Haugen, Aage

    )-DNA adducts detected by SFS and the BPDE co-migrating spot detected by 32P-postlabeling. We have also analyzed the relationship between adduct levels and TP53 mutations. By postlabeling diagonal radioactive zone (DRZ) adducts were detected in 37 of 39 (95%) lung tissues from lung cancer patients and the...... levels in lung tissue and TP53 mutations....

  14. High prevalence of impaired glucose homeostasis and myopathy in asymptomatic and oligosymptomatic 3243A>G mitochondrial DNA mutation-positive subjects

    DEFF Research Database (Denmark)

    Frederiksen, Anja Lisbeth; Jeppesen, Tina Dysgaard; Vissing, John;

    2009-01-01

    INTRODUCTION: The point mutation of 3243A>G mtDNA is the most frequent cause of mitochondrial diabetes, often presenting as the syndrome maternally inherited diabetes and deafness (MIDD). The mutation may also cause myopathy, ataxia, strokes, ophthalmoplegia, epilepsy, and cardiomyopathy in various...

  15. Homoplasmy of the G7444A mtDNA and heterozygosity of the GJB2 c.35delG mutations in a family with hearing loss

    DEFF Research Database (Denmark)

    Kokotas, Haris; Grigoriadou, Maria; Li, Yang;

    2011-01-01

    Mitochondrial mutations have been shown to be responsible for syndromic as well as non-syndromic hearing loss. The G7444A mitochondrial DNA mutation affects COI/the precursor of tRNA(Ser(UCN)), encoding the first subunit of cytochrome oxidase. Here we report on the first Greek family with the G74...

  16. Effects of carbonic anhydrase-related protein VIII on human cells harbouring an A8344G mitochondrial DNA mutation.

    Science.gov (United States)

    Wang, Tze-Kai; Cheng, Che-Kun; Chi, Tang-Hao; Ma, Yi-Shing; Wu, Shi-Bei; Wei, Yau-Huei; Hsieh, Mingli

    2014-04-01

    MERRF (myoclonus epilepsy associated with ragged-red fibres) is a maternally inherited mitochondrial encephalomyopathy with various syndromes involving both muscular and nervous systems. The most common mutation in MERRF syndrome, the A8344G mutation in mtDNA, has been associated with severe defects in the respiratory function of mitochondria. In the present study, we show that there is a significant decrease in CA8 (carbonic anhydrase-related protein VIII) in cybrids harbouring the MERRF A8344G mutation. CA8 deficiency and mutations were found to be associated with a distinctive lifelong gait disorder in wdl (Waddles) mice and novel syndromes characterized by cerebellar ataxia and mental retardation in humans. The results of the present study showed that overexpression of CA8 in MERRF cybrids significantly decreased cell death induced by STS (staurosporine) treatment, suggesting a protective function of CA8 in cells harbouring the A8344G mutation of mtDNA. Interestingly, an increase in the formation of LC3-II (microtubule-associated protein 1 light chain 3-II) was found in the cybrids with down-regulated CA8 expression, suggesting that reduced expression of CA8 leads to autophagy activation. Furthermore, cybrids exhibiting down-regulated CA8 showed increased cytosolic Ca2+ signals and reduced levels of phospho-Akt compared with those in the cybrids with overexpressed CA8, indicating that phospho-Akt is involved in the protection of cells by CA8. Our findings suggest that CA8 is involved in the autophagic pathway and may have a protective role in cultured cells from patients with MERRF. Targeting CA8 and the downstream autophagic pathway might help develop therapeutic agents for treatment of MERRF syndrome in the future. PMID:24476000

  17. Negative effect of the M184V mutation in HIV-1 reverse transcriptase on initiation of viral DNA synthesis

    International Nuclear Information System (INIS)

    The M184V mutation in HIV reverse transcriptase (RT) is associated with high-level resistance against the nucleoside inhibitor lamivudine as well as diminished viral replication capacity. We have previously demonstrated that HIV variants containing the M184V mutation were relatively unable to successfully undergo compensatory mutagenesis following deletion of an A-rich loop located upstream of the primer binding site (PBS). To understand the mechanisms involved, we synthesized viral RNA templates containing different compensatory mutations that were emergent during the long-term culture of the A-rich loop-deleted viruses. These templates were then used in cell-free reverse transcription initiation assays and in tRNA primer placement assays performed with either recombinant wild-type RT or recombinant RT containing the M184V substitution. The results showed that the RNA template that contained the A-rich loop deletion was impaired in ability to initiate reverse transcription and that the presence of the M184V substitution in RT amplified this effect. Clearance from pausing at position +3 during synthesis of viral DNA was identified as a sensitive step in this reaction that could not be efficiently bypassed with the M184V mutant enzyme. Increased efficiency of initiation was seen with the deleted RNA templates that also contained mutations identified in the revertant viruses, provided that these mutations facilitated formation of a competent binary tRNA/RNA complex. These findings provide biochemical evidence that initiation of tRNALys3-primed DNA synthesis is an important rate-limiting step in reverse transcription that correlates with viral replication fitness

  18. Mutational analysis of Mycobacterium UvrD1 identifies functional groups required for ATP hydrolysis, DNA unwinding, and chemomechanical coupling.

    Science.gov (United States)

    Sinha, Krishna Murari; Glickman, Michael S; Shuman, Stewart

    2009-05-19

    Mycobacterial UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3' to 5' DNA helicase. The UvrD1 motor domain resembles that of the prototypal superfamily I helicases UvrD and PcrA. Here we performed a mutational analysis of UvrD1 guided by the crystal structure of a DNA-bound Escherichia coli UvrD-ADP-MgF(3) transition state mimetic. Alanine scanning and conservative substitutions identified amino acids essential for both ATP hydrolysis and duplex unwinding, including those implicated in phosphohydrolase chemistry via transition state stabilization (Arg308, Arg648, Gln275), divalent cation coordination (Glu236), or activation of the nucleophilic water (Glu236, Gln275). Other residues important for ATPase/helicase activity include Phe280 and Phe72, which interact with the DNA 3' single strand tail. ATP hydrolysis was uncoupled from duplex unwinding by mutations at Glu609 (in helicase motif V), which contacts the ATP ribose sugar. Introducing alanine in lieu of the adenine-binding "Q motif" glutamine (Gln24) relaxed the substrate specificity in NTP hydrolysis, e.g., eliciting a gain of function as a UTPase/TTPase, although the Q24A mutant still relied on ATP/dATP for duplex unwinding. Our studies highlight the role of the Q motif as a substrate filter and the contributions of adenosine-binding residues as couplers of NTP hydrolysis to motor activity. The Ku-binding function of UvrD1 lies within its C-terminal 270 amino acid segment. Here we found that deleting the 90 amino acid C-terminal domain, which is structurally uncharacterized, diminished DNA unwinding, without affecting ATP hydrolysis or binding to the DNA helicase substrate, apparently by affecting the strength of the UvrD1-Ku interaction. PMID:19317511

  19. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair

    International Nuclear Information System (INIS)

    C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair

  20. Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase η from Arabidopsis thaliana

    International Nuclear Information System (INIS)

    DNA polymerase η belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Polη homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Polη activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates

  1. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

    Science.gov (United States)

    Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

    2015-01-01

    Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent “gold standard”. Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients. PMID:26562020

  2. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

    Directory of Open Access Journals (Sweden)

    Julia Stadler

    Full Text Available Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution, at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

  3. MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR

    Institute of Scientific and Technical Information of China (English)

    Jian-yong WANG; Yang-shun GU; Jing WANG; Yi TONG; Ying WANG; Jun-bing SHAO; Ming QI

    2008-01-01

    Objective:Leber's hereditary optic neuropathY (LHON)is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA(mtDNA).Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing.This study aims to develop a minor groove binder(MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction(PCR).Methods:Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation,with 20 normal individuals as a control group at the same time.A real-time PCR involving two MGB probes was used to detect the mtDNA 11778 mutation and heteroplasmy.A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones.Results:All 48 LHON patients and their matemal relatives were positive for mtDNA 11778 mutation in our assay,27 heteroplasmic and 21 homoplasmic.Eighteen cases did not show an occurrence of the disease,while 9 developed the disease among the 27 heteroplasmic mutation cases.Eleven did not show an occurrence of the disease,while 10 cases developed the disease among 21 homoplasmic mutation cases.There was a significant difierence in the incidence between the heteroplasmic and the homoplasmic mutation types.The time needed for running a real-time PCR assay was only 80 min.Conclusion:This real-time PCR assay is a rapid,reliable method for mtDNA mutation detection as well as heteroplasmy quantification.Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.

  4. Prevalence of Germline Mutations in Genes Engaged in DNA Damage Repair by Homologous Recombination in Patients with Triple-Negative and Hereditary Non-Triple-Negative Breast Cancers.

    Directory of Open Access Journals (Sweden)

    Pawel Domagala

    Full Text Available This study sought to assess the prevalence of common germline mutations in several genes engaged in the repair of DNA double-strand break by homologous recombination in patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers. Tumors deficient in this type of DNA damage repair are known to be especially sensitive to DNA cross-linking agents (e.g., platinum drugs and to poly(ADP-ribose polymerase (PARP inhibitors.Genetic testing was performed for 36 common germline mutations in genes engaged in the repair of DNA by homologous recombination, i.e., BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D, in 202 consecutive patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers.Thirty five (22.2% of 158 patients in the triple-negative group carried mutations in genes involved in DNA repair by homologous recombination, while 10 (22.7% of the 44 patients in the hereditary non-triple-negative group carried such mutations. Mutations in BRCA1 were most frequent in patients with triple-negative breast cancer (18.4%, and mutations in CHEK2 were most frequent in patients with hereditary non-triple-negative breast cancers (15.9%. In addition, in the triple-negative group, mutations in CHEK2, NBN, and ATM (3.8% combined were found, while mutations in BRCA1, NBN, and PALB2 (6.8% combined were identified in the hereditary non-triple-negative group.Identifying mutations in genes engaged in DNA damage repair by homologous recombination other than BRCA1/2 can substantially increase the proportion of patients with triple-negative breast cancer and hereditary non-triple-negative breast cancer who may be eligible for therapy using PARP inhibitors and platinum drugs.

  5. A rapid method for sequencing of rRNA gene(s) amplified by polymerase chain reaction using an automated DNA sequencer

    OpenAIRE

    Dwivedi, P.P.; Patel, B.K.C.; Rees, G.N.; Ollivier, Bernard

    1996-01-01

    A method for DNA sequencing of ribosomal RNA (rRNA) genes, amplified by polymerase chain reaction (PCR), using internal primers, designed on the basis of conserved regions of rRNA genes for determining a near complete sequence (99%) of the gene using an automated DNA sequencer (Applied Biosystem Incorporation, USA) is described. The procedure is extremely rapid as cloning of the gene is not required for sequence determination. In addition time consuming steps such as ethanol precipitation and...

  6. Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection

    Directory of Open Access Journals (Sweden)

    Frey Daniel

    2010-08-01

    Full Text Available Abstract Background Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products. Results Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in E.coli cells. Variable tags are available both in N-terminal or C-terminal position. A newly developed β-lactamase (ΔW290 selection cassette contains a segment inside the β-lactamase open reading frame encoding a stretch of hydrophilic amino acids that result in a T7 promoter when back-translated. This position of the promoter permits positive selection and attenuated expression of fusion proteins with C-terminal tags. We have tested eight vectors by inserting six target sequences of variable length, provenience and function. The target proteins were cloned, expressed and detected using an automated Tecan Freedom Evo II liquid handling work station. Only two colonies had to be picked to score with 85% correct inserts while 80% of those were positive in expression tests. Conclusions Our results establish co-transformation and positive/negative selection cloning in conjunction with the provided vectors and selection cassettes as an automatable alternative to commercialized high-throughput cloning systems like Gateway® or ligase-independent cloning (LIC .

  7. Response to immunotherapy in a patient with adult onset Leigh syndrome and T9176C mtDNA mutation.

    Science.gov (United States)

    Chuquilin, Miguel; Govindarajan, Raghav; Peck, Dawn; Font-Montgomery, Esperanza

    2016-09-01

    Leigh syndrome is a mitochondrial disease caused by mutations in different genes, including ATP6A for which no known therapy is available. We report a case of adult-onset Leigh syndrome with response to immunotherapy. A twenty year-old woman with baseline learning difficulties was admitted with progressive behavioral changes, diplopia, headaches, bladder incontinence, and incoordination. Brain MRI and PET scan showed T2 hyperintensity and increased uptake in bilateral basal ganglia, respectively. Autoimmune encephalitis was suspected and she received plasmapheresis with clinical improvement. She was readmitted 4 weeks later with dysphagia and aspiration pneumonia. Plasmapheresis was repeated with resolution of her symptoms. Given the multisystem involvement and suggestive MRI changes, genetic testing was done, revealing a homoplasmic T9176C ATPase 6 gene mtDNA mutation. Monthly IVIG provided clinical improvement with worsening when infusions were delayed. Leigh syndrome secondary to mtDNA T9176C mutations could have an autoimmune mechanism that responds to immunotherapy. PMID:27408822

  8. Mutations in mitochondrial DNA associated with hypertension%高血压相关的线粒体DNA突变

    Institute of Scientific and Technical Information of China (English)

    薛凌; 陈红; 孟燕子; 王燕; 卢中秋; 吕建新; 管敏鑫

    2011-01-01

    线粒体DNA(mtDNA)突变是高血压发病的分子机制之一.已经报道的与原发性高血压相关的mtDNA突变包括:tRNAMetA4435G,tRNAMet/tRNAGln A4401G,tRNAIleA4263G,T4291C和A4295G突变.这些高血压相关的mtDNA突变改变了相应的线粒体tRNA的结构,导致线粒体tRNA的代谢障碍.而线粒体tRNAs的代谢缺陷则影响蛋白质合成,造成氧化磷酸化缺陷,降低ATP的合成,增加活性氧的产生.因此,线粒体的功能缺陷可能在高血压的发生发展中起一定的作用.mtDNA突变发病的组织特异性则可能与线粒体tRNAs的代谢以及核修饰基因相关.目前发现的这些高血压相关的mtDNA突变则应该作为今后高血压诊断的遗传风险因子,高血压相关的线粒体功能缺陷的深入研究也将进一步诠释母系遗传高血压的分子致病机制,为高血压的预防、控制和治疗提供依据,文章对高血压相关的mtDNA突变进行了综述.%Mutations in mitochondria! DNA (mtDNA) are one of the molecular bases of hypertension. Among these, the tKNAMel A4435G, tRNAMe7tRNAGto A4401Q tRNA11' A4263Q T4291C and A4295G mutations have been reported to be associated with essential hypertension. These mutations alter the structure of the corresponding mitochondrial tRNAs and cause failures in tRNA metabolism. These shortages of these tRNAs lead to an impairment of mitochondrial protein synthesis and a failure in the oxidative phosphorylation function. These result in a deficit in ATP synthesis and an increase of generation of reactive oxygen species. As a result, these mitochondrial dysfunctions may contribute to the development of hypertension. Furthermore, the tissue specificity of these pathogenic mtDNA mutations might be associated with tRNA metabolism and nuclear modifier genes. These mtDNA mutations should be considered as inherited risk factors for future molecular diagnosis. Thus, these findings provide new insights into the molecular mechanism, management and

  9. Comparative Evaluation of a Commercially Available Automated System for Extraction of Viral DNA from Whole Blood: Application to Monitoring of Epstein-Barr Virus and Cytomegalovirus Load ▿

    OpenAIRE

    Pillet, Sylvie; Bourlet, Thomas; Pozzetto, Bruno

    2009-01-01

    The NucliSENS easyMAG automated system was compared to the column-based Qiagen method for Epstein-Barr virus (EBV) or cytomegalovirus (CMV) DNA extraction from whole blood before viral load determination using the corresponding R-gene amplification kits. Both extraction techniques exhibited a total agreement of 81.3% for EBV and 87.2% for CMV.

  10. Mutation patterns of mtDNA : empirical inferences for the coding region

    OpenAIRE

    Santos, Cristina; Montiel, Rafael; Arruda, Adriana; Álvarez, Luis; Aluja, Ma. Pilar; Lima, Manuela

    2008-01-01

    Background: Human mitochondrial DNA (mtDNA) has been extensively used in population and evolutionary genetics studies. Thus, a valid estimate of human mtDNA evolutionary rate is important in many research fields. The small number of estimations performed for the coding region of the molecule, showed important differences between phylogenetic and empirical approaches. We analyzed a portion of the coding region of mtDNA (tRNALeu, ND1 and tRNAIle genes), using individuals belonging to extended f...

  11. Mutation patterns of mtDNA: Empirical inferences for the coding region

    OpenAIRE

    Alvarez Luis; Arruda Adriana; Montiel Rafael; Santos Cristina; Aluja Maria; Lima Manuela

    2008-01-01

    Abstract Background Human mitochondrial DNA (mtDNA) has been extensively used in population and evolutionary genetics studies. Thus, a valid estimate of human mtDNA evolutionary rate is important in many research fields. The small number of estimations performed for the coding region of the molecule, showed important differences between phylogenetic and empirical approaches. We analyzed a portion of the coding region of mtDNA (tRNALeu, ND1 and tRNAIle genes), using individuals belonging to ex...

  12. Two families with Leber's hereditary optic neuropathy carrying G11778A and T14502C mutations with haplogroup H2a2a1 in mitochondrial DNA.

    Science.gov (United States)

    Qiao, Chen; Wei, Tanwei; Hu, Bo; Peng, Chunyan; Qiu, Xueping; Wei, Li; Yan, Ming

    2015-08-01

    The mitochondrial haplogroup has been reported to affect the clinical expression of Leber's hereditary optic neuropathy (LHON). The present study aimed to investigate the interaction between mutations and the haplogroup of mitochondrial DNA (mtDNA) in families. Two unrelated families with LHON were enrolled in the study, and clinical, genetic and molecular characterizations were determined in the affected and unaffected family members. Polymerase chain reaction direct sequencing was performed using 24 pairs of overlapping primers for whole mtDNA to screen for mutations and haplogroup. Bioinformatics analysis was performed to evaluate the pathogenic effect of these mtDNA mutations and the haplogroup. The G11778A mutation was identified in the two families. In addition, the members of family 2 exhibited the T14502C mutation and those in family 1 exhibited the T3394C and T14502C mutations, which were regarded as secondary mutations. The penetrance of visual loss in families 1 and 2 were 30.8 and 33.3%, respectively. In addition, the two families were found to be in the H2a2a1 haplogroup. In this limited sample size, it was demonstrated that the H2a2a1 haplogroup had a possible protective effect against LHON. Additional modifying factors, including environmental factors, lifestyle, estrogen levels and nuclear genes may also be important in LHON. PMID:25936877

  13. Protective effect of curcumin and chlorophyllin against DNA mutation induced by cyclophosphamide or benzo[a]pyrene

    Energy Technology Data Exchange (ETDEWEB)

    Ibrahim, M.A.; Elbehairy, A.M.; Ghoneim, M.A.; Amer, H.A. [Cairo Univ., Giza (Egypt). Biochemistry Dept. and Biotechnology Center

    2007-03-15

    The current study was carried out to evaluate the potency of curcumin and chlorophyllin as natural antioxidants to reduce the oxidative stress markers induced by cyclophosphamide (CP) and benzo[a]pyrene [B(a)P] which were used as free radical inducers. For this purpose, 126 male albino rats were used. The animals were assigned into 4 main groups: negative control group; oxidant-treated group (subdivided into two subgroups: cyclophosphamide- treated group and benzo[a]pyrene-treated group); curcumin-treated group; and chlorophyllin-treated group. Liver samples were collected after two days post the oxidant inoculation and at the end of the experimental period (10 weeks). These samples were examined for determination of liver microsomal malondialdehyde (MDA), DNA fragmentation, restriction fragment length polymorphism (RFLP) and 8-hydroxy deoxyguanosine (8-OHdG) concentration. Both CP and B(a)P caused increments in DNA fragmentation percentages, liver microsomal MDA, concentration of 8-OHdG and induced point mutation. Treatment of rats with either curcumin or chlorophyllin revealed lower DNA fragmentation percentages, liver microsomal MDA concentration, concentration of 8-OHdG and prevented induction of mutations, i. e., reversed the oxidative stress induced by CP and B(a)P and proved that they were capable of protecting rats against the oxidative damage evoked by these oxidants. (orig.)

  14. The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

    Science.gov (United States)

    Jiang, Pingping; Jin, Xiaofen; Peng, Yanyan; Wang, Meng; Liu, Hao; Liu, Xiaoling; Zhang, Zengjun; Ji, Yanchun; Zhang, Juanjuan; Liang, Min; Zhao, Fuxin; Sun, Yan-Hong; Zhang, Minglian; Zhou, Xiangtian; Chen, Ye; Mo, Jun Qin; Huang, Taosheng; Qu, Jia; Guan, Min-Xin

    2016-02-01

    Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disorder. Nuclear modifier genes are proposed to modify the phenotypic expression of LHON-associated mitochondrial DNA (mtDNA) mutations. By using an exome sequencing approach, we identified a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene encoding mitochondrial tyrosyl-tRNA synthetase, which interacts with m.11778G>A mutation to cause visual failure. We performed functional assays by using lymphoblastoid cell lines derived from members of Chinese families (asymptomatic individuals carrying m.11778G>A mutation, or both m.11778G>A and heterozygous p.191Gly>Val mutations and symptomatic subjects harboring m.11778G>A and homozygous p.191Gly>Val mutations) and controls lacking these mutations. The 191Gly>Val mutation reduced the YARS2 protein level in the mutant cells. The aminoacylated efficiency and steady-state level of tRNA(Tyr) were markedly decreased in the cell lines derived from patients both carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The failure in tRNA(Tyr) metabolism impaired mitochondrial translation, especially for polypeptides with high content of tyrosine codon such as ND4, ND5, ND6 and COX2 in cells lines carrying homozygous YARS2 p.191Gly>Val and m.11778G>A mutations. The YARS2 p.191Gly>Val mutation worsened the respiratory phenotypes associated with m.11778G>A mutation, especially reducing activities of complexes I and IV. The respiratory deficiency altered the efficiency of mitochondrial ATP synthesis and increased the production of reactive oxygen species. Thus, mutated YARS2 aggravates mitochondrial dysfunctions associated with the m.11778G>A mutation, exceeding the threshold for the expression of blindness phenotype. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between mtDNA mutation and mutated nuclear-modifier YARS2. PMID:26647310

  15. A novel, double mutation in DNA gyrase A of Escherichia coli conferring resistance to quinolone antibiotics.

    OpenAIRE

    Truong, Q C; Nguyen Van, J C; Shlaes, D; Gutmann, L; Moreau, N J

    1997-01-01

    A spontaneous Escherichia coli mutant, named Q3, resistant to nalidixic acid was obtained from a previously described clinical isolate of E. coli, Q2, resistant to fluoroquinolones but susceptible to nalidixic acid (E. Cambau, F. Bordon, E. Collatz, and L. Gutmann, Antimicrob. Agents Chemother. 37:1247-1252, 1993). Q3 harbored the mutation Asp82Gly in addition to the Gly81Asp mutation of Q2. The different mutations leading to Gly81Asp, Asp82Gly, and Gly81AspAsp82Gly were introduced into the g...

  16. Translesion DNA polymerases Pol , Pol , Pol , Pol and Rev1 are not essential for repeat-induced point mutation in Neurospora crassa

    Indian Academy of Sciences (India)

    Ranjan Tamuli; C Ravindran; Durgadas P Kasbekar

    2006-12-01

    Pol , Pol , Pol , Pol and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. We show however, that the translesion polymerases are dispensable for repeat-induced point mutation (RIP) in Neurospora crassa. Additionally, we demonstrate that the upr-1 gene, which encodes the catalytic subunit of Pol , is a highly polymorphic locus in Neurospora.

  17. A comparative study on low-energy ion beam and neutralized beam modifications of naked DNA and biological effect on mutation

    Energy Technology Data Exchange (ETDEWEB)

    Sarapirom, S.; Thongkumkoon, P.; Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Anuntalabhochai, S. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, L.D., E-mail: yuld@fnrf.science.cmu.ac.th [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand)

    2012-02-01

    DNA conformation change or damage induced by low-energy ion irradiation has been of great interest owing to research developments in ion beam biotechnology and ion beam application in biomedicine. Mechanisms involved in the induction of DNA damage may account for effect from implanting ion charge. In order to check this effect, we used both ion beam and neutralized beam at keV energy to bombard naked DNA. Argon or nitrogen ion beam was generated and extracted from a radiofrequency (RF) ion source and neutralized by microwave-driven plasma in the beam path. Plasmid DNA pGFP samples were irradiated with the ion or neutralized beam in vacuum, followed by gel electrophoresis to observe changes in the DNA conformations. It was revealed that the ion charge played a certain role in inducing DNA conformation change. The subsequent DNA transfer into bacteria Escherichia coli (E. coli) for mutation analysis indicated that the charged ion beam induced DNA change had high potential in mutation induction while neutralized beam did not. The intrinsic reason was attributed to additional DNA deformation and contortion caused by ion charge exchange effect so that the ion beam induced DNA damage could hardly be completely repaired, whereas the neutralized beam induced DNA change could be more easily recoverable owing to absence of the additional DNA deformation and contortion.

  18. Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

    Energy Technology Data Exchange (ETDEWEB)

    Holland, P.; Hollis, T

    2010-01-01

    In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a 'searching' mode and 'repair' mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  19. Structural and mutational analysis of Escherichia coli AlkB provides insight into substrate specificity and DNA damage searching.

    Directory of Open Access Journals (Sweden)

    Paul J Holland

    Full Text Available BACKGROUND: In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG iron(II dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA and 3-methylcytosine (3-meC lesions, but it also repairs 1-methylguanine (1-meG and 3-methylthymine (3-meT at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. METHODOLOGY/PRINCIPAL FINDINGS: We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. CONCLUSIONS/SIGNIFICANCE: A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a "searching" mode and "repair" mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  20. ATRX mutations and glioblastoma: Impaired DNA damage repair, alternative lengthening of telomeres, and genetic instability.

    Science.gov (United States)

    Koschmann, Carl; Lowenstein, Pedro R; Castro, Maria G

    2016-05-01

    Alpha thalassemia/mental retardation syndrome X-linked (ATRX) is mutated in nearly a third of pediatric glioblastoma (GBM) patients. We developed an animal model of ATRX-deficient GBM. Using this model combined with analysis of multiple human glioma genome-wide datasets, we determined that ATRX mutation leads to genetic instability, impaired non-homologous end joining, and alternate lengthening of telomeres (ALT). PMID:27314101

  1. CpG dinucleotides and the mutation rate of non-CpG DNA

    OpenAIRE

    Walser, Jean-Claude; Ponger, Loïc; Furano, Anthony V

    2008-01-01

    The neutral mutation rate is equal to the base substitution rate when the latter is not affected by natural selection. Differences between these rates may reveal that factors such as natural selection, linkage, or a mutator locus are affecting a given sequence. We examined the neutral base substitution rate by measuring the sequence divergence of ∼30,000 pairs of inactive orthologous L1 retrotransposon sequences interspersed throughout the human and chimpanzee genomes. In contrast to other st...

  2. Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

    OpenAIRE

    Jesus Gonzalez-Bosquet; Jacob Calcei; Wei, Jun S.; Montserrat Garcia-Closas; Sherman, Mark E.; Stephen Hewitt; Joseph Vockley; Jolanta Lissowska; Yang, Hannah P.; Javed Khan; Stephen Chanock

    2011-01-01

    Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounde...

  3. Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples.

    Science.gov (United States)

    Stangegaard, Michael; Frøslev, Tobias G; Frank-Hansen, Rune; Hansen, Anders J; Morling, Niels

    2011-04-01

    We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Männedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpFℓSTR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid handler leading to the reduction of manual work, and increased quality and throughput. PMID:21609694

  4. 5-hydroxymethylcytosine marks regions with reduced mutation frequency in human DNA

    Science.gov (United States)

    Tomkova, Marketa; McClellan, Michael; Kriaucionis, Skirmantas; Schuster-Boeckler, Benjamin

    2016-01-01

    CpG dinucleotides are the main mutational hot-spot in most cancers. The characteristic elevated C>T mutation rate in CpG sites has been related to 5-methylcytosine (5mC), an epigenetically modified base which resides in CpGs and plays a role in transcription silencing. In brain nearly a third of 5mCs have recently been found to exist in the form of 5-hydroxymethylcytosine (5hmC), yet the effect of 5hmC on mutational processes is still poorly understood. Here we show that 5hmC is associated with an up to 53% decrease in the frequency of C>T mutations in a CpG context compared to 5mC. Tissue specific 5hmC patterns in brain, kidney and blood correlate with lower regional CpG>T mutation frequency in cancers originating in the respective tissues. Together our data reveal global and opposing effects of the two most common cytosine modifications on the frequency of cancer causing somatic mutations in different cell types. DOI: http://dx.doi.org/10.7554/eLife.17082.001 PMID:27183007

  5. Control region mutations and the 'common deletion' are frequent in the mitochondrial DNA of patients with esophageal squamous cell carcinoma

    International Nuclear Information System (INIS)

    North central China has some of the highest rates of esophageal squamous cell carcinoma in the world with cumulative mortality surpassing 20%. Mitochondrial DNA (mtDNA) accumulates more mutations than nuclear DNA and because of its high abundance has been proposed as a early detection device for subjects with cancer at various sites. We wished to examine the prevalence of mtDNA mutation and polymorphism in subjects from this high risk area of China. We used DNA samples isolated from tumors, adjacent normal esophageal tissue, and blood from 21 esophageal squamous cell carcinoma cases and DNA isolated from blood from 23 healthy persons. We completely sequenced the control region (D-Loop) from each of these samples and used a PCR assay to assess the presence of the 4977 bp common deletion. Direct DNA sequencing revealed that 7/21 (33%, 95% CI = 17–55%) tumor samples had mutations in the control region, with clustering evident in the hyper-variable segment 1 (HSV1) and the homopolymeric stretch surrounding position 309. The number of mutations per subject ranged from 1 to 16 and there were a number of instances of heteroplasmy. We detected the 4977 bp 'common deletion' in 92% of the tumor and adjacent normal esophageal tissue samples examined, whereas no evidence of the common deletion was found in corresponding peripheral blood samples. Control region mutations were insufficiently common to warrant attempts to develop mtDNA mutation screening as a clinical test for ESCC. The common deletion was highly prevalent in the esophageal tissue of cancer cases but absent from peripheral blood. The potential utility of the common deletion in an early detection system will be pursued in further studies

  6. Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice

    Science.gov (United States)

    Araki, Ryoko; Fujimori, Akira; Hamatani, Kiyohiro; Mita, Kazuei; Saito, Toshiyuki; Mori, Masahiko; Fukumura, Ryutaro; Morimyo, Mitsuoki; Muto, Masahiro; Itoh, Masahiro; Tatsumi, Kouichi; Abe, Masumi

    1997-01-01

    The severe combined immune deficiency (SCID) mouse was reported as an animal model for human immune deficiency. Through the course of several studies, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene came to be considered a candidate for the SCID-responsible gene. We isolated an ORF of the murine DNA-PKcs gene from SCID mice and their parent strain C.B-17 mice and determined the DNA sequences. The ORF of the murine DNA-PKcs gene contained 4128-aa residues and had 78.9% homology with the human DNA-PKcs gene. A particularly important finding is that a T to A transversion results in the substitution of termination codon in SCID mice for the Tyr-4046 in C.B-17 mice. No other mutation was detected in the ORF of the gene. The generality of this transversion was confirmed using four individual SCID and wild-type mice. The substitution took place in the phosphatidylinositol 3-kinase domain, and the mutated gene encodes the truncated products missing 83 residues of wild-type DNA-PKcs products. Furthermore, the quantity of DNA-PKcs transcript in wild-type and SCID cells was almost equal. These observations indicate that the DNA-PKcs gene is the SCID-responsible gene itself and that the detected mutation leads to the SCID aberration. PMID:9122213

  7. Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

    Energy Technology Data Exchange (ETDEWEB)

    Dinsart, C.; Cornelis, J.J.; Klein, B.; van der Eb, A.J.; Rommelaere, J.

    1984-02-01

    Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.

  8. Does Ataxia Telangiectasia Mutated (ATM) protect testicular and germ cell DNA integrity by regulating the redox status?

    Science.gov (United States)

    Godschalk, Roger W L; Vanhees, Kimberly; Maas, Lou; Drittij, Marie-Jose; Pachen, Daniëlle; van Doorn-Khosrovani, Sahar van Waalwijk; van Schooten, Frederik J; Haenen, Guido R M M

    2016-08-01

    A balanced redox homeostasis in the testis is essential for genetic integrity of sperm. Reactive oxygen species can disturb this balance by oxidation of glutathione, which is regenerated using NADPH, formed by glucose-6-phosphate dehydrogenase (G6PDH). G6PDH is regulated by the Ataxia Telangiectasia Mutated (Atm) protein. Therefore, we studied the redox status and DNA damage in testes and sperm of mice that carried a deletion in Atm. The redox status in heterozygote mice, reflected by glutathione levels and antioxidant capacity, was lower than in wild type mice, and in homozygotes the redox status was even lower. The redox status correlated with oxidative DNA damage that was highest in mice that carried Atm deletions. Surprisingly, G6PDH activity was highest in homozygotes carrying the deletion. These data indicate that defective Atm reduces the redox homeostasis of the testis and genetic integrity of sperm by regulating glutathione levels independently from G6PDH activity. PMID:27318254

  9. Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

    International Nuclear Information System (INIS)

    The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in different affected pedigrees. Here we describe two large Chinese families that show a striking difference in the penetrance of LHON, in which 53.3% and 15.0% of members were affected (P < 0.02), respectively. Analysis of the complete mtDNA genome of the two families revealed the presence of the primary mutation G11778A and several other variants suggesting the same haplogroup status G2a. The family with higher penetrance contained a previously described secondary mutation G13708A, which presents a polymorphism in normal Chinese samples and does not affect in vivo mitochondrial oxidative metabolism as described in a previous study. Evolutionary analysis failed to indicate any putatively pathogenic mutation that cosegregated with G11778A in these two pedigrees. Our results suggest that the variable penetrance of LHON in the two Chinese families is independent of both their mtDNA haplotype background and a secondary mutation G13708A. As a result, it is likely that unknown nuclear gene involvement and/or other factors contribute to the strikingly different penetrance of LHON

  10. Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

    Energy Technology Data Exchange (ETDEWEB)

    Wang Huawei [Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)]|[Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091 (China); Jia Xiaoyun; Ji Yanli [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060 (China); Kong Qingpeng [State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China); Zhang Qingjiong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060 (China)], E-mail: qingjiongzhang@yahoo.com; Yao Yonggang [Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)]|[State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China)], E-mail: ygyaozh@yahoo.com; Zhang Yaping [Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091 (China)]|[State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China)

    2008-08-25

    The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in different affected pedigrees. Here we describe two large Chinese families that show a striking difference in the penetrance of LHON, in which 53.3% and 15.0% of members were affected (P < 0.02), respectively. Analysis of the complete mtDNA genome of the two families revealed the presence of the primary mutation G11778A and several other variants suggesting the same haplogroup status G2a. The family with higher penetrance contained a previously described secondary mutation G13708A, which presents a polymorphism in normal Chinese samples and does not affect in vivo mitochondrial oxidative metabolism as described in a previous study. Evolutionary analysis failed to indicate any putatively pathogenic mutation that cosegregated with G11778A in these two pedigrees. Our results suggest that the variable penetrance of LHON in the two Chinese families is independent of both their mtDNA haplotype background and a secondary mutation G13708A. As a result, it is likely that unknown nuclear gene involvement and/or other factors contribute to the strikingly different penetrance of LHON.

  11. MUTATIONS THAT PROBE THE COOPERATIVE ASSEMBLY OF O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE (AGT) COMPLEXES†

    OpenAIRE

    Adams, Claire A.; Fried, Michael G.

    2011-01-01

    O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts present in DNA that has been exposed to alkylating agents. AGT binds DNA cooperatively and models of cooperative complexes predict that residues 1–7 of one protein molecule and 163–169 of a neighboring protein are closely juxtaposed. To test these models we used directed mutagenesis to substitute triplets of alanine for triplets of native residues across these two sequences. Six of eight d...

  12. Mitochondrial DNA mutations. Brain developmental and ageing consequences, and possible treatments

    OpenAIRE

    Ross, Jaime M.

    2012-01-01

    Ageing is a complex process that involves cellular senescence, a gradual loss of tissue homeostasis, and decline in organ function. Abundant evidence implicates mitochondria in ageing suggesting: (i) accumulation of mitochondrial DNA (mtDNA) damage, (ii) progressive respiratory chain dysfunction, and (iii) increased reactive oxygen species production. The “Mitochondrial Theory of Aging”, first proposed by Denham Harman in 1972, suggests that damage to mtDNA slowly accumulates with time and ca...

  13. Does tumorigenesis select for or against mutations of the DNA repair-associated genes BRCA2 and MRE11?: Considerations from somatic mutations in microsatellite unstable (MSI gastrointestinal cancers

    Directory of Open Access Journals (Sweden)

    Elghalbzouri-Maghrani Elhaam

    2006-01-01

    Full Text Available Abstract Background The BRCA2 and MRE11 proteins participate in the repair of double-strand DNA breaks by homologous recombination. Germline BRCA2 mutations predispose to ovarian, breast and pancreatic cancer, while a germline MRE11 mutation is associated with an ataxia telangiectasia-like disorder. Somatic mutations of BRCA2 are rare in typical sporadic cancers. In tumors having microsatellite instability (MSI, somatic truncating mutations in a poly [A] tract of BRCA2 are reported on occasion. Results We analyzed gastrointestinal MSI cancers by whole gene BRCA2 sequencing, finding heterozygous truncating mutations in seven (47% of 15 patients. There was no cellular functional defect in RAD51 focus-formation in three heterozygously mutated lines studied, although other potential functions of the BRCA2 protein could still be affected. A prior report of mutations in primary MSI tumors affecting the IVS5-(5–15 poly [T] tract of the MRE11 gene was confirmed and extended by analysis of the genomic sequence and protein expression in MSI cancer cell lines. Statistical analysis of the published MRE11 mutation rate in MSI tumors did not provide evidence for a selective pressure favoring biallelic mutations at this repeat. Conclusion Perhaps conflicting with common suspicions, the data are not compatible with selective pressures during tumorigenesis promoting the functional loss of BRCA2 and MRE11 in MSI tumors. Instead, these data fit closely with an absence of selective pressures acting on BRCA2 and MRE11 gene status during tumorigenesis.

  14. Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations

    Directory of Open Access Journals (Sweden)

    Pavlov Youri I

    2004-05-01

    Full Text Available Abstract Background DNA polymerase ε (Pol ε is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol ε active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect. Results Purified four-subunit Y831A Pol ε turns over more deoxynucleoside triphosphates to deoxynucleoside monophosphates than does wild-type Pol ε, suggesting altered coordination between the polymerase and exonuclease active sites. The pol2-Y831A mutation suppresses the mutator effect of the pol2-4 mutation in the exonuclease active site that abolishes proofreading by Pol ε, as measured in haploid strain with the pol2-Y831A,4 double mutation. Analysis of mutation rates in diploid strains reveals that the pol2-Y831A allele is recessive to pol2-4. In addition, the mutation rates of strains with the pol2-4 mutation in combination with active site mutator mutations in Pol δ and Pol α suggest that Pol ε may proofread certain errors made by Pol α and Pol δ during replication in vivo. Conclusions Our data suggest that Y831A replacement in Pol ε reduces replication fidelity and its participation in chromosomal replication, but without eliminating an additional function that is essential for viability. This suggests that other polymerases can substitute for certain functions of polymerase ε.

  15. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.

    Science.gov (United States)

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9. PMID:27609304

  16. Congenital encephalomyopathy and adult-onset myopathy and diabetes mellitus: Different phenotypic associations of a new heteroplasmic mtDNA tRNA glutamic acid mutation

    Energy Technology Data Exchange (ETDEWEB)

    Hanna, M.G.; Nelson, I.; Sweeney, M.G.; Cooper, J.M.; Watkins, P.J.; Morgan-Hughes, J.A.; Harding, A.E. [Kings College Hospital, London (United Kingdom)

    1995-05-01

    We report the clinical, biochemical, and molecular genetic findings in a family with an unusual mitochondrial disease phenotype harboring a novel mtDNA tRNA glutamic acid mutation at position 14709. The proband and his sister presented with congenital myopathy and mental retardation and subsequently developed cerebellar ataxia. Other family members had either adult-onset diabetes mellitus with muscle weakness or adult-onset diabetes mellitus alone. Ragged-red and cytochrome c oxidase (COX)-negative fibers were present in muscle biopsies. Biochemical studies of muscle mitochondria showed reduced complex I and IV activities. The mtDNA mutation was heteroplasmic in blood and muscle in all matrilineal relatives analyzed. Primary myoblast, but not fibroblast, cultures containing high proportions of mutant mtDNA exhibited impaired mitochondrial translation. These observations indicate that mtDNA tRNA point mutations should be considered in the differential diagnosis of congenital myopathy. In addition they illustrate the diversity of phenotypes associated with this mutation in the same family and further highlight the association between mtDNA mutations and diabetes mellitus. 43 refs., 4 figs., 1 tab.

  17. Mitochondrial DNA point 13731 mutation in spinocerebellar ataxia%线粒体DNA13731点突变与脊髓小脑性共济失调相关性研究

    Institute of Scientific and Technical Information of China (English)

    王栋慧; 王进

    2009-01-01

    Objective To study the possible relationship between the point mutation in mitochondrial DNA (mtDNA) and the progression spinocerebellar ataxia(SCA).Methods Polymerase chain reaction(PCR) was used to amplify the mtDNA segments of these patients and their relatives individuals,40 volunteers. The mtDNA segment lied in the above mtDNA ND5 gene. For PCR products of rhe mtDNA segment,single strand conformation polymorphism(SSCP) was executed to detect mutations and the abnormal segments were sequenced.Results We had found a new mtDNA mutation in segments of mtDNA point 13731(T>C),was identified in 1 patient and 1 presymptomatic relatives.Conclusion A new point mutation of detected mitochondrial DNA may be lated to SCA.%目的 探索线粒体DNA(mtDNA)突变位点与脊髓小脑性共济失调(SCA)的关系.方法 采用聚合酶链反应(PCR)对基因确诊的四个SCA家系10例患者及其亲属共34例与40例健康对照的线粒体ND5基因片段进行扩增,扩增产物进行单链构象多态性分析(SSCP),对SSCP出现异常的样本进行相应mtDNA片段测序.结果 在一家系的1名确诊患者及1名症状前患者检测到mtDNA13731(T>C)点突变.结论 脊髓小脑性共济失调的发生、发展可能与mtDNA突变有关.

  18. The co-occurrence of mtDNA mutations on different oxidative phosphorylation subunits, not detected by haplogroup analysis, affects human longevity and is population specific

    DEFF Research Database (Denmark)

    Raule, Nicola; Sevini, Federica; Li, Shengting;

    2014-01-01

    To re-examine the correlation between mtDNA variability and longevity, we examined mtDNAs from samples obtained from over 2200 ultranonagenarians (and an equal number of controls) collected within the framework of the GEHA EU project. The samples were categorized by high-resolution classification......, while about 1300 mtDNA molecules (650 ultranonagenarians and an equal number of controls) were completely sequenced. Sequences, unlike standard haplogroup analysis, made possible to evaluate for the first time the cumulative effects of specific, concomitant mtDNA mutations, including those that per se...... analysis, suggests that mitochondrial DNA variation does affect human longevity, but its effect is heavily influenced by the interaction between mutations concomitantly occurring on different mtDNA genes....

  19. DNA hybridization in nanostructural molecular assemblies enables detection of gene mutations without a fluorescent probe.

    Science.gov (United States)

    Maruyama, Tatsuo; Park, Lian-Chun; Shinohara, Toshimitsu; Goto, Masahiro

    2004-01-01

    We have developed a simple single nucleotide polymorphisms (SNPs) analysis utilizing DNA hybridization in nanostructural molecular assemblies. The novel technique enables the detection of a single-base mismatch in a DNA sequence without a fluorescent probe. This report describes for the first time that DNA hybridization occurs in the nanostructural molecular assemblies (termed reverse micelles) formed in an organic medium. The restricted nanospace in the reverse micelles amplifies the differences in the hybridization rate between mismatched and perfectly matched DNA probes. For a model system, we hybridized a 20-mer based on the p53 gene sequence to 20-mer complementary oligonucleotides with various types of mismatches. Without any DNA labeling or electrochemical apparatus, we successfully detected the various oligonucleotide mismatches by simply measuring the UV absorbance at 260 nm. PMID:14715007

  20. Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma.

    Science.gov (United States)

    Asmar, Fazila; Punj, Vasu; Christensen, Jesper; Pedersen, Marianne T; Pedersen, Anja; Nielsen, Anders B; Hother, Christoffer; Ralfkiaer, Ulrik; Brown, Peter; Ralfkiaer, Elisabeth; Helin, Kristian; Grønbæk, Kirsten

    2013-12-01

    The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; Pcells (P=1.5×10(-30)). Surprisingly, gene expression profiling showed that only 11% of the hypermethylated genes were down-regulated, among which there were several genes previously suggested to be tumor suppressors. A meta-analysis suggested that the 35 hypermethylated and down-regulated genes are associated with the activated B-cell-like type of diffuse large B-cell lymphoma in other studies. In conclusion, our data suggest that TET2 mutations may cause aberrant methylation mainly of genes involved in hematopoietic development, which are silenced but poised for activation in human embryonic stem cells. PMID:23831920

  1. DNA Variations in Oculocutaneous Albinism: An Updated Mutation List and Current Outstanding Issues in Molecular Diagnostics

    OpenAIRE

    Simeonov, Dimitre R.; Wang, Xinjing; Wang, Chen; Sergeev, Yuri; Dolinska, Monika; Bower, Matthew; Fischer, Roxanne; Winer, David; Dubrovsky, Genia; BALOG, JOAN Z.; Huizing, Marjan; Hart, Rachel; Zein, Wadih M.; William A Gahl; Brooks, Brian P.

    2013-01-01

    Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. There are four types of OCA, caused by mutations in TYR (OCA-1), OCA2 (OCA-2), TYRP1 (OCA-3), or SLC45A2 (OCA-4). Here we report 22 novel mutations; 14 from a cohort of 61 patients seen as part of the NIH OCA Natural History Study and 8 from a prior study at the University of Minnesota. We also include a comprehensive list of almost 600 previously reported OCA muta...

  2. Difficult diagnosis of the fragile X syndrome made possible by direct detection of DNA mutations.

    OpenAIRE

    Tarleton, J; Wong, S.; Heitz, D.; Schwartz, C.

    1992-01-01

    Genetic recombination near the fragile X locus (Xq27.3) has frequently been a problem in linkage studies of families in which the fragile X is segregating. This case report illustrates the resolution of a difficult situation in a fragile X family for whom cytogenetic studies were inconclusive and where recombination had twice confounded attempts at prenatal DNA diagnosis by RFLP analysis. Using a newly developed DNA probe, StB12.3, for direct detection of DNA instability in the fragile X locu...

  3. Ionizing radiation-induced mutation of human cells with different DNA repair capacities

    International Nuclear Information System (INIS)

    We have observed significant differences in the response to ionizing radiation of two closely related human cell lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by α-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and α-particles. WTK1 is also more mutable by α-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while α-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-induced tk- mutants of TK6, were not induced significantly by α-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes

  4. Ionizing radiation-induced mutation of human cells with different DNA repair capacities

    Energy Technology Data Exchange (ETDEWEB)

    Amundson, S.A.; Chen, D.J.

    1994-12-31

    We have observed significant differences in the response to ionizing radiation of two closely related human cell lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by {alpha}-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and {alpha}-particles. WTK1 is also more mutable by {alpha}-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while {alpha}-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-induced tk- mutants of TK6, were not induced significantly by {alpha}-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes.

  5. Mathematical modelling of the automated FADU assay for the quantification of DNA strand breaks and their repair in human peripheral mononuclear blood cells

    International Nuclear Information System (INIS)

    Cells continuously undergo DNA damage from exogenous agents like irradiation or genotoxic chemicals or from endogenous radicals produced by normal cellular metabolic activities. DNA strand breaks are one of the most common genotoxic lesions and they can also arise as intermediates of DNA repair activity. Unrepaired DNA damage can lead to genomic instability, which can massively compromise the health status of organisms. Therefore it is important to measure and quantify DNA damage and its repair. We have previously published an automated method for measuring DNA strand breaks based on fluorimetric detection of alkaline DNA unwinding [1], and here we present a mathematical model of the FADU assay, which enables to an analytic expression for the relation between measured fluorescence and the number of strand breaks. Assessment of the formation and also the repair of DNA strand breaks is a crucial functional parameter to investigate genotoxicity in living cells. A reliable and convenient method to quantify DNA strand breakage is therefore of significant importance for a wide variety of scientific fields, e.g. toxicology, pharmacology, epidemiology and medical sciences

  6. Heart tissue of harlequin (hq)/Big Blue mice has elevated reactive oxygen species without significant impact on the frequency and nature of point mutations in nuclear DNA

    International Nuclear Information System (INIS)

    Age is a major risk factor for heart disease, and cardiac aging is characterized by elevated mitochondrial reactive oxygen species (ROS) with compromised mitochondrial and nuclear DNA integrity. To assess links between increased ROS levels and mutations, we examined in situ levels of ROS and cII mutation frequency, pattern and spectrum in the heart of harlequin (hq)/Big Blue mice. The hq mouse is a model of premature aging with mitochondrial dysfunction and increased risk of oxidative stress-induced heart disease with the means for in vivo mutation detection. The hq mutation produces a significant downregulation in the X-linked apoptosis-inducing factor gene (Aif) impairing both the antioxidant and oxidative phosphorylation functions of AIF. Brain and skin of hq disease mice have elevated frequencies of point mutations in nuclear DNA and histopathology characterized by cell loss. Reports of associated elevations in ROS in brain and skin have mixed results. Herein, heart in situ ROS levels were elevated in hq disease compared to AIF-proficient mice (p < 0.0001) yet, mutation frequency and pattern were similar in hq disease, hq carrier and AIF-proficient mice. Heart cII mutations were also assessed 15 days following an acute exposure to an exogenous ROS inducer (10 mg paraquat/kg). Acute paraquat exposure with a short mutant manifestation period was insufficient to elevate mutation frequency or alter mutation pattern in the post-mitotic heart tissue of AIF-proficient mice. Paraquat induction of ROS requires mitochondrial complex I and thus is likely compromised in hq mice. Results of this preliminary survey and the context of recent literature suggest that determining causal links between AIF deficiency and the premature aging phenotypes of specific tissues is better addressed with assay of mitochondrial ROS and large-scale changes in mitochondrial DNA in specific cell types.

  7. Heart tissue of harlequin (hq)/Big Blue mice has elevated reactive oxygen species without significant impact on the frequency and nature of point mutations in nuclear DNA

    Energy Technology Data Exchange (ETDEWEB)

    Crabbe, Rory A. [Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 (Canada); Hill, Kathleen A., E-mail: khill22@uwo.ca [Department of Biology, University of Western Ontario, London, Ontario, N6A 5B7 (Canada)

    2010-09-10

    Age is a major risk factor for heart disease, and cardiac aging is characterized by elevated mitochondrial reactive oxygen species (ROS) with compromised mitochondrial and nuclear DNA integrity. To assess links between increased ROS levels and mutations, we examined in situ levels of ROS and cII mutation frequency, pattern and spectrum in the heart of harlequin (hq)/Big Blue mice. The hq mouse is a model of premature aging with mitochondrial dysfunction and increased risk of oxidative stress-induced heart disease with the means for in vivo mutation detection. The hq mutation produces a significant downregulation in the X-linked apoptosis-inducing factor gene (Aif) impairing both the antioxidant and oxidative phosphorylation functions of AIF. Brain and skin of hq disease mice have elevated frequencies of point mutations in nuclear DNA and histopathology characterized by cell loss. Reports of associated elevations in ROS in brain and skin have mixed results. Herein, heart in situ ROS levels were elevated in hq disease compared to AIF-proficient mice (p < 0.0001) yet, mutation frequency and pattern were similar in hq disease, hq carrier and AIF-proficient mice. Heart cII mutations were also assessed 15 days following an acute exposure to an exogenous ROS inducer (10 mg paraquat/kg). Acute paraquat exposure with a short mutant manifestation period was insufficient to elevate mutation frequency or alter mutation pattern in the post-mitotic heart tissue of AIF-proficient mice. Paraquat induction of ROS requires mitochondrial complex I and thus is likely compromised in hq mice. Results of this preliminary survey and the context of recent literature suggest that determining causal links between AIF deficiency and the premature aging phenotypes of specific tissues is better addressed with assay of mitochondrial ROS and large-scale changes in mitochondrial DNA in specific cell types.

  8. Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

    Directory of Open Access Journals (Sweden)

    Ellis David

    2009-08-01

    Full Text Available Abstract Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. Results The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96% isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78% fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates, G448E (n = 3, G307S (n = 3, K143R (n = 3 and Y123H, S405F and R467K (each n = 1. DNA sequencing revealed a novel substitution, G450V, in one isolate. Conclusion The sensitive RCA

  9. A fully automated 384 capillary array for DNA sequencer. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Li, Qingbo; Kane, T

    2003-03-20

    Phase I SpectruMedix has successfully developed an automatic 96-capillary array DNA prototype based on the multiplexed capillary electrophoresis system originated from Ames Laboratory-USDOE, Iowa State University. With computer control of all steps involved in a 96-capillary array running cycle, the prototype instrument (the SCE9600) is now capable of sequencing 450 base pairs (bp) per capillary, or 48,000 bp per instrument run within 2 hrs. Phase II of this grant involved the advancement of the core 96 capillary technologies, as well as designing a high density 384 capillary prototype. True commercialization of the 96 capillary instrument involved finalization of the gel matrix, streamlining the instrument hardware, creating a more reliable capillary cartridge, and further advancement of the data processing software. Together these silos of technology create a truly commercializable product (the SCE9610) capable of meeting the operation needs of the sequencing centers.

  10. Genetic signatures from amplification profiles characterize DNA mutation in somatic and radiation-induced sports of chrysanthemum

    International Nuclear Information System (INIS)

    The chrysanthemum (Dendranthema grandiflora Tzvelev.) cultivars 'Dark Charm', 'Salmon Charm', 'Coral Charm' and 'Dark Bronze Charm' are either radiation-induced mutants or spontaneous sports of 'Charm' and constitute a family or series of plants that primarily differ in flower color. These cultivars, which were difficult to differentiate genetically by DNA amplification fingerprinting (DAF), were easily identified by using arbitrary signatures from amplification profiles (ASAP). Genomic DNA was first amplified with three standard octamer arbitrary primers, all of which produced monomorphic profiles. Products from each of these DNA fingerprints were subsequently reamplified using four minihairpin decamer primers. The 12 primer combinations produced signatures containing approximately 37% polymorphic character loci, which were used to estimate genetic relationships between cultivars. Forty-six (32%) unique amplification products were associated with individual cultivars. The number of ASAP polymorphisms detected provided an estimate of the mutation rate in the mutant cultivars, ranging from 0.03% to 1.6% of nucleotide changes within an average of 18 kb of arbitrary amplified DAF sequence. The ASAP technique permits the clear genetic identification of somatic mutants and radiation-induced sports that are genetically highly homogeneous and should facilitate marker assisted breeding and protection of plant breeders rights of varieties or cultivars

  11. High prevalence of impaired glucose homeostasis and myopathy in asymptomatic and oligosymptomatic 3243A>G mitochondrial DNA mutation-positive subjects

    DEFF Research Database (Denmark)

    Frederiksen, A.L.; Jeppesen, T.D.; Vissing, J.;

    2009-01-01

    INTRODUCTION: The point mutation of 3243A>G mtDNA is the most frequent cause of mitochondrial diabetes, often presenting as the syndrome maternally inherited diabetes and deafness (MIDD). The mutation may also cause myopathy, ataxia, strokes, ophthalmoplegia, epilepsy, and cardiomyopathy in various...... healthy controls were subjected to an oral glucose tolerance test. Twenty-six adult 3243A>G carriers with unknown myopathy status and 17 healthy controls had a maximal cycle test and a muscle biopsy performed. The mutation loads were quantified in blood and muscle biopsies and correlated to the clinical...

  12. Mitochondrial DNA mutation-elicited oxidative stress, oxidative damage, and altered gene expression in cultured cells of patients with MERRF syndrome.

    Science.gov (United States)

    Wu, Shi-Bei; Ma, Yi-Shing; Wu, Yu-Ting; Chen, Yin-Chiu; Wei, Yau-Huei

    2010-06-01

    Myoclonic epilepsy and ragged-red fibers (MERRF) syndrome is a rare disorder characterized by myoclonus, muscle weakness, cerebellar ataxia, heart conduction block, and dementia. It has been documented that 80-90% of the patients with MERRF syndrome are caused by the A8344G mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA). We and other investigators have reported that the mtDNA mutation results in not only inefficient generation of adenosine triphosphate but also increased production of reactive oxygen species (ROS) in cultured cells harboring A8344G mutation of mtDNA. In addition, we found an imbalance in the gene expression of antioxidant enzymes in the skin fibroblasts of MERRF patients. The mRNA, protein, and enzyme activity levels of manganese-superoxide dismutase were increased, but those of Cu,Zn-SOD, catalase, and glutathione peroxidase did not show significant changes. Recently, we showed that the excess ROS could damage voltage-dependent anion channel, prohibitin, Lon protease, and aconitase in the MERRF cells. Moreover, there was a dramatic increase in the gene expression and activity of matrix metalloproteinase 1, which may contribute to the cytoskeleton remodeling involved in the weakness and atrophy of muscle commonly seen in MERRF patients. Taken together, we suggest that mtDNA mutation-elicited oxidative stress, oxidative damage, and altered gene expression are involved in the pathogenesis and progression of MERRF syndrome. PMID:20411357

  13. Use of spontaneously mutated human DNA as competitive internal standard for nucleic acid quantification by reverse transcription-polymerase chain reaction (RT-PCR)

    International Nuclear Information System (INIS)

    Quantification of gene expression is of increasing interest in many medical sciences. Methods based on reverse transcription-polymerase chain reactions (RT-PCRs) are timesaving and require only very small amounts of RNA. A limiting factor, however, is the significant fluctuation in the efficacy of reverse transcription as well in the polymerase chain reactions. Various external and internal standards have been suggested for correcting these fluctuations. We describe a novel way of creating an internal standard for assessing the expression of type VII collagen in human cells. The total RNA of a patient with hereditary 'epidermilysis bulosa dystrophica' associated with a homozygous T to A point mutation in type VII collagen gene was reverse transcribed and a 382bp fragment of type VII collagen cDNA containing the mutation was amplified. The mutated cDNA, unlike normal type VII collagen cDNA could be cleaved by 'Ear I' endonuclease into 244bp and 138bp fragments. Semiquantitative PCR was performed with the mutated cDNA as internal standard and the studied cDNA sample in the same tube in the presence of α32P-labelled dCTP. The reaction was followed by 'Ear I' digestion, electrophoresis on a polyacrylamide gel and exposure to a X-ray film. In conclusion, we describe a timesaving method for creating internal standards for semiquantitative RT-PCR. (author). 12 refs, 3 figs

  14. Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmentosum patients

    International Nuclear Information System (INIS)

    The UV component of sunlight is the major carcinogen involved in the etiology of skin cancers. The authors have studied the rare, hereditary syndrome xeroderma pigmentosum (XP), which is characterized by a very high incidence of cutaneous tumors on exposed skin at an early age, probably due to a deficiency in excision repair of UV-induced lesions. It is interesting to determine the UV mutation spectrum in XP skin tumors in order to correlate the absence of repair of specific DNA lesions and the initiation of skin tumors. The p53 gene is frequently mutated in human cancers and represents a good target for studying mutation spectra since there are >100 potential sites for phenotypic mutations. Using reverse transcription-PCR and single-strand conformation polymorphism to analyze >40 XP skin tumors (mainly basal and squamous cell carcinomas), the authors have found that 40% (17 out of 43) contained at least one point mutation on the p53 gene. All the mutations were located at dipyrimidine sites, essentially at CC sequences, which are hot spots for UV-induced DNA lesions. Sixty-one percent of these mutations were tandem CC → TT mutations considered to be unique to UV-induced lesions; these mutations are not observed in internal human tumors. All the mutations, except two, must be due to translesion synthesis of unrepaired dipyrimidine lesions left on the nontranscribed strand. These results show the existence of preferential repair of UV lesions [either pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts] on the transcribed strand in human tissues

  15. A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

    OpenAIRE

    Houtteman, S W; Elder, R T

    1993-01-01

    Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting...

  16. Alternative mechanisms of telomere lengthening: permissive mutations, DNA repair proteins and tumorigenic progression

    OpenAIRE

    Sandy Gocha1, April Renee; Harris, Julia; Groden, Joanna

    2012-01-01

    Telomeres protect chromosome termini to maintain genomic stability and regulate cellular lifespan. Maintenance of telomere length is required for neoplastic cells after the acquisition of mutations that deregulate cell cycle control and increase cellular proliferation, and can occur through expression of the enzyme telomerase or in a telomerase-independent manner termed alternative lengthening of telomeres (ALT). The precise mechanisms that govern the activation of ALT or telomerase in tumor ...

  17. Protective action of DNA preparations on the survival of cells and yield of 8-azaguanine resistant mutations in X-irradiated cell culture of chinese hamsters

    International Nuclear Information System (INIS)

    A DNA preparation (molecular weight 19.6-21.0x1O6 daltons) administered to cell culture of Chinese hamsters in concentrations of 100 to 122 μg/ml 60 minutes before and in the course of 3 days after X-irradiation (600 R) decreased the lethality of irradiated cells and reduced induction of 8-azaguanine resistant genic mutations. DNA preparations with the concentrations under study had no toxic action on cells and were not mutagenous

  18. Prevalence of mutations in HBV DNA polymerase gene associated with nucleos(tide resistance in treatment-naive patients with Chronic Hepatitis B in Central China

    Directory of Open Access Journals (Sweden)

    Youyun Zhao

    2016-04-01

    Full Text Available Abstract Objective There are a lot of disagreements in the studies on hepatitis B virus (HBV DNA polymerase mutation rate associated with nucleos(tide analogues (NAs in treatment-naive chronic hepatitis B (CHB patients. This is the first study aimed to investigate the prevalence of spontaneous HBV resistance mutations in Central China. Methods This study included treatment-naive patients with CHB from June 2012 to May 2015 receiving care at the Institute of Liver Disease in Central China. All patients completed a questionnaire covering different aspects, such as family medical history, course of liver disease, medication history, alcohol use, among others. Mutations in HBV DNA polymerase associated with NAs resistance were detected using INNO-LiPA assay. Results 269 patients were infected with HBV genotype B (81.4%, C (17.9%, and both B and C (0.7%. Mutations in HBV DNA polymerase were detected in 24 patients (8.9% including rtM204I/V (n = 6, rtN236T (n = 5, rtM250V (n = 2, rtL180M (n = 2, rtT184G (n = 1, rtM207I (n = 1, rtS202I (n = 1, rtM204V/I & rtL180M (n = 5, and rtM204I & rtM250V (n = 1. Conclusion Spontaneous HBV resistance mutations in HBV DNA polymerase were found in treatment-naive patients with CHB in Central China. These findings suggest that we should analyze HBV DNA polymerase resistance mutation associated with NAs before giving antiviral therapy such as lamivudine (LAM, adefovir (ADV, and telbivudine (LdT.

  19. 线粒体DNA突变合并肾脏损害%Mitochondrial DNA mutation and renal involvement

    Institute of Scientific and Technical Information of China (English)

    谢红浪; 许书添; 何群鹏; 杨柳; 郭锦洲; 桂兰兰; 陈惠萍; 刘志红

    2012-01-01

    目的:探讨3例线粒体病合并慢性肾脏病(CKD)患者的临床特点及预后. 方法:2011年5月至2012年9月在南京军区南京总医院全军肾脏病研究所住院的CKD患者中共3例经线粒体DNA(mtDNA)基因检测确诊线粒体病,分析其肾外受累及肾脏损害的临床表现和肾活检病理特点,并观察其预后. 结果:(1)3例患者中2例青少年(14岁),另1例30岁,全部患者均消瘦(体质量指数13 ~ 17 kg/m2).例1生长发育迟缓,例3为早产儿.例1、例2有糖尿病家族史,例2有尿毒症家族史.(2)3例患者病程3~7月,临床均表现为水肿、蛋白尿和胱抑素C增高,肾小管损伤明显,均无高血压和镜下血尿.例1和例3组织学示局灶节段性肾小球硬化(FSGS),例2肾小球轻度系膜增生伴小动脉节段透明变性.(3)3例患者中糖尿病(2例)、高乳酸血症(2例)、听力消失或下降(3例)、癫痫发作(1例)、脑梗塞(1例)、智力障碍(2例)、视野缺失(1例),心律失常(1例).(4)3例患者均经基因测序并证实mtDNA 3243 A>G突变位点,例2母亲和弟弟也检出与患者相同突变位点.(5)经治疗3例均停免疫抑制剂,补充辅酶Q10和左卡尼汀等治疗,蛋白尿部分缓解,血清肌酐稳定. 结论:本文首次在国内报道3例伴肾脏损害的线粒体病患者,提示这类患者并非罕见,临床除肾脏受累外均伴明显的心脏和中枢神经系统损害.临床医师应加强对此类疾病的认识.%Objective : Mitochondrial cytopathy is a heterogeneous disease with multiple organ system involvement. We report 3 mitochondrial cytopathy patients with mitochondrial DNA (mtDNA) point mutation and renal involvement. Methodology: Clinical feature and family history were collected. Renal biopsy was performed. DNA was isolated from peripheral blood leukocytes of the patients. Polymerase chain reaction was performed to amplify the mtDNA. Sequencing analysis was used to detect the presence of any point mutation of mtDNA

  20. Evaluation of AgClNPs@SBA-15/IL nanoparticle-induced oxidative stress and DNA mutation in Escherichia coli.

    Science.gov (United States)

    Karimi, Farrokh; Dabbagh, Somayyeh; Alizadeh, Sina; Rostamnia, Sadegh

    2016-08-01

    The bactericidal effects of silver nanoparticles have been demonstrated in the past years. Recently, the new antimicrobial compounds of silver nanoparticles with different formulations have been developed. In this work, AgClNPs@SBA-15/IL as a new compound of Ag nanoparticles, was synthesized and characterized by XRD, TEM, SEM, FTIR, and EDX. The antibacterial activity and the molecular mechanism effects of AgClNPs@SBA-15/IL nanoparticles (SNPs) on Escherichia coli DH5α cells were investigated by analyzing the growth inhibitory, H2O2 level, catalase activity, DNA mutation, and plasmid copy number following treatment with AgClNPs@SBA-15/IL nanoparticles. In experimental results, the minimum inhibitory concentration (MIC) was observed in 75 μg/ml and the antibacterial efficacy (ABE) in CFU analysis was estimated 95.3 %. In bacterial cells treated with 75 and 100 μg/ml, H2O2 level significantly increased and catalase activity decreased compared with control. The random amplified polymorphic DNA (RAPD) was used to evaluate the effect of AgClNPs@SBA-15/IL nanoparticles in DNA damages and mutation in E. coli genome. RADP-PCR results indicated different banding patterns including appearance or disappearance of bands and differences in their intensity. Cluster analysis of the RAPD-PCR results based on genetic similarity showed genetic difference between E. coli cells treated with AgClNPs@SBA-15/IL nanoparticles, and control and phylogenetic tree were divided to two clusters. Plasmid copy number analysis indicated that after 8 h incubation of E. coli cells with 50, 75, and 100 μg/ml AgClNPs@SBA-15/IL nanoparticles, copy number of pET21a (+) significantly decreased compared with control which indicating DNA replication inhibition by Ag nanoparticles. In conclusion, the results of this study indicated that AgClNPs@SBA-15/IL nanoparticles can be used as an effective bactericidal agent against bacterial cells. PMID:27209037

  1. DNA Methylation and Somatic Mutations Converge on the Cell Cycle and Define Similar Evolutionary Histories in Brain Tumors.

    Science.gov (United States)

    Mazor, Tali; Pankov, Aleksandr; Johnson, Brett E; Hong, Chibo; Hamilton, Emily G; Bell, Robert J A; Smirnov, Ivan V; Reis, Gerald F; Phillips, Joanna J; Barnes, Michael J; Idbaih, Ahmed; Alentorn, Agusti; Kloezeman, Jenneke J; Lamfers, Martine L M; Bollen, Andrew W; Taylor, Barry S; Molinaro, Annette M; Olshen, Adam B; Chang, Susan M; Song, Jun S; Costello, Joseph F

    2015-09-14

    The evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution. PMID:26373278

  2. Mutations in yeast Rad51 that partially bypass the requirement for Rad55 and Rad57 in DNA repair by increasing the stability of Rad51–DNA complexes

    OpenAIRE

    Fortin, Gary S.; Symington, Lorraine S.

    2004-01-01

    Yeast Rad51 promotes homologous pairing and strand exchange in vitro, but this activity is inefficient in the absence of the accessory proteins, RPA, Rad52, Rad54 and the Rad55–Rad57 heterodimer. A class of rad51 alleles was isolated that suppresses the requirement for RAD55 and RAD57 in DNA repair, but not the other accessory factors. Five of the six mutations isolated map to the region of Rad51 that by modeling with RecA corresponds to one of the DNA-binding sites. The other mutation is in ...

  3. Obtaining insurance after DNA diagnostics: a survey among hypertrophic cardiomyopathy mutation carriers

    NARCIS (Netherlands)

    I. Christiaans; T.M. Kok; I.M. van Langen; E. Birnie; G.J. Bonsel; A.A.M. Wilde; E.M.A. Smets

    2010-01-01

    Hypertrophic cardiomyopathy (HCM) is a common hereditary heart disease associated with increased mortality. Disclosure of DNA test results may have social implications such as low access to insurance. In the Netherlands, insurance companies are restricted in the use of genetic information of their c

  4. Obtaining insurance after DNA diagnostics : A survey among hypertrophic cardiomyopathy mutation carriers

    NARCIS (Netherlands)

    Christiaans, Imke; Kok, Tjitske M.; Van Langen, Irene M.; Birnie, Erwin; Bonsel, Gouke J.; Wilde, Arthur A. M.; Smets, Ellen M. A.

    2010-01-01

    Hypertrophic cardiomyopathy (HCM) is a common hereditary heart disease associated with increased mortality. Disclosure of DNA test results may have social implications such as low access to insurance. In the Netherlands, insurance companies are restricted in the use of genetic information of their c

  5. Desmin common mutation is associated with multi-systemic disease manifestations and depletion of mitochondria and mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Elizabeth eMcCormick

    2015-06-01

    Full Text Available Desmin (DES is a major muscle scaffolding protein that also functions to anchor mitochondria. Pathogenic DES mutations, however, have not previously been recognized as a cause of multi-systemic mitochondrial disease. Here, we describe a 45-year-old man who presented to The Children’s Hospital of Philadelphia Mitochondrial-Genetics Diagnostic Clinic for evaluation of progressive cardiac, neuromuscular, gastrointestinal, and mood disorders. Muscle biopsy at age 45 was remarkable for cytoplasmic bodies, as well as ragged red fibers and SDH positive/COX negative fibers that were suggestive of a mitochondrial myopathy. Muscle also showed significant reductions in mitochondrial content (16% of control mean for citrate synthase activity and mitochondrial DNA (35% of control mean. His family history was significant for cardiac conduction defects and myopathy in multiple maternal relatives. Multiple single gene and panel-based sequencing studies were unrevealing. Whole exome sequencing identified a known pathogenic p.S13F mutation in DES that had previously been associated with desmin-related myopathy. Desmin-related myopathy is an autosomal dominant disorder characterized by right ventricular hypertrophic cardiomyopathy, myopathy, and arrhythmias. However, neuropathy, gastrointestinal dysfunction, and depletion of both mitochondria and mitochondrial DNA have not previously been widely recognized in this disorder. Recognition that mitochondrial dysfunction occurs in desmin-related myopathy clarifies the basis for the multi-systemic manifestations, as are typical of primary mitochondrial disorders. Understanding the mitochondrial pathophysiology of desmin-related myopathy highlights the possibility of new therapies for the otherwise untreatable and often fatal class of disease. We postulate that drug treatments aimed at improving mitochondrial biogenesis or reducing oxidative stress may be effective therapies to ameliorate the effects of desmin

  6. The resistance of Micrococcus radiodurans to killing and mutation by agents which damage DNA

    International Nuclear Information System (INIS)

    The resistance of Micrococcus radiodurans to the lethal and mutagenic action of ultraviolet (UV) light, ionising (γ) radiation, mitomycin C (MTC), nitrous acid (NA), hydroxylamine (HA), N-methyl-N'-nitro-N-nitrosoguanidine (NG), ethylmethanesulphonate (EMS) and β-propiolactone (βPL) has been compared with that of Escherichia coli B/r. M. radiodurans was much more resistant than E. coli B/r to the lethal effects of UV light (by a factor of 33), γ-radiation (55), NG (15) and NA (62), showed intermediate resistance to MTC (4) and HA (7), but was sensitive to EMS (1) and βPL (2). M. radiodurans was very resistant to mutagens producing damage which can be repaired by a recombination system, indicating that it possesses an extremely efficient recombination repair mechanism. Both species were equally sensitive to mutation to trimethoprim resistance by NG, but M. radiodurans was more resistant than E. coli B/r to the other mutagens tested, being non-mutable by UV light, γ-radiation, MTC and HA, and only slightly sensitive to mutation by NA, EMS, and βPL. The resistance of M. radiodurans to mutation by UV light, γ-radiation and MTC is consistent with an hypothesis that recombination repair in M. radiodurans is accurate since these mutagens may depend on an 'error-prone' recombination system for their mutagenic effect in E. coli B/r. However, because M. radiodurans is also resistant to mutagens such as HA and EMS, which are mutagenic in E. coli in the absence of an 'error-prone' system, we propose that all the mutagens tested may have a common mode of action in E. coli B/r, but that this mutagenic pathway is missing in M. radiodurans

  7. Multiple symmetric lipomas with high levels of mtDNA with the tRNA(Lys) A-->G(8344) mutation as the only manifestation of disease in a carrier of myoclonus epilepsy and ragged-red fibers (MERRF) syndrome.

    OpenAIRE

    Holme, E; Larsson, N G; Oldfors, A; Tulinius, M; Sahlin, P; Stenman, G

    1993-01-01

    We have investigated the morphology, cytogenetics, and the fraction of mtDNA with the tRNA(Lys) A-->G(8344) mutation in three lipomas in a carrier of this mutation. The son of the patient had myoclonus epilepsy and ragged-red fibers syndrome. The fraction of mtDNA with the tRNA(Lys) mutation varied between 62% and 80% in cultured skin fibroblasts, lymphocytes, normal adipose tissue, and muscle. In the three lipomas the mean fraction of mutated mtDNA was 90%, 94%, and 94%. Ultrastructural exam...

  8. A Point Mutation in DNA Polymerase β (POLB) Gene Is Associated with Increased Progesterone Receptor (PR) Expression and Intraperitoneal Metastasis in Gastric Cancer

    Science.gov (United States)

    Tan, Xiaohui; Wu, Xiaoling; Ren, Shuyang; Wang, Hongyi; Li, Zhongwu; Alshenawy, Weaam; Li, Wenmei; Cui, Jiantao; Luo, Guangbin; Siegel, Robert S.; Fu, Sidney W.; Lu, Youyong

    2016-01-01

    Increased expression of progesterone receptor (PR) has been reported in gastric cancer (GC). We have previously identified a functional T889C point mutation in DNA polymerase beta (POLB), a DNA repair gene in GC. To provide a detailed analysis of molecular changes associated with the mutation, human cDNA microarrays focusing on 18 signal transduction pathways were used to analyze differential gene expression profiles between GC tissues with T889C mutant in POLB gene and those with wild type. Among the differentially expressed genes, notably, PR was one of the significantly up-regulated genes in T889C mutant POLB tissues, which were subsequently confirmed in POLB gene transfected AGS cell line. Interestingly, patients with T889C mutation and PR positivity were associated with higher incidence of intraperitoneal metastasis (IM). In vitro studies indicate that PR expression was upregulated in AGS cell line when transfected with T889C mutant expression vector. Cotransfection of T889C mutant allele and PR gene induced cell migration in the cell line. These data demonstrated that T889C mutation-associated PR overexpression results in increased IM. Therefore, T889C mutation-associated PR overexpression may serve as a biomarker for an adverse prognosis for human GC.

  9. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

    Science.gov (United States)

    Loera-Castañeda, Verónica; Sandoval-Ramírez, Lucila; Pacheco Moisés, Fermín Paul; Macías-Islas, Miguel Ángel; Alatorre Jiménez, Moisés Alejandro; González-Renovato, Erika Daniela; Cortés-Enríquez, Fernando; Célis de la Rosa, Alfredo; Velázquez-Brizuela, Irma E.

    2014-01-01

    Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS). Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III) forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II) in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12%) harbored the A8027G polymorphism and three of them were early onset (EO) AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn't been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD. PMID:24701363

  10. Digital PCR for quantification of recurrent and potentially actionable somatic mutations in circulating free DNA from patients with diffuse large B-cell lymphoma.

    Science.gov (United States)

    Camus, Vincent; Sarafan-Vasseur, Nasrin; Bohers, Elodie; Dubois, Sydney; Mareschal, Sylvain; Bertrand, Philippe; Viailly, Pierre-Julien; Ruminy, Philippe; Maingonnat, Catherine; Lemasle, Emilie; Stamatoullas, Aspasia; Picquenot, Jean-Michel; Cornic, Marie; Beaussire, Ludivine; Bastard, Christian; Frebourg, Thierry; Tilly, Hervé; Jardin, Fabrice

    2016-09-01

    Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy harboring frequent targetable activating somatic mutations. Emerging evidence suggests that circulating cell-free DNA (cfDNA) can be used to detect somatic variants in DLBCL using Next-Generation Sequencing (NGS) experiments. In this proof-of-concept study, we chose to develop simple and valuable digital PCR (dPCR) assays for the detection of recurrent exportin-1 (XPO1) E571K, EZH2 Y641N, and MYD88 L265P mutations in DLBCL patients, thereby identifying patients most likely to potentially benefit from targeted therapies. We demonstrated that our dPCR assays were sufficiently sensitive to detect rare XPO1, EZH2, and MYD88 mutations in plasma cfDNA, with a sensitivity of 0.05%. cfDNA somatic mutation detection by dPCR seems to be a promising technique in the management of DLBCL, in addition to NGS experiments. PMID:26883583

  11. Impact of a novel homozygous mutation in nicotinamide nucleotide transhydrogenase on mitochondrial DNA integrity in a case of familial glucocorticoid deficiency

    Directory of Open Access Journals (Sweden)

    Yasuko Fujisawa

    2015-06-01

    General significance: By studying a family affected with a novel point mutation in the NNT gene, a gene–dose response was found for various mitochondrial outcomes providing for novel insights into the role of NNT in the maintenance of mtDNA integrity beyond that described for preventing oxidative stress.

  12. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers

    DEFF Research Database (Denmark)

    Osorio, Ana; Milne, Roger L; Kuchenbaecker, Karoline;

    2014-01-01

    Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of th...

  13. Low-energy (30 keV) carbon ion induced mutation spectrum in the LacZα gene of M13mp18 double-stranded DNA

    International Nuclear Information System (INIS)

    Double-stranded M13mp18 DNA was irradiated with 30 keV carbon ions in dry state under vacuum to investigate the low-energy heavy ion induced mutation spectra. When the irradiated DNA was used to transfect Escherichia coli JM105, 3.6-5.7-fold increases in mutation frequency were observed, in contrast to the spontaneous group. Sequences of the 92 induced mutants showed that the carbon ions in this study could induce an interesting mutation spectrum in the lacZα gene. One-base mutations (96.8%) and base pair substitutions (56.4%) were predominant, most of which involved G:C base pairs (90.6%), especially G:C → T:A transversions (49.6%) and G:C → A:T transitions (39.6%). This is similar to the spectra induced by γ-rays in the same ds M13, wild type E. coli system. We also found a considerable amount of carbon ion induced one-base deletion (38.5%) and the mutation sites distribution on the target lacZα gene was obviously non-random. We compared this study with previous data employing γ-rays to discuss the possible causes of the mutation spectrum

  14. Optimization and preparation of FRET-based sensor for detection of DNA mutations

    Czech Academy of Sciences Publication Activity Database

    Datinská, Vladimíra; Týčová, Anna; Klepárník, Karel; Belšánová, B.; Minárik, M.; Foret, František

    Elsevier, 2015. s. 2.058. [International Conference on Bio-Sensing Technology /4./. 10.05.2015-13.05.2015, Lisbon] R&D Projects: GA TA ČR(CZ) TA02010672; GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : FRET * quantum dot * DNA Subject RIV: CB - Analytical Chemistry, Separation https://elsevier.conference-services.net/secureProgramme.asp?conferenceID=3808&uID=703758&action=secure_prog_titles

  15. Somatic mutations in glioblastoma are associated with methylguanine-DNA methyltransferase methylation

    OpenAIRE

    McDonald, Kerrie L.; Tabone, Tania; Nowak, Anna K; Erber, Wendy N.

    2015-01-01

    The high level of methylguanine-DNA methyltransferase (MGMT) in glioblastoma is responsible for resistance to alkylating agents, such as temozolomide (TMZ). In glioblastomas with a methylated MGMT promoter, MGMT deficiency is presumed, resulting in an enhanced effect of TMZ. The aim of the present study was to investigate whether genomic alterations work synergistically with MGMT methylation status and contribute to the response to treatment and overall prognosis in glioblastoma. The current ...

  16. Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions

    International Nuclear Information System (INIS)

    Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m2, each of the mutants showed 2, the rate in AA8 was similar to that at 6 J/m2, but the rates in the mutants were significantly higher (approx. 20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both the hprt and aprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, here CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV

  17. Impact of Somatic Mutations in the D-Loop of Mitochondrial DNA on the Survival of Oral Squamous Cell Carcinoma Patients

    OpenAIRE

    Lin, Jin-Ching; Wang, Chen-Chi; Jiang, Rong-San; Wang, Wen-yi; Liu, Shih-An

    2015-01-01

    Objectives The aim of this study was to investigate somatic mutations in the D-loop of mitochondrial DNA (mtDNA) and their impact on survival in oral squamous cell carcinoma patients. Materials and Methods Surgical specimen confirmed by pathological examination and corresponding non-cancerous tissues were collected from 120 oral squamous cell carcinoma patients. The sequence in the D-loop of mtDNA from non-cancerous tissues was compared with that from paired cancer samples and any sequence di...

  18. Identification of BRCA1/2 founder mutations in Southern Chinese breast cancer patients using gene sequencing and high resolution DNA melting analysis.

    Directory of Open Access Journals (Sweden)

    Ava Kwong

    Full Text Available BACKGROUND: Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China. METHODOLOGY/PRINCIPAL FINDINGS: A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM analysis. Among the 451 probands analyzed, 69 (15.3% deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations (c.470_471delCT, c.3342_3345delAGAA, c.5406+1_5406+3delGTA and c.981_982delAT accounted for 34.5% (10/29 of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations (c.2808_2811delACAA, c.3109C>T, c.7436_7805del370 and c.9097_9098insA accounted for 40% (16/40 of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated. CONCLUSION: In this study, our findings suggest that BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment.

  19. Segregation and manifestations of the mtDNA tRNA[sup Lys] A[r arrow]G[sup (8344)] mutation of myoclonus epilepsy and ragged-red fibers (MERRF) syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Larsson, N.G.; Tulinius, M.H.; Holme, E.; Oldfors, A.; Andersen, O.; Wahlstroem, J. (Univ. of Gothenburg (Sweden)); Aasly, J. (Univ. of Trondheim (Norway))

    1992-12-01

    The authors have studied the segregation and manifestations of the tRNA[sup Lys] A[r arrow]G[sup (8344)] mutation of mtDNA. Three unrelated patients with myoclonus epilepsy and ragged-red fibers (MERRF) syndrome were investigated, along with 30 of their maternal relatives. Mutated mtDNA was not always found in the offspring of women carrying the tRNA[sup Lys] mutation. Four women had 10%-33% of mutated mtDNA in lymphocytes, and no mutated mtDNA was found in 7 of their 14 investigated children. The presence of mutated mtDNA was excluded at a level of 3:1,000. Five women had a proportion of 43%-73% mutated mtDNA in lymphocytes, and mutated mtDNA was found in all their 12 investigated children. This suggests that the risk for transmission of mutated mtDNA to the offspring increases if high levels are present in the mother and that, above a threshold level of 35%-40%, it is very likely that transmission will occur to all children. The three patients with MERRF syndrone had, in muscle, both 94%-96% mutated mtDNA and biochemical and histochemical evidence of a respiratory-chain dysfunction. Four relatives had a proportion of 61%-92% mutated mtDNA in muscle, and biochemical measurements showed a normal respiratory-chain function in muscle in all cases. These findings suggest that >92% of mtDNA with the tRNA[sup Lys] mutation in muscle is required to cause a respiratory-chain dysfunction that can be detected by biochemical methods. There was a positive correlation between the levels of mtDNA with the tRNA[sup Lys] mutation in lymphocytes and the levels in muscle, in all nine investigated cases. The levels of mutated mtDNA were higher in muscle than in lymphocytes in all cases. 30 refs., 3 figs., 5 tabs.

  20. TET2 Mutations Affect Non-CpG Island DNA Methylation at Enhancers and Transcription Factor-Binding Sites in Chronic Myelomonocytic Leukemia.

    Science.gov (United States)

    Yamazaki, Jumpei; Jelinek, Jaroslav; Lu, Yue; Cesaroni, Matteo; Madzo, Jozef; Neumann, Frank; He, Rong; Taby, Rodolphe; Vasanthakumar, Aparna; Macrae, Trisha; Ostler, Kelly R; Kantarjian, Hagop M; Liang, Shoudan; Estecio, Marcos R; Godley, Lucy A; Issa, Jean-Pierre J

    2015-07-15

    TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here, we report on DNA methylomes in TET2 wild-type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites [28,114 sites in CpG islands (CGI) and 57,020 in non-CpG islands (NCGI)]. TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. In contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and nonpromoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1 and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor-binding sites. PMID:25972343

  1. The mtDNA NARP mutation activates the actin-Nrf2 signaling of antioxidant defenses

    International Nuclear Information System (INIS)

    An efficient handling of superoxides by antioxidant defenses is a crucial issue for cells with respiratory chain deficient mitochondria. We used human cultured skin fibroblasts to delineate the mechanism controlling the expression of antioxidant defenses in the case of a severe ATPase deficiency resulting from an 8993T>G mutation in the mitochondrial ATPase6 gene. We observed the nuclear translocation of the transcription factor Nrf2 associated with thinning of the actin stress fibers. The mobilization of the Nrf2 signaling pathway could be mimicked by a chemical blockade of the ATPase with a specific inhibitor, oligomycin. Interestingly enough, Nrf2 nuclear translocation was not observed in the case of a severe cytochrome oxidase deficiency, indicating that studying the status of this signaling pathway could throw some light on the importance of the oxidative insult associated with different respiratory chain defects

  2. Discriminating somatic and germline mutations in tumor DNA samples without matching normals.

    Science.gov (United States)

    Hiltemann, Saskia; Jenster, Guido; Trapman, Jan; van der Spek, Peter; Stubbs, Andrew

    2015-09-01

    Tumor analyses commonly employ a correction with a matched normal (MN), a sample from healthy tissue of the same individual, in order to distinguish germline mutations from somatic mutations. Since the majority of variants found in an individual are thought to be common within the population, we constructed a set of 931 samples from healthy, unrelated individuals, originating from two different sequencing platforms, to serve as a virtual normal (VN) in the absence of such an associated normal sample. Our approach removed (1) >96% of the germline variants also removed by the MN sample and (2) a large number (2%-8%) of additional variants not corrected for by the associated normal. The combination of the VN with the MN improved the correction for polymorphisms significantly, with up to ∼30% compared with MN and ∼15% compared with VN only. We determined the number of unrelated genomes needed in order to correct at least as efficiently as the MN is about 200 for structural variations (SVs) and about 400 for single-nucleotide variants (SNVs) and indels. In addition, we propose that the removal of common variants with purely position-based methods is inaccurate and incurs additional false-positive somatic variants, and more sophisticated algorithms, which are capable of leveraging information about the area surrounding variants, are needed for optimal accuracy. Our VN correction method can be used to analyze any list of variants, regardless of sequencing platform of origin. This VN methodology is available for use on our public Galaxy server. PMID:26209359

  3. Influence of anoxia on the induction of mutations by phenylalanine radicals during γ-irradiation of plasmid DNA in aqueous solution

    International Nuclear Information System (INIS)

    When DNA is irradiated in aqueous solution, most of the damage is inflicted by water-derived radicals. This is called the indirect effect of ionizing radiation. However in whole cells not only the primary formed water radicals play a role, because some cellular compounds form secondary radicals which can also damage DNA. It is known that the amino acid phenylalanine is able to react with water radicals, resulting in the production of secondary phenylalanine radicals which can damage and inactivate DNA. In a previous study the influence of the presence of phenylalanine during γ-irradiation of DNA in aqueous solution under oxic conditions was studied. Under anoxic irradiation conditions different amounts and types of reactive water-derived radicals are formed compared to oxic conditions and also different phenylalanine radicals are formed. Therefore, this study examines the influence of the presence of phenylalanine under anoxic conditions on the γ-radiation-induced mutation spectrum. The results indicate that phenylalanine radicals are damaging to DNA, but less effective compared to primary water radicals. On the mutational level, in the presence of phenylalanine radicals under anoxic conditions, the amount of mutations on G:C base pairs was significantly decreased as compared to oxic conditions. Furthermore, the results of this study indicate that nucleotide excision repair is involved in repair of both inactivating and mutagenic damage induced by phenylalanine radicals under anoxic conditions

  4. A de novo frameshift mutation in chromodomain helicase DNA-binding domain 8 (CHD8): A case report and literature review.

    Science.gov (United States)

    Merner, Nancy; Forgeot d'Arc, Baudouin; Bell, Scott C; Maussion, Gilles; Peng, Huashan; Gauthier, Julie; Crapper, Liam; Hamdan, Fadi F; Michaud, Jacques L; Mottron, Laurent; Rouleau, Guy A; Ernst, Carl

    2016-05-01

    Mutations in chromodomain helicase DNA-binding domain 8 (CHD8) have been identified in independent genotyping studies of autism spectrum disorder. To better understand the phenotype associated with CHD8 mutations, we genotyped all CHD8 exons in carefully assessed cohorts of autism (n = 142), schizophrenia (SCZ; n = 143), and intellectual disability (ID; n = 94). We identified one frameshift mutation, seven non-synonymous variants, and six synonymous variants. The frameshift mutation, p.Asn2092Lysfs*2, which creates a premature stop codon leading to the loss of 212 amino acids of the protein, was from an autism case on whom we present multiple clinical assessments and pharmacological treatments spanning more than 10 years. RNA and protein analysis support a model where the transcript generated from the mutant allele results in haploinsufficiency of CHD8. This case report supports the association of CHD8 mutations with classical autism, macrocephaly, infantile hypotonia, speech delay, lack of major ID, and psychopathology in late adolescence caused by insufficient dosage of CHD8. Review of 16 other CHD8 mutation cases suggests that clinical features and their severity vary considerably across individuals; however, these data support a CHD8 mutation syndrome, further highlighting the importance of genomic medicine to guide clinical assessment and treatment. PMID:26789910

  5. PhyloBot: A Web Portal for Automated Phylogenetics, Ancestral Sequence Reconstruction, and Exploration of Mutational Trajectories

    Science.gov (United States)

    Hanson-Smith, Victor; Johnson, Alexander

    2016-01-01

    The method of phylogenetic ancestral sequence reconstruction is a powerful approach for studying evolutionary relationships among protein sequence, structure, and function. In particular, this approach allows investigators to (1) reconstruct and “resurrect” (that is, synthesize in vivo or in vitro) extinct proteins to study how they differ from modern proteins, (2) identify key amino acid changes that, over evolutionary timescales, have altered the function of the protein, and (3) order historical events in the evolution of protein function. Widespread use of this approach has been slow among molecular biologists, in part because the methods require significant computational expertise. Here we present PhyloBot, a web-based software tool that makes ancestral sequence reconstruction easy. Designed for non-experts, it integrates all the necessary software into a single user interface. Additionally, PhyloBot provides interactive tools to explore evolutionary trajectories between ancestors, enabling the rapid generation of hypotheses that can be tested using genetic or biochemical approaches. Early versions of this software were used in previous studies to discover genetic mechanisms underlying the functions of diverse protein families, including V-ATPase ion pumps, DNA-binding transcription regulators, and serine/threonine protein kinases. PhyloBot runs in a web browser, and is available at the following URL: http://www.phylobot.com. The software is implemented in Python using the Django web framework, and runs on elastic cloud computing resources from Amazon Web Services. Users can create and submit jobs on our free server (at the URL listed above), or use our open-source code to launch their own PhyloBot server. PMID:27472806

  6. PhyloBot: A Web Portal for Automated Phylogenetics, Ancestral Sequence Reconstruction, and Exploration of Mutational Trajectories.

    Science.gov (United States)

    Hanson-Smith, Victor; Johnson, Alexander

    2016-07-01

    The method of phylogenetic ancestral sequence reconstruction is a powerful approach for studying evolutionary relationships among protein sequence, structure, and function. In particular, this approach allows investigators to (1) reconstruct and "resurrect" (that is, synthesize in vivo or in vitro) extinct proteins to study how they differ from modern proteins, (2) identify key amino acid changes that, over evolutionary timescales, have altered the function of the protein, and (3) order historical events in the evolution of protein function. Widespread use of this approach has been slow among molecular biologists, in part because the methods require significant computational expertise. Here we present PhyloBot, a web-based software tool that makes ancestral sequence reconstruction easy. Designed for non-experts, it integrates all the necessary software into a single user interface. Additionally, PhyloBot provides interactive tools to explore evolutionary trajectories between ancestors, enabling the rapid generation of hypotheses that can be tested using genetic or biochemical approaches. Early versions of this software were used in previous studies to discover genetic mechanisms underlying the functions of diverse protein families, including V-ATPase ion pumps, DNA-binding transcription regulators, and serine/threonine protein kinases. PhyloBot runs in a web browser, and is available at the following URL: http://www.phylobot.com. The software is implemented in Python using the Django web framework, and runs on elastic cloud computing resources from Amazon Web Services. Users can create and submit jobs on our free server (at the URL listed above), or use our open-source code to launch their own PhyloBot server. PMID:27472806

  7. Analysis of forensic SNPs in the canine mtDNA HV1 mutational hotspot region.

    Science.gov (United States)

    Baute, Danielle T; Satkoski, Jessica A; Spear, Theresa F; Smith, David G; Dayton, Melody R; Malladi, Venkat S; Goyal, Vivek; Kou, Alexander; Kinaga, Jennifer L; Kanthaswamy, Sreetharan

    2008-11-01

    A 60 bp sequence variation hotspot in the canine mitochondrial DNA hypervariable region 1 was evaluated for its use in forensic investigations. Nineteen haplotypes containing 18 single nucleotide polymorphisms were observed among laboratory-generated and GenBank-derived domestic dog sequences representing five regional localities in the U.S. Samples from the different localities were highly variable with the levels of intra-population variability being similar among the populations studied. AMOVA further confirmed that there was no significant genetic structuring of the populations. Assays using these haplotypes were robust, canid specific and portend a rapid method for correctly excluding individual dogs as noncontributors of forensic evidence. Species-specificity of the primers was confirmed by means of in-tube polymerase chain reaction of human and cat DNA and in-silico assessment of the genomes of several animal species. Breed-specific fragments were not detected among the common haplotypes but there is evidence that this assay may be capable of differentiating domestic dog, wolf, and coyote sequences. PMID:18808373

  8. Retrospective molecular detection of Transthyretin Met 111 mutation in a Danish kindred with familial amyloid cardiomyopathy, using DNA from formalin-fixed and paraffin-embedded tissues.

    Science.gov (United States)

    Nordvåg, B Y; Ranløv, I; Riise, H M; Husby, G; el-Gewely, M R

    1993-10-01

    Severe familial amyloid cardiomyopathy (FAC) in a Danish kindred is associated with a specific mutation (Met for Leu 111) in the transthyretin (TTR) gene. The mutation causes the loss of a DdeI restriction site in the gene, allowing molecular diagnostic studies. We studied formalin-fixed, paraffin-embedded tissues, up to 39 years old, from 29 family members of this kindred. DNA was partially purified from deparaffinized tissue sections and a DNA sequence of the TTR gene flanking the mutation site was amplified by the polymerase chain reaction (PCR), followed by restriction enzyme analysis. Amplified DNA was obtained from tissues representing 23 of the 29 persons. Ten out of the 23 family members were found to carry the TTR Met 111 mutation, whereas 13 were not affected. The results were consistent with known clinical data and with corresponding serum TTR examinations. This retrospective study shows that archival tissues can be used to confirm the diagnosis and disease pattern in members of families affected by hereditary diseases. PMID:8406434

  9. Evaluation of the metabolic fate of munitions material (TNT & RDX) in plant systems. Initial assessment of plant DNA mutation spectra as a biomarker

    Energy Technology Data Exchange (ETDEWEB)

    Leung, F.; Cataldo, D.A.; Fellows, R.J.; Jarrell, A.E.; Harvey, S.D.

    1995-09-01

    Munitions material can enter the environment as a result of manufacturing activities and field usage. Predictor methodologies, or biomarkers would enhance evaluation of environmental impacts. The goal of this exploratory study deoxyribonucleic acid (DNA) mutation frequency as a biomarker for munitions exposure. The approach e resolution of an effective repetitive sequence probe for the identification of characteristic mutations, and (2) the development of a testing media [a clonal cell line of carrot (Daucus carota) spension cells]. Commercially available probes demonstrated marginal resolution therefore a low-C{sub o}t library was then constructed. Three colonies from the low-C{sub o}t DNA library were screened and the DNA isolates sequenced. A suspension culture of carrot (Daucus carota) was developed. A mutation spectra experiment was initiated at a 10-mg TNT/L exposure concentration with the attempt to clone over 1500 single TNT-exposed cells. Over the following six months greater than 98% of the initially isolated cells were unable to survive and produce micro calluses. The remaining calli were too few to be statistically significant and the experiment was terminated. The biomarker concept itself remains to be disproved, but the need for large numbers of uniform clones to differentiate true mutations suggest that more direct techniques using whole tissues need to be developed.

  10. Mitochondrial DNA from colorectal cancer cells induces the mutation of NIH3T3 cells%大肠癌线粒体DNA诱导NIH3T3细胞D-环突变

    Institute of Scientific and Technical Information of China (English)

    姬宏莉; 姬宏娟; 宋卫兵; 马永全; 杜芳; 王辉; 肖冰

    2011-01-01

    Objective To investigate the mtDNA mutation in NIH3T3 cell, which was transfected with mutated mtDNA from colorectal cancer cell line. Methods Recombinant eukaryotic vector, expressing plasmid of the mutated mtDNA, was transfected into NIH3T3 cell using Lipofection 2000 TM and screened by G418. The mutated mtDNA of transfected NIH3T3 cell was detected by PCR. Results Mutation and polymorphism was observed in NIH3T3 cell transfected with mutated mtDNA. Conclusion MtDNA mutations in colorectal cancer cell affects NIH3T3 cell mtDNA loci, though further study is required.%目的 观察突变的大肠癌细胞线粒体DNA(mtDNA)转染 NIH3T3 细胞后转染细胞mtDNA D-环突变特性.方法 通过脂质体法(Lipofection 2000TM) 将大肠癌细胞突变的 mtDNA 真核表达载体转染 NIH3T3细胞,利用 G418 抗性筛选克隆细胞;用PCR法检测转染细胞线粒体突变情况.结果 突变mtDNA 导致转染细胞 mtDNA 的突变和多态性变化.结论 突变的大肠癌 mtDNA可致NIH3T3细胞mtDNA位点变化,但具体的机制和过程有待于进一步研究.

  11. Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma

    Science.gov (United States)

    Harlé, Alexandre; Salleron, Julia; Franczak, Claire; Dubois, Cindy; Filhine-Tressarieu, Pierre; Leroux, Agnès; Merlin, Jean-Louis

    2016-01-01

    Background Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies. Methods Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM) PCR, Real-time allele-specific amplification (RT-ASA) PCR, Next generation sequencing (NGS), immunohistochemistry (IHC) and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays. Results BRAF mutations were found in 28(47.5%), 29(49.2%), 31(52.5%), 29(49.2%) and 27(45.8%) samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2%) samples were found bearing a c.1799T>A (p.Val600Glu) mutation, three (9.4%) with a c.1798_1799delinsAA (p.Val600Lys) mutation and one with c.1789_1790delinsTC (p.Leu597Ser) mutation. Two samples were found bearing complex mutations. Conclusions HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays. PMID:27111917

  12. Novel FOXF1 mutations in sporadic and familial cases of alveolar capillary dysplasia with misaligned pulmonary veins imply a role for its DNA binding domain.

    Science.gov (United States)

    Sen, Partha; Yang, Yaping; Navarro, Colby; Silva, Iris; Szafranski, Przemyslaw; Kolodziejska, Katarzyna E; Dharmadhikari, Avinash V; Mostafa, Hasnaa; Kozakewich, Harry; Kearney, Debra; Cahill, John B; Whitt, Merrissa; Bilic, Masha; Margraf, Linda; Charles, Adrian; Goldblatt, Jack; Gibson, Kathleen; Lantz, Patrick E; Garvin, A Julian; Petty, John; Kiblawi, Zeina; Zuppan, Craig; McConkie-Rosell, Allyn; McDonald, Marie T; Peterson-Carmichael, Stacey L; Gaede, Jane T; Shivanna, Binoy; Schady, Deborah; Friedlich, Philippe S; Hays, Stephen R; Palafoll, Irene Valenzuela; Siebers-Renelt, Ulrike; Bohring, Axel; Finn, Laura S; Siebert, Joseph R; Galambos, Csaba; Nguyen, Lananh; Riley, Melissa; Chassaing, Nicolas; Vigouroux, Adeline; Rocha, Gustavo; Fernandes, Susana; Brumbaugh, Jane; Roberts, Kari; Ho-Ming, Luk; Lo, Ivan F M; Lam, Stephen; Gerychova, Romana; Jezova, Marta; Valaskova, Iveta; Fellmann, Florence; Afshar, Katayoun; Giannoni, Eric; Muhlethaler, Vincent; Liang, Jinlong; Beckmann, Jacques S; Lioy, Janet; Deshmukh, Hitesh; Srinivasan, Lakshmi; Swarr, Daniel T; Sloman, Melissa; Shaw-Smith, Charles; van Loon, Rosa Laura; Hagman, Cecilia; Sznajer, Yves; Barrea, Catherine; Galant, Christine; Detaille, Thierry; Wambach, Jennifer A; Cole, F Sessions; Hamvas, Aaron; Prince, Lawrence S; Diderich, Karin E M; Brooks, Alice S; Verdijk, Robert M; Ravindranathan, Hari; Sugo, Ella; Mowat, David; Baker, Michael L; Langston, Claire; Welty, Stephen; Stankiewicz, Pawel

    2013-06-01

    Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Nonpulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and 10 deletions, we have identified an additional 38 novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, 20 missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic. We report four familial cases of which three show maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA-binding domain, indicating its plausible role in FOXF1 function. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis. PMID:23505205

  13. [The analysis of mitochondrial DNA haplogroups and variants for Leber's hereditary optic neuropathy in Chinese families carrying the m.14484T >C mutation].

    Science.gov (United States)

    Meng, Xiangjuan; Zhu, Jinping; Gao, Min; Zhang, Sai; Zhao, Fuxin; Zhang, Juanjuan; Liu, Xiaoling; Wei, Qiping; Tong, Yi; Zhang, Minglian; Qu, Jia; Guan, Minxin

    2014-04-01

    The m.14484T>C mutation in mitochondrial ND6 gene (MT-ND6) is a primary mutation underlying the development of Leber's hereditary optic neuropathy (LHON) , but by itself not enough to cause visual loss. To explore the role of mitochondrial haplogroups on the expression of LHON for the people carrying the m.14484T>C mutation, we performed systematic and extended mutational screening of MT-ND6 gene in a cohort of 1177 Han Chinese patients with LHON. A total of 67 affected subjects carried the homoplasmic m.14484T>C mutation, accounting for 5.7% of this LHON population. The penetrances of optic neuropathy among 51 pedigrees carrying the m.14484T>C mutation ranged from 5.6% to 100.0%, with the average of 21.5%. The sequence analysis of entire mitochondrial genomes of 51 probands exhibited distinct sets of polymorphisms belonging to 18 Eastern Asian haplogroups. The frequencies of haplogroup A and haplogroup F were sig-nificantly less in the LHON mtDNA samples than those in 106 Chinese controls. On the other hand, the haplogroup M10a accounted for 9.8% of the patient's mtDNA samples but was absent in 106 Chinese controls. Strikingly, the average pene-trance (46.13%) of optic neuropathy for the pedigrees carrying mitochondrial haplogroup M10a was higher than those car-rying other mtDNA haplogroups. These observations indicated that mitochondrial haplogroup M10a may increase the risk of visual loss. PMID:24846978

  14. Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma

    Science.gov (United States)

    Kloss-Brandstätter, Anita; Weissensteiner, Hansi; Erhart, Gertraud; Schäfer, Georg; Forer, Lukas; Schönherr, Sebastian; Pacher, Dominic; Seifarth, Christof; Stöckl, Andrea; Fendt, Liane; Sottsas, Irma; Klocker, Helmut; Huck, Christian W.; Rasse, Michael; Kronenberg, Florian; Kloss, Frank R.

    2015-01-01

    Background Oral squamous cell carcinoma (OSCC) is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA) mutations in primary oral tumors, recurrences and metastases. Methods We performed an in-depth validation of mtDNA next-generation sequencing (NGS) on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb) from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood) collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base). Results We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases. Conclusions We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer. PMID:26262956

  15. Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma.

    Directory of Open Access Journals (Sweden)

    Anita Kloss-Brandstätter

    Full Text Available Oral squamous cell carcinoma (OSCC is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA mutations in primary oral tumors, recurrences and metastases.We performed an in-depth validation of mtDNA next-generation sequencing (NGS on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base.We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases.We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer.

  16. DNA glycosylases involved in base excision repair may be associated with cancer risk in BRCA1 and BRCA2 mutation carriers.

    Directory of Open Access Journals (Sweden)

    Ana Osorio

    2014-04-01

    Full Text Available Single Nucleotide Polymorphisms (SNPs in genes involved in the DNA Base Excision Repair (BER pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase, and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2 gene (HR: 1.09, 95% CI (1.03-1.16, p = 2.7 × 10(-3 for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95%CI: 1.03-1.21, p = 4.8 × 10(-3. DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.

  17. Mitochondrial DNA D-loop AG/TC transition mutation in cortical neurons of mice after long-term exposure to nucleoside analogues.

    Science.gov (United States)

    Zhang, Yulin; Wang, Bishi; Liang, Qi; Qiao, Luxin; Xu, Bin; Zhang, Hongwei; Yang, Sufang; Chen, Jun; Guo, Hongliang; Wu, Jian; Chen, Dexi

    2015-10-01

    With the wide application of combined antiretroviral therapy, the prognosis of human immunodeficiency virus (HIV)-1 infected patient has been significantly improved. However, long-term administration of antiretroviral drugs can result in various drug-associated toxicities. Among them, nucleoside analogues were confirmed to inhibit DNA polymerase gamma, resulting in mitochondrial toxicity. Our previous study indicated that long-term exposure of mice to nucleoside analogue could induce mitochondria DNA (mtDNA) loss in cortical neurons. Herein, we further identify mitochondrial toxicity of four nucleoside analogues (zidovudine (AZT), stavudine (D4T), lamivudine (3TC), and didanosine (DDI)) by cloning and sequencing mtDNA D-loop region in mice neurons captured with laser capture microdissection. The results showed that mutation of neuronal mtDNA D-loop sequences increased in mice treated with each of the four nucleoside analogues for 4 months and D4T and DDI induced more severe D-loop lesion than the other two nucleoside analogues. The major type of D-loop point mutations induced by four nucleoside analogues was transition, in particular of "A→G" and "T→C" transition, but the point transition sites were variable. Our findings suggest that long-term exposure to nucleoside analogue can result in mtDNA D-loop region lesion in mouse cortical neurons. PMID:26015313

  18. Recombination-Independent Recognition of DNA Homology for Repeat-Induced Point Mutation (RIP) Is Modulated by the Underlying Nucleotide Sequence.

    Science.gov (United States)

    Gladyshev, Eugene; Kleckner, Nancy

    2016-05-01

    Haploid germline nuclei of many filamentous fungi have the capacity to detect homologous nucleotide sequences present on the same or different chromosomes. Once recognized, such sequences can undergo cytosine methylation or cytosine-to-thymine mutation specifically over the extent of shared homology. In Neurospora crassa this process is known as Repeat-Induced Point mutation (RIP). Previously, we showed that RIP did not require MEI-3, the only RecA homolog in Neurospora, and that it could detect homologous trinucleotides interspersed with a matching periodicity of 11 or 12 base-pairs along participating chromosomal segments. This pattern was consistent with a mechanism of homology recognition that involved direct interactions between co-aligned double-stranded (ds) DNA molecules, where sequence-specific dsDNA/dsDNA contacts could be established using no more than one triplet per turn. In the present study we have further explored the DNA sequence requirements for RIP. In our previous work, interspersed homologies were always examined in the context of a relatively long adjoining region of perfect homology. Using a new repeat system lacking this strong interaction, we now show that interspersed homologies with overall sequence identity of only 36% can be efficiently detected by RIP in the absence of any perfect homology. Furthermore, in this new system, where the total amount of homology is near the critical threshold required for RIP, the nucleotide composition of participating DNA molecules is identified as an important factor. Our results specifically pinpoint the triplet 5'-GAC-3' as a particularly efficient unit of homology recognition. Finally, we present experimental evidence that the process of homology sensing can be uncoupled from the downstream mutation. Taken together, our results advance the notion that sequence information can be compared directly between double-stranded DNA molecules during RIP and, potentially, in other processes where homologous

  19. The DNA Ligase IV Syndrome R278H Mutation Impairs B Lymphopoiesis via Error-Prone Nonhomologous End-Joining.

    Science.gov (United States)

    Park, Jihye; Welner, Robert S; Chan, Mei-Yee; Troppito, Logan; Staber, Philipp B; Tenen, Daniel G; Yan, Catherine T

    2016-01-01

    Hypomorphic mutations in the nonhomologous end-joining (NHEJ) DNA repair protein DNA ligase IV (LIG4) lead to immunodeficiency with varying severity. In this study, using a murine knock-in model, we investigated the mechanisms underlying abnormalities in class switch recombination (CSR) associated with the human homozygous Lig4 R278H mutation. Previously, we found that despite the near absence of Lig4 end-ligation activity and severely reduced mature B cell numbers, Lig4(R278H/R278H) (Lig4(R/R)) mice exhibit only a partial CSR block, producing near normal IgG1 and IgE but substantially reduced IgG3, IgG2b, and IgA serum levels. In this study, to address the cause of these abnormalities, we assayed CSR in Lig4(R/R) B cells generated via preassembled IgH and IgK V region exons (HL). This revealed that Lig4(R278H) protein levels while intact exhibited a higher turnover rate during activation of switching to IgG3 and IgG2b, as well as delays in CSR kinetics associated with defective proliferation during activation of switching to IgG1 and IgE. Activated Lig4(R/R)HL B cells consistently accumulated high frequencies of activation-induced cytidine deaminase-dependent IgH locus chromosomal breaks and translocations and were more prone to apoptosis, effects that appeared to be p53-independent, as p53 deficiency did not markedly influence these events. Importantly, NHEJ instead of alternative end-joining (A-EJ) was revealed as the predominant mechanism catalyzing robust CSR. Defective CSR was linked to failed NHEJ and residual A-EJ access to unrepaired double-strand breaks. These data firmly demonstrate that Lig4(R278H) activity renders NHEJ to be more error-prone, and they predict increased error-prone NHEJ activity and A-EJ suppression as the cause of the defective B lymphopoiesis in Lig4 patients. PMID:26608917

  20. Rational design of point mutation-selective antisense DNA targeted to codon 12 of Ha-ras mRNA in human cells.

    Science.gov (United States)

    Duroux, I; Godard, G; Boidot-Forget, M; Schwab, G; Hélène, C; Saison-Behmoaras, T

    1995-09-11

    Antisense oligodeoxynucleotides targeted to Ha-ras mRNA have been designed to discriminate between the codon 12-mutated oncogene and the normal proto-oncogene. An in vitro assay using two different sources of RNase H (rabbit reticulocyte lysates and nuclear extract from HeLa cells) was used to characterize oligonucleotide binding to normal and mutated Ha-ras mRNA. Short oligonucleotides (12- or 13mers) centered on the mutation had a very high discriminatory efficiency. Longer oligonucleotides (16mers) did not discriminate efficiently between the mutated and the normal mRNA. We have tested the efficacy of dodecanucleotides to induce RNase H cleavage of the full-length mRNA, moving the target sequence from the loop to the stem region which is formed in the vicinity of mutated codon 12. The most selective oligonucleotides were centered on the mutation which is located near the junction between the loop and stem regions even though they were less efficient at inducing RNase H cleavage than those targeted to the loop region. The 12mer antisense oligonucleotide with the highest discriminatory power was selected for cell culture studies. This oligonucleotide inhibited the proliferation of a human cell line which had been transformed with the mutated Ha-ras gene (HBL100ras1) but had no effect on the parental cell line which was transfected with the vector DNA (HBL 100neo) and expressed only the normal Ha-ras gene. Growth inhibition of HBL100ras1 cells was associated with specific ablation of targeted Ha-ras mRNA as shown by RT-PCR. These results show that 'in vitro' evaluation using an RNase H assay allowed us to select an antisense oligonucleotide which elicited a selectivity towards point-mutated Ha-ras mRNA when added at 10 microM concentration to the culture medium of cells expressing wild type and mutated Ha-ras mRNA. PMID:7567450

  1. Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.

    Directory of Open Access Journals (Sweden)

    Raphael Roduit

    Full Text Available BACKGROUND: NR2E3 (PNR is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S- cone syndrome (ESCS and, more recently, with autosomal dominant retinitis pigmentosa (adRP. NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD. The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2. NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients.

  2. cDNA analyses of CAPN3 enhance mutation detection and reveal a low prevalence of LGMD2A patients in Denmark

    DEFF Research Database (Denmark)

    Duno, M.; Sveen, M.L.; Schwartz, M.;

    2008-01-01

    Calpainopathy or limb-girdle muscular dystrophy type 2A (LGMD2A) is generally recognized as the most prevalent form of recessive LGMD and is caused by mutations in the CAPN3 gene. Out of a cohort of 119 patients fulfilling clinical criteria for LGMD2, referred to our neuromuscular clinic, 46 were......, only a single heterozygous mutation could be identified both at the genomic level and on full-length CAPN3 cDNA. All three patients exhibited a highly abnormal western blot for calpain-3 and clinical characteristics of LGMD2A. Only three of the genetically confirmed LGMD2A patients were of Danish...

  3. Genetic study of a membrane protein: DNA sequence alterations due to 17 lamB point mutations affecting adsorption of phage lambda.

    OpenAIRE

    Clément, J M; Lepouce, E; Marchal, C.; Hofnung, M

    1983-01-01

    Gene lamB encodes the outer membrane receptor for phage lambda in Escherichia coli K12. We have determined the DNA sequence alterations of 17 lamB point mutations which result in resistance to phage lambda h+. The mutations correspond to four phenotypic classes according to the pattern of growth of three phages which use the lambda receptor: lambda h (a one-step host-range derivative of lambda h+), lambda hh* (a two-step host-range derivative of lambda h+) and K10 (another lambdoid phage). Fo...

  4. Frameshift mutations in infectious cDNA clones of Citrus tristeza virus: a strategy to minimize the toxicity of viral sequences to Escherichia coli

    International Nuclear Information System (INIS)

    The advent of reverse genetics revolutionized the study of positive-stranded RNA viruses that were amenable for cloning as cDNAs into high-copy-number plasmids of Escherichia coli. However, some viruses are inherently refractory to cloning in high-copy-number plasmids due to toxicity of viral sequences to E. coli. We report a strategy that is a compromise between infectivity of the RNA transcripts and toxicity to E. coli effected by introducing frameshift mutations into 'slippery sequences' near the viral 'toxicity sequences' in the viral cDNA. Citrus tristeza virus (CTV) has cDNA sequences that are toxic to E. coli. The original full-length infectious cDNA of CTV and a derivative replicon, CTV-ΔCla, cloned into pUC119, resulted in unusually limited E. coli growth. However, upon sequencing of these cDNAs, an additional uridinylate (U) was found in a stretch of U's between nts 3726 and 3731 that resulted in a change to a reading frame with a stop codon at nt 3734. Yet, in vitro produced RNA transcripts from these clones infected protoplasts, and the resulting progeny virus was repaired. Correction of the frameshift mutation in the CTV cDNA constructs resulted in increased infectivity of in vitro produced RNA transcripts, but also caused a substantial increase of toxicity to E. coli, now requiring 3 days to develop visible colonies. Frameshift mutations created in sequences not suspected to facilitate reading frame shifting and silent mutations introduced into oligo(U) regions resulted in complete loss of infectivity, suggesting that the oligo(U) region facilitated the repair of the frameshift mutation. Additional frameshift mutations introduced into other oligo(U) regions also resulted in transcripts with reduced infectivity similarly to the original clones with the +1 insertion. However, only the frameshift mutations introduced into oligo(U) regions that were near and before the toxicity region improved growth and stability in E. coli. These data demonstrate that

  5. Mutation in the bimD gene of Aspergillus nidulans confers a conditional mitotic block and sensitivity to DNA damaging agents

    International Nuclear Information System (INIS)

    Mutation in the bimD gene of Aspergillus nidulans results in a mitotic block in anaphase characterized by a defective mitosis. Mutation in bimD also confers, at temperatures permissive for the mitotic arrest phenotype, an increased sensitivity to DNA damaging agents, including methyl methanesulfonate and ultraviolet light. In order to better understand the relationship between DNA damage and mitotic progression, the authors cloned the bimD gene from Aspergillus. A cosmid containing the bimD gene was identified among pools of cosmids by cotransformation with the nutritional selective pyrG gene of a strain carrying the recessive, temperature-sensitive lethal bimD6 mutation. The bimD gene encodes a predicted polypeptide of 166,000 daltons in mass and contains amino acid sequence motifs similar to those found in some DNA-binding transcription factors. These sequences include a basic domain followed by a leucine zipper, which together are called a bZIP motif, and a carboxyl-terminal domain enriched in acidic amino acids. Overexpression of the wild-type bimD protein resulted in an arrest of the nuclear division cycle that was reversible and determined to be in either the G1 or S phase of the cell cycle. The data suggest that bimD may play an essential regulatory role relating to DNA metabolism which is required for a successful mitosis. 7l refs., 7 figs., 1 tab

  6. Parkinson's disease brain mitochondria have impaired respirasome assembly, age-related increases in distribution of oxidative damage to mtDNA and no differences in heteroplasmic mtDNA mutation abundance

    Directory of Open Access Journals (Sweden)

    Keeney Paula M

    2009-09-01

    Full Text Available Abstract Background Sporadic Parkinson's disease (sPD is a nervous system-wide disease that presents with a bradykinetic movement disorder and is frequently complicated by depression and cognitive impairment. sPD likely has multiple interacting causes that include increased oxidative stress damage to mitochondrial components and reduced mitochondrial bioenergetic capacity. We analyzed mitochondria from postmortem sPD and CTL brains for evidence of oxidative damage to mitochondrial DNA (mtDNA, heteroplasmic mtDNA point mutations and levels of electron transport chain proteins. We sought to determine if sPD brains possess any mtDNA genotype-respiratory phenotype relationships. Results Treatment of sPD brain mtDNA with the mitochondrial base-excision repair enzyme 8-oxyguanosine glycosylase-1 (hOGG1 inhibited, in an age-dependent manner, qPCR amplification of overlapping ~2 kbase products; amplification of CTL brain mtDNA showed moderate sensitivity to hOGG1 not dependent on donor age. hOGG1 mRNA expression was not different between sPD and CTL brains. Heteroplasmy analysis of brain mtDNA using Surveyor nuclease® showed asymmetric distributions and levels of heteroplasmic mutations across mtDNA but no patterns that statistically distinguished sPD from CTL. sPD brain mitochondria displayed reductions of nine respirasome proteins (respiratory complexes I-V. Reduced levels of sPD brain mitochondrial complex II, III and V, but not complex I or IV proteins, correlated closely with rates of NADH-driven electron flow. mtDNA levels and PGC-1α expression did not differ between sPD and CTL brains. Conclusion PD brain mitochondria have reduced mitochondrial respiratory protein levels in complexes I-V, implying a generalized defect in respirasome assembly. These deficiencies do not appear to arise from altered point mutational burden in mtDNA or reduction of nuclear signaling for mitochondrial biogenesis, implying downstream etiologies. The origin of age

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

    Science.gov (United States)

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

    2016-08-01

    In childhood, several rare genetic diseases have overlapping symptoms and signs, including those regarding growth alterations, thus the differential diagnosis is sometimes difficult. The proband, aged 3 years, was suspected to have Silver-Russel syndrome because of intrauterine growth retardation, postnatal growth retardation, typical facial dysmorphic features, macrocephaly, body asymmetry, and bilateral fifth finger clinodactyly. Other features were left atrial and ventricular enlargement and patent foramen ovale. Total X-ray skeleton showed hypoplasia of the twelfth rib bilaterally and of the coccyx, slender long bones with thick cortex, and narrow medullary channels. The genetic investigation did not confirm Silver-Russel syndrome. At the age of 5 the patient developed an additional sign: hepatomegaly. Array CGH revealed a 147 kb deletion (involving TRIM 37 and SKA2 genes) on one allele of chromosome 17, inherited from his mother. These results suggested Mulibrey nanism. The clinical features were found to fit this hypothesis. Sequencing of the TRIM 37 gene showed a single base change at a splicing locus, inherited from his father that provoked a truncated protein. The combined use of Array CGH and DNA sequencing confirmed diagnosis of Mulibrey nanism. The large deletion involving the SKA2 gene, along with the increased frequency of malignant tumours in mulibrey patients, suggests closed monitoring for cancer of our patient and his mother. Array CGH should be performed as first tier test in all infants with multiple anomalies. The clinician should reconsider the clinical features when the genetics suggests this. © 2016 Wiley Periodicals, Inc. PMID:27256967

  8. deconstructSigs: delineating mutational processes in single tumors distinguishes DNA repair deficiencies and patterns of carcinoma evolution

    OpenAIRE

    Rosenthal, R; McGranahan, N.; Herrero, J.; Taylor, B S; Swanton, C.

    2016-01-01

    Background Analysis of somatic mutations provides insight into the mutational processes that have shaped the cancer genome, but such analysis currently requires large cohorts. We develop deconstructSigs, which allows the identification of mutational signatures within a single tumor sample. Results Application of deconstructSigs identifies samples with DNA repair deficiencies and reveals distinct and dynamic mutational processes molding the cancer genome in esophageal adenocarcinoma compared t...

  9. UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

    International Nuclear Information System (INIS)

    We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in φX174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

  10. UVA activation of N-dialkylnitrosamines releasing nitric oxide, producing strand breaks as well as oxidative damages in DNA, and inducing mutations in the Ames test

    Energy Technology Data Exchange (ETDEWEB)

    Arimoto-Kobayashi, Sakae, E-mail: arimoto@cc.okayama-u.ac.jp [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan); Sano, Kayoko; Machida, Masaki; Kaji, Keiko; Yakushi, Keiko [Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima, Okayama 700-8530 (Japan)

    2010-09-10

    We investigated the photo-mutagenicity and photo-genotoxicity of N-dialkylnitrosamines and its mechanisms of UVA activation. With simultaneous irradiation of UVA, photo-mutagenicity of seven N-dialkylnitrosamines was observed in Ames bacteria (Salmonella typhimurium TA1535) in the absence of metabolic activation. Mutagenicity of pre-irradiated N-dialkylnitrosamines was also observed with S. typhimurium hisG46, TA100, TA102 and YG7108 in the absence of metabolic activation. UVA-mediated mutation with N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) decreased by adding either the NO or OH radical scavenger. When superhelical DNA was irradiated with N-dialkylnitrosamines, nicked circular DNA appeared. Ten N-dialkylnitrosamines examined produced strand breaks in the treated DNA in the presence of UVA. The level of single-strand breaks in {phi}X174 DNA mediated by N-nitrosomorpholine (NMOR) and UVA decreased by adding either a radical scavenger or superoxide dismutase. When calf thymus DNA was treated with N-dialkylnitrosamines (NDMA, NDEA, NMOR, N-nitrosopyrrolidine (NPYR) and N-nitrosopiperidine (NPIP)) and UVA, the ratio of 8-oxodG/dG in the DNA increased. Action spectra were obtained to determine if nitrosamine acts as a sensitizer of UVA. Both mutation frequency and NO formation were highest at the absorption maximum of nitrosamines, approximately 340 nm. The plots of NO formation and mutation frequency align with the absorption curve of NPYR, NMOR and NDMA. A significant linear correlation between the optical density of N-dialkynitrosamines at 340 nm and NO formation in each irradiated solution was revealed by ANOVA. We would like to propose the hypothesis that the N-nitroso moiety of N-dialkylnitrosamines absorbs UVA photons, UVA-photolysis of N-dialkylnitrosamines brings release of nitric oxide, and subsequent production of alkyl radical cations and active oxygen species follow as secondary events, which cause DNA strand breaks, oxidative and

  11. The SWI/SNF Subunit INI1 Contains an N-Terminal Winged Helix DNA Binding Domain that Is a Target for Mutations in Schwannomatosis.

    Science.gov (United States)

    Allen, Mark D; Freund, Stefan M V; Zinzalla, Giovanna; Bycroft, Mark

    2015-07-01

    SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromatin. In mammals they play a central role in regulating gene expression during differentiation and proliferation. Mutations in SWI/SNF subunits are among the most frequent gene alterations in cancer. The INI1/hSNF5/SMARCB1 subunit is mutated in both malignant rhabdoid tumor, a highly aggressive childhood cancer, and schwannomatosis, a tumor-predisposing syndrome characterized by mostly benign tumors of the CNS. Here, we show that mutations in INI1 that cause schwannomatosis target a hitherto unidentified N-terminal winged helix DNA binding domain that is also present in the BAF45a/PHF10 subunit of the SWI/SNF complex. The domain is structurally related to the SKI/SNO/DAC domain, which is found in a number of metazoan chromatin-associated proteins. PMID:26073604

  12. Algorithm for prediction of tumour suppressor p53 affinity for binding sites in DNA

    OpenAIRE

    Veprintsev, Dmitry B.; Fersht, Alan R.

    2008-01-01

    The tumour suppressor p53 is a transcription factor that binds DNA in the vicinity of the genes it controls. The affinity of p53 for specific binding sites relative to other DNA sequences is an inherent driving force for specificity, all other things being equal. We measured the binding affinities of systematically mutated consensus p53 DNA-binding sequences using automated fluorescence anisotropy titrations. Based on measurements of the effects of every possible single base-pair substitution...

  13. Deletion mutations of bacteriophage

    International Nuclear Information System (INIS)

    Resolution of mutation mechanism with structural changes of DNA was discussed through the studies using bacteriophage lambda. One of deletion mutations inductions of phage lambda is the irradiation of ultraviolet ray. It is not clear if the inductions are caused by errors in reparation of ultraviolet-induced damage or by the activation of int gene. Because the effective site of int gene lies within the regions unnecessary for existing, it is considered that int gene is connected to deletion mutations induction. A certain system using prophage complementarity enables to detect deletion mutations at essential hereditary sites and to solve the relations of deletion mutations with other recombination system, DNA reproduction and repairment system. Duplication and multiplication of hereditary elements were discussed. If lambda deletion mutations of the system, which can control recombination, reproduction and repairment of added DNA, are constructed, mutations mechanism with great changes of DNA structure can be solved by phage lambda. (Ichikawa, K.)

  14. Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location

    OpenAIRE

    Yauk, Carole; Polyzos, Aris; Rowan-Carroll, Andrea; Somers, Christopher M.; Godschalk, Roger W.; Van Schooten, Frederik J.; Berndt, M. Lynn; Pogribny, Igor P.; Koturbash, Igor; Williams, Andrew; Douglas, George R.; Kovalchuk, Olga

    2008-01-01

    Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysi...

  15. The protective roles of phosphorylated heat shock protein 27 in human cells harboring myoclonus epilepsy with ragged-red fibers A8344G mtDNA mutation.

    Science.gov (United States)

    Chen, Hsueh-Fu; Chen, Chin-Yi; Lin, Ting-Hui; Huang, Zhao-Wei; Chi, Tang-Hao; Ma, Yi-Shing; Wu, Shi-Bei; Wei, Yau-Huei; Hsieh, Mingli

    2012-08-01

    Mitochondrial DNA (mtDNA) mutations are associated with a large number of neuromuscular diseases. Myoclonus epilepsy with ragged-red fibers (MERRF) syndrome is a mitochondrial disease inherited through the maternal lineage. The most common mutation in MERRF syndrome, the A8344G mutation of mtDNA, is associated with severe defects in mitochondrial protein synthesis, which impair the assembly and function of the respiratory chain. We have previously shown that there is a decreased level of heat shock protein 27 (HSP27) in lymphoblastoid cells derived from a MERRF patient and in cytoplasmic hybrids (cybrids) harboring the A8344G mutation of mtDNA. In the present study, we found a dramatic decrease in the level of phosphorylated HSP27 (p-HSP27) in the mutant cybrids. Even though the steady-state level of p-HSP27 was reduced in the mutant cybrids, normal phosphorylation and dephosphorylation were observed upon exposure to stress, indicating normal kinase and phosphatase activities. To explore the roles that p-HSP27 may play, transfection experiments with HSP27 mutants, in which three specific serines were replaced with alanine or aspartic acid, showed that the phosphomimicking HSP27 desensitized mutant cybrids to apoptotic stress induced by staurosporine (STS). After heat shock stress, p-HSP27 was found to enter the nucleus immediately, and with a prolonged interval of recovery, p-HSP27 returned to the cytoplasm in wild-type cybrids but not in mutant cybrids. The translocation of p-HSP27 was correlated with cell viability, as shown by the increased number of apoptotic cells after p-HSP27 returned to the cytoplasm. In summary, our results demonstrate that p-HSP27 provides significant protection when cells are exposed to different stresses in the cell model of MERRF syndrome. Therapeutic agents targeting anomalous HSP27 phosphorylation might represent a potential treatment for mitochondrial diseases. PMID:22742457

  16. Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations.

    OpenAIRE

    Elledge, S J; Mulligan, J T; Ramer, S W; Spottswood, M; Davis, R W

    1991-01-01

    This work describes a multifunctional phage lambda expression vector system, lambda YES, designed to facilitate gene isolation from eukaryotes by complementation of Escherichia coli and Saccharomyces cerevisiae mutations. lambda YES vectors have a selection for cDNA inserts using an oligo adaptor strategy and are capable of expressing genes in both E. coli and S. cerevisiae. They also allow conversion from phage lambda to plasmid clones by using the cre-lox site-specific recombination system,...

  17. Automated direct sequencing of the iduronate-2 sulfatase gene reveals a vast spectrum of mutations causing Hunter syndrome (mucopolysaccharidosis type II) and a {open_quotes}hot spot{close_quotes} at R468

    Energy Technology Data Exchange (ETDEWEB)

    Whitley, C.B; Jonsson, J.J.; Aronovich, E.L. [Univ. of Minnesota Medical School, Minneapolis, MN (United States)

    1994-09-01

    Hunter syndrome is an X-linked recessive, lethal disease resulting from deficiency of iduronate-2-sulfatase (IDS) catalytic activity. Because of low reproductive fitness, most affected individuals are expected to have new mutations. Most of such defects are anticipated to be single base pair (bp) changes; however, several previous studies utilizing Southern analysis of RT-PCR have identified numerous large gene deletions in patients having the {open_quotes}severe form{close_quotes} with neurologic disease. To investigate the spectrum of IDS mutations, we have developed a method of automated direct sequencing of RT-PCR products representing the entire IDS coding region. Of 19 patients studied by this approach, only 1 had an IDS coding region which did not contain a mutation; 1 had a single bp insertion; 1 had a 2 bp deletion; and 13 had single-base substitutions. Of the 13 having single base substitutions, 2 resulted in aberrant splicing. Only 1 patient had a complete gene deletion; in view of previous reports, there was a surprising lack of major gene deletions. Notably, a CpG dinucleotide at R468 was identified as a {open_quotes}hot spot{close_quotes} for mutation. Five unrelated individuals had substitutions at this site which thus accounted for 28% of all mutations in this series: R468W (3 patients) and R468Q (2 patients). MspI digestion provided a method of rapid diagnosis and determination of heterozygote status for such R468 mutations. Genotype-phenotype correlations in this R468 group are not yet possible because of confounding information, i.e., there are both {open_quotes}mild{close_quotes} and {open_quotes}severe{close_quotes} patients in this group and some have co-existing neurologic diseases. This approach of gene sequencing appears to be necessary, and sufficient, to characterize the vast spectrum of mutations in Hunter syndrome.

  18. Mutation-Induced Population Shift in the MexR Conformational Ensemble Disengages DNA Binding: A Novel Mechanism for MarR Family Derepression.

    Science.gov (United States)

    Anandapadamanaban, Madhanagopal; Pilstål, Robert; Andresen, Cecilia; Trewhella, Jill; Moche, Martin; Wallner, Björn; Sunnerhagen, Maria

    2016-08-01

    MexR is a repressor of the MexAB-OprM multidrug efflux pump operon of Pseudomonas aeruginosa, where DNA-binding impairing mutations lead to multidrug resistance (MDR). Surprisingly, the crystal structure of an MDR-conferring MexR mutant R21W (2.19 Å) presented here is closely similar to wild-type MexR. However, our extended analysis, by molecular dynamics and small-angle X-ray scattering, reveals that the mutation stabilizes a ground state that is deficient of DNA binding and is shared by both mutant and wild-type MexR, whereas the DNA-binding state is only transiently reached by the more flexible wild-type MexR. This population shift in the conformational ensemble is effected by mutation-induced allosteric coupling of contact networks that are independent in the wild-type protein. We propose that the MexR-R21W mutant mimics derepression by small-molecule binding to MarR proteins, and that the described allosteric model based on population shifts may also apply to other MarR family members. PMID:27427478

  19. Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

    Directory of Open Access Journals (Sweden)

    Yu-Fen Lin

    Full Text Available We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

  20. High quality DNA obtained with an automated DNA extraction method with 70+ year old formalin-fixed celloidin-embedded (FFCE) blocks from the indiana medical history museum

    OpenAIRE

    Niland, Erin E; McGuire, Audrey; Cox, Mary H; Sandusky, George E

    2012-01-01

    DNA and RNA have been used as markers of tissue quality and integrity throughout the last few decades. In this research study, genomic quality DNA of kidney, liver, heart, lung, spleen, and brain were analyzed in tissues from post-mortem patients and surgical cancer cases spanning the past century. DNA extraction was performed on over 180 samples from: 70+ year old formalin-fixed celloidin-embedded (FFCE) tissues, formalin-fixed paraffin-embedded (FFPE) tissue samples from surgical cases and ...

  1. A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

    Directory of Open Access Journals (Sweden)

    Gao Chen

    2012-02-01

    Full Text Available Abstract Background The human papillomavirus (HPV E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. Results CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. Conclusions These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

  2. A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity

    OpenAIRE

    Debrand, Emmanuel; Lykoudi, Alexandra; Bradshaw, Elizabeth; Allen, Stephanie K.

    2015-01-01

    Introduction Non-invasive prenatal diagnosis (NIPD) makes use of cell-free fetal DNA (cffDNA) in the mother’s bloodstream as an alternative to invasive sampling methods such as amniocentesis or CVS, which carry a 0.5–1% risk of fetal loss. We describe a droplet digital PCR (ddPCR) assay designed to inform the testing options for couples whose offspring are at risk of suffering from cystic fibrosis via compound heterozygosity. By detecting the presence or absence of the paternal mutation in th...

  3. The DNA ND4 12026 A to G mutation of mitochondria and type 2 diabetes%线粒体DNA ND4 12026基因突变与糖尿病

    Institute of Scientific and Technical Information of China (English)

    秦跃娟; 于永春; 史京衡; 吴松华

    2001-01-01

    目的研究线粒体DNA ND4 12026位点在中国上海地区非肥胖2型糖尿病患者中的突变情况,并探讨该位点突变的临床特征。方法采用PCR-SSCP、PCR-RFLP及PCR产物直接测序等技术检测线粒体DNA、ND4片段的突变情况。结果发现286例糖尿病患者血样中有12例患者mtDNA ND4基因上12026位点存在AtoG的点突变,导致异亮氨酸错义突变成缬氨酸,而在242例非糖尿病对照组个体中仅1例存在同样突变。对该位点突变的临床分析发现起病年龄低,58%有家族史,需要胰岛素治疗。结论12026位点A to G的突变可能与糖尿病的发生有关,并有一定的临床特征。%Objective To investigate the prevalence of mutation at position 12026 of mitochondrial ND4 gene in Chinese diabetes and analyze the clinical characteristics of these patients. Methods Using PCR-SSCP, PCR-RFLP and PCR product direct sequencing technique, we analyzed the mutation of mitochondrial DNA(mtDNA) ND4 gene.Results A to G mutation at position 12026 of mitochondrial DNA ND4 were found in 12 diabetics (12/286) and one in the control subjects, which was resulted from misplacement of isoleucine by valine. The patients with mutation exhibited characteristics of early onset, more family histories(58 % ) and needed insulin therapy more frequently than the patients without the mutation. Conclusion We infer that the mutation at position 12026 is related to the development of diabetes. (Shanghai Med J, 2001,24: 210-213)

  4. Automated Extraction and Quantification of Human Cytomegalovirus DNA in Whole Blood by Real-Time PCR Assay

    OpenAIRE

    Mengelle, C.; Sandres-Sauné, K.; Pasquier, C; Rostaing, L.; Mansuy, J.-M.; Marty, M.; Da Silva, I.; Attal, M.; Massip, P.; Izopet, J.

    2003-01-01

    The measurement of human cytomegalovirus (HCMV) DNA in blood is becoming the standard method for monitoring HCMV infection in immune-suppressed and unsuppressed patients. As various blood compartments can be used, we have compared the HCMV DNA measured in whole blood (WB), peripheral blood leukocytes (PBL), and plasma by real-time PCR. We tested 286 samples: HCMV DNA was extracted automatically from WB and PBL with the MagNA Pure instrument (Roche Molecular Biochemicals) and manually from pla...

  5. Heterozygous PALB2 c.1592delT mutation channels DNA double-strand break repair into error-prone pathways in breast cancer patients.

    Science.gov (United States)

    Obermeier, K; Sachsenweger, J; Friedl, T W P; Pospiech, H; Winqvist, R; Wiesmüller, L

    2016-07-21

    Hereditary heterozygous mutations in a variety of DNA double-strand break (DSB) repair genes have been associated with increased breast cancer risk. In the Finnish population, PALB2 (partner and localizer of BRCA2) represents a major susceptibility gene for female breast cancer, and so far, only one mutation has been described, c.1592delT, which leads to a sixfold increased disease risk. PALB2 is thought to participate in homologous recombination (HR). However, the effect of the Finnish founder mutation on DSB repair has not been investigated. In the current study, we used a panel of lymphoblastoid cell lines (LCLs) derived from seven heterozygous female PALB2 c.1592delT mutation carriers with variable health status and six wild-type matched controls. The results of our DSB repair analysis showed that the PALB2 mutation causes specific changes in pathway usage, namely increases in error-prone single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) compared with wild-type LCLs. These data indicated haploinsufficiency regarding the suppression of error-prone DSB repair in PALB2 mutation carriers. To the contrary, neither reduced HR activities, nor impaired RAD51 filament assembly, nor sensitization to PARP inhibition were consistently observed. Expression of truncated mutant versus wild-type PALB2 verified a causal role of PALB2 c.1592delT in the shift to error-prone repair. Discrimination between healthy and malignancy-presenting PALB2 mutation carriers revealed a pathway shift particularly in the breast cancer patients, suggesting interaction of PALB2 c.1592delT with additional genomic lesions. Interestingly, the studied PALB2 mutation was associated with 53BP1 accumulation in the healthy mutation carriers but not the patients, and 53BP1 was limiting for error-prone MMEJ in patients but not in healthy carriers. Our study identified a rise in error-prone DSB repair as a potential threat to genomic integrity in heterozygous PALB2 mutation carriers

  6. Application of DNA based marker mutations for improvement of cereals and other sexually reproduced crop plants. Proceedings of a final research co-ordination meeting

    International Nuclear Information System (INIS)

    The Co-ordinated Research Programme (CRP) on the Application of DNA Based Marker Mutations for Improvement of Cereals and Other Sexually Reproduced Crop Plants represents the first of three CRPs dealing with the application of molecular markers to mutations and plant breeding and was implemented between 1992 and 1996. A second companion CRP entitled Use of Novel DNA Fingerprinting Techniques for the Detection and Characterization of Genetic Variation in Vegetatively Propagated Crops devoted to the application of molecular markers in vegetatively propagated crops species was implemented between 1993 and 1997. One positive consequence of these two CRPs has been the implementation of a third CRP entitled Radioactively Labeled DNA Probes for Crop Improvement, which began in 1995 and aims to provide enabling technologies, in the form of probes and primers, to laboratories in developing countries. The rapid development of molecular marker technologies has also resulted in a dramatic increase in request from developing Member States for technical co-operation projects utilizing molecular markers to improve local varieties for biotic and abiotic stresses and other traits of relevance. With the intensified use of induced mutations in genetic studies, it will be important to continue the important work of understanding induced mutations at the molecular level. Evidence of the progress made in implementing molecular marker technologies in laboratories around the world is presented in this publication, which contains the results presented by the participants at the fourth and final Research Co-ordination Meeting of the CRP held in Vienna, 4-8 November 1996. The FAO and IAEA wish to express their sincere appreciation to the participants of the meeting for their work during the project period resulting in the summary and scientific reports presented in this publication

  7. A comparative investigation of DNA strand breaks, sister chromatid exchanges and K-ras gene mutations induced by cadmium salts in cultured human cells

    International Nuclear Information System (INIS)

    Cadmium (Cd) is a toxic heavy metal of continuing occupational and environmental concern with a wide variety of adverse effects. Several studies have shown that cadmium produces DNA strand breaks, DNA-protein cross-links, oxidative DNA damage, chromosomal aberrations, dysregulation of gene expression resulting in enhanced proliferation, depressed apoptosis and/or altered DNA repair. This study was undertaken to investigate the ability of cadmium chloride (CdCl2) and cadmium sulphate (CdSO4) to induce point mutations in codon 12 of the K-ras protooncogene assessed by polymerase chain reaction-single strand conformation polymorphisms (PCR-SSCP) and RFLP-enriched PCR methods. Also their genotoxic effects were analyzed by the comet assay and sister chromatid exchanges test. The human lung fibroblast cell line MRC-5 was used for the experiments. Sister chromatid exchanges assay (SCEs) frequencies were significantly increased in cells exposed to cadmium salts in relation to controls (p < 0.001). Despite the slow increment observed in the three comet parameters considered when cells were treated with cadmium chloride, significant differences between groups were only found in the variable comet moment (CM) (p < 0.005). On the other hand, when cells were exposed to cadmium sulphate, the Kruskal-Wallis test showed highly significant differences between groups for migration, tail moment and comet moment parameters (p < 0.001). Nevertheless, a null or weak point mutation induction in K-ras protooncogene was detected using polymerase chain reaction-low ionic strength-single strand conformation polymorphisms (PCR-LIS-SSCP) and RFLP-enriched PCR methods when cells were treated with cadmium salts. Thus, inorganic cadmium produces genotoxicity in human lung fibroblast MRC-5 cells, in the absence of significant point mutation of the K-ras gene

  8. The impact of SF3B1 mutations in CLL on the DNA-damage response

    DEFF Research Database (Denmark)

    Te Raa, G D; Derks, I A M; Navrkalova, V;

    2015-01-01

    Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part assoc...

  9. A new mtDNA mutation in the tRNA[sup Lys] gene associated with myoclonic epilepsy and ragged-red fibers (MERRF)

    Energy Technology Data Exchange (ETDEWEB)

    Silvestri, G.; Moraes, C.T.; Shanske, S.; DiMauro, S. (Columbia Univ. College of Physicians and Surgeons, New York, NY (United States)); Oh, S.J. (Univ. of Alabama, Birmingham (United States))

    1992-12-01

    Myoclonic epilepsy with ragged-red fibers (MERRF) has been associated with an A[r arrow]G transition at mtDNA nt 8344, within a conserved region of the tRNA[sup Lys] gene. Although the 8344 mutation is highly prevalent in patients with MERRF, it is not observed in 10%-20% of the cases, suggesting genetic heterogeneity. The authors have sequenced the tRNA[sup Lys] gene of five MERRF patients lacking the common 8344 mutation. One of these showed a novel T[r arrow]C transition at nucleotide position 8356, disrupting a highly conserved base pair in the T[Psi]C stem. The mutant mtDNA population was essentially homoplasmic in muscle but was heteroplasmic in blood (47%). Neither 20 patients with other mitochondrial diseases nor 25 controls carried this mutation. These findings suggest that tRNA[sup Lys] alterations may play a specific role in the pathogenesis of MERRF syndrome. 21 refs., 4 figs.

  10. Low-dose DNA damage and replication stress responses quantified by optimized automated single-cell image analysis

    DEFF Research Database (Denmark)

    Mistrik, Martin; Oplustilova, Lenka; Lukas, Jiri;

    2009-01-01

    advantages and applicability of this technique. Our present data on assessment of low radiation doses, repair kinetics, spontaneous DNA damage in cancer cells, as well as constitutive and replication stress-induced HR events and their dependence on upstream factors within the DDR machinery document the......Maintenance of genome integrity is essential for homeostasis and survival as impaired DNA damage response (DDR) may predispose to grave pathologies such as neurodegenerative and immunodeficiency syndromes, cancer and premature aging. Therefore, accurate assessment of DNA damage caused by...... environmental or metabolic genotoxic insults is critical for contemporary biomedicine. The available physical, flow cytometry and sophisticated scanning approaches to DNA damage estimation each have some drawbacks such as insufficient sensitivity, limitation to analysis of cells in suspension, or high costs and...

  11. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

    OpenAIRE

    Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

    2015-01-01

    Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no appar...

  12. Only male matrilineal relatives with Leber's hereditary optic neuropathy in a large Chinese family carrying the mitochondrial DNA G11778A mutation

    International Nuclear Information System (INIS)

    We report here the characterization of a five-generation large Chinese family with Leber's hereditary optic neuropathy (LHON). Very strikingly, six affected individuals of 38 matrilineal relatives (17 females/21 males) are exclusively males in this Chinese family. These matrilineal relatives in this family exhibited late-onset/progressive visual impairment with a wide range of severity, ranging from blindness to normal vision. The age of onset in visual impairment varies from 17 to 30 years. Sequence analysis of the complete mitochondrial genome in this pedigree revealed the presence of the G11778A mutation in ND4 gene and 29 other variants. This mitochondrial genome belongs to the Southern Chinese haplogroup B5b. We showed that the G11778A mutation is present at near homoplasmy in matrilineal relatives of this Chinese family but not in 164 Chinese controls. Incomplete penetrance of LHON in this family indicates the involvement of modulatory factors in the phenotypic expression of visual dysfunction associated with the G11778A mutation. However, none of other mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence. Thus, nuclear modifier gene(s) or environmental factor(s) seem to account for the penetrance and phenotypic variability of LHON in this Chinese family carrying the G11778A mutation

  13. MUTATION SPECTRA IN SALMONELLA OF SUNLIGHT, WHITE FLUORESCENT LIGHT, AND LIGHT FROM TANNING SALON BEDS: INDUCTION OF TANDEM MUTATIONS AND ROLE OF DNA REPAIR

    Science.gov (United States)

    We evaluated the mutagenicity of sunlight (SUN), uncovered cool white fluorescent light (FLR), and light from a tanning salon bed (TAN) at the base-substitution allele hisG46 of Salmonella in four DNA repair backgrounds (wild type, uvrB, pKM101, and uvrB+pKM101). Approximately 80...

  14. Collateral damage: Spread of repeat-induced point mutation from a duplicated DNA sequence into an adjoining single-copy gene in Neurospora crassa

    Indian Academy of Sciences (India)

    Meenal Vyas; Durgadas P Kasbekar

    2005-02-01

    Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered in Neurospora crassa. RIP occurs during a sexual cross and induces numerous G : C to A : T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of the erg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1.5 or 2 kb) and located at defined distances (0, 0.5, 1 or 2 kb) upstream or downstream of the erg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency of erg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into the erg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0.1–0.8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterial hph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation.

  15. Mutation of the mouse Syce1 gene disrupts synapsis and suggests a link between synaptonemal complex structural components and DNA repair.

    Directory of Open Access Journals (Sweden)

    Ewelina Bolcun-Filas

    2009-02-01

    Full Text Available In mammals, the synaptonemal complex is a structure required to complete crossover recombination. Although suggested by cytological work, in vivo links between the structural proteins of the synaptonemal complex and the proteins of the recombination process have not previously been made. The central element of the synaptonemal complex is traversed by DNA at sites of recombination and presents a logical place to look for interactions between these components. There are four known central element proteins, three of which have previously been mutated. Here, we complete the set by creating a null mutation in the Syce1 gene in mouse. The resulting disruption of synapsis in these animals has allowed us to demonstrate a biochemical interaction between the structural protein SYCE2 and the repair protein RAD51. In normal meiosis, this interaction may be responsible for promoting homologous synapsis from sites of recombination.

  16. Novel mutations and DNA-based screening in non-Jewish carriers of Tay-Sachs disease.

    OpenAIRE

    Akerman, B R; Natowicz, M R; Kaback, M M; Loyer, M.; Campeau, E; Gravel, R A

    1997-01-01

    We have evaluated the feasibility of using PCR-based mutation screening for non-Jewish enzyme-defined carriers identified through Tay-Sachs disease-prevention programs. Although Tay-Sachs mutations are rare in the general population, non-Jewish individuals may be screened as spouses of Jewish carriers or as relatives of probands. In order to define a panel of alleles that might account for the majority of mutations in non-Jewish carriers, we investigated 26 independent alleles from 20 obligat...

  17. Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons

    OpenAIRE

    Yao, Fei; Ariyaratne, Pramila N.; Hillmer, Axel M.; Lee, Wah Heng; Li, Guoliang; Teo, Audrey S. M.; Woo, Xing Yi; Zhang, Zhenshui; Chen, Jieqi P.; Poh, Wan Ting; Zawack, Kelson F. B.; Chan, Chee Seng; Leong, See Ting; Neo, Say Chuan; Choi, Poh Sum D.

    2012-01-01

    Structural variations (SVs) contribute significantly to the variability of the human genome and extensive genomic rearrangements are a hallmark of cancer. While genomic DNA paired-end-tag (DNA-PET) sequencing is an attractive approach to identify genomic SVs, the current application of PET sequencing with short insert size DNA can be insufficient for the comprehensive mapping of SVs in low complexity and repeat-rich genomic regions. We employed a recently developed procedure to generate PET s...

  18. A pedigree of Leber's hereditary optic neuropathy with mtDNA14484 mutation%伴有mtDNA14484位点突变的Leber病一大家系

    Institute of Scientific and Technical Information of China (English)

    童绎; 王影; 姜枫; 刘斌; 张守康; 杨薇

    2008-01-01

    Objective To analyze a pedigree of Leber' s hereditary optic neuropathy, and its penetrance, anticipation, and spontaneous eyesight improvement, and its relationship with mitochondrial DNA mutation. Methods Eighteen members in the tinnily were undergone routine visual check. Five cases were taken visual evoked potential and visual field examination. DNA sequencing was performed on6 eases to check the mtDNA11778,3460 and 14484 loci.Results (1)The offsprings from the first wife in the first generation showed decreased acuity of the two eyes, which was optic atrophy identified by funduseopy. (2) The mtDNA had mutation at position 14484, but not at positions 11778 and 3460. Conclusion The pedigree showed a typical maternal inheritance of Leber' s hereditary optic neuropathy. It was eaused by mtDNA 14484 mutation.%目的 研究Leber病一大家系的遗传因素,探讨其外显率、遗传早现、自发视力恢复,以及与mtDNA的关联性.方法 对家系中18例患者进行调查,并行视力、眼底等常规检查;5例患者作了视觉诱发电位(visual evoked potential,VEP)及视野检查;应用聚合酶链反应对6例患者外周血液进行mtDNA11778、3460及144843个位点检查.结果 (1)本家系第Ⅰ代娶二妻,前妻后代皆无明显诱因出现双眼视力下降,经眼底镜检查符合视神经萎缩诊断.后妻无眼病,其后代均无眼病.(2)6例患者的mtDNA检查显示11778、3460位点未发现突变,而在14484位点出现同质性突变.结论 该家系Leber病呈典型母系遗传,该病的临床表现可能与mtDNA14484位点突变密切关联.

  19. High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation

    Directory of Open Access Journals (Sweden)

    Kramme Stefanie

    2008-05-01

    Full Text Available Abstract Background Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. Due to its high environmental stability and infectivity it is regarded as a category B biological weapon agent. In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis. Endocarditis represents the most common form of chronic Q-fever. In humans serology is the gold standard for diagnosis but is inadequate for early case detection. In order to serve as a diagnostic tool in an eventual biological weapon attack or in local epidemics we developed a real-time 5'nuclease based PCR assay with an internal control system. To facilitate high-throughput an automated extraction procedure was evaluated. Results To determine the minimum number of copies that are detectable at 95% chance probit analysis was used. Limit of detection in blood was 2,881 copies/ml [95%CI, 2,188–4,745 copies/ml] with a manual extraction procedure and 4,235 copies/ml [95%CI, 3,143–7,428 copies/ml] with a fully automated extraction procedure, respectively. To demonstrate clinical application a total of 72 specimens of animal origin were compared with respect to manual and automated extraction. A strong correlation between both methods was observed rendering both methods suitable. Testing of 247 follow up specimens of animal origin from a local Q-fever epidemic rendered real-time PCR more sensitive than conventional PCR. Conclusion A sensitive and thoroughly evaluated real-time PCR was established. Its high-throughput mode may show a useful approach to rapidly screen samples in local outbreaks for other organisms relevant for humans or animals. Compared to a conventional PCR assay sensitivity of real-time PCR was higher after testing samples from a local Q-fever outbreak.

  20. Elevated incidence of polyp formation in APC(Min/⁺Msh2⁻/⁻ mice is independent of nitric oxide-induced DNA mutations.

    Directory of Open Access Journals (Sweden)

    Antoaneta Belcheva

    Full Text Available Gut microbiota has been linked to a number of human diseases including colon cancer. However, the mechanism through which gut bacteria influence colon cancer development and progression remains unclear. Perturbation of the homeostasis between the host immune system and microbiota leads to inflammation and activation of macrophages which produce large amounts of nitric oxide that acts as a genotoxic effector molecule to suppress bacterial growth. However, nitric oxide also has genotoxic effects to host cells by producing mutations that can predispose to colon cancer development. The major DNA lesions caused by nitric oxide are 8oxoG and deamination of deoxycytosine bases. Cellular glycosylases that belong to the base excision repair pathway have been demonstrated to repair these mutations. Recent evidence suggests that the mismatch repair pathway (MMR might also repair nitric oxide-induced DNA damage. Since deficiency in MMR predisposes to colon cancer, we hypothesized that MMR-deficient colon epithelial cells are incapable of repairing nitric-oxide induced genetic lesions that can promote colon cancer. Indeed, we found that the MMR pathway repairs nitric oxide-induced DNA mutations in cell lines. To test whether nitric oxide promotes colon cancer, we genetically ablated the inducible nitric oxide synthase (iNOS or inhibited iNOS activity in the APC(Min/+Msh2(-/- mouse model of colon cancer. However, despite the fact that nitric oxide production was strongly reduced in the colon using both approaches, colon cancer incidence was not affected. These data show that nitric oxide and iNOS do not promote colon cancer in APC(Min/+Msh2(-/- mice.

  1. The mitochondrial ND1 m.3337G>A mutation associated to multiple mitochondrial DNA deletions in a patient with Wolfram syndrome and cardiomyopathy

    Energy Technology Data Exchange (ETDEWEB)

    Mezghani, Najla [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Mnif, Mouna [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Mkaouar-Rebai, Emna, E-mail: emna_mkaouar@mail2world.com [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Kallel, Nozha [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Salem, Ikhlass Haj [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Charfi, Nadia; Abid, Mohamed [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Fakhfakh, Faiza [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia)

    2011-07-29

    Highlights: {yields} We reported a patient with Wolfram syndrome and dilated cardiomyopathy. {yields} We detected the ND1 mitochondrial m.3337G>A mutation in 3 tested tissues (blood leukocytes, buccal mucosa and skeletal muscle). {yields} Long-range PCR amplification revealed the presence of multiple mitochondrial deletions in the skeletal muscle. {yields} The deletions remove several tRNA and protein-coding genes. -- Abstract: Wolfram syndrome (WFS) is a rare hereditary disorder also known as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). It is a heterogeneous disease and full characterization of all clinical and biological features of this disorder is difficult. The wide spectrum of clinical expression, affecting several organs and tissues, and the similarity in phenotype between patients with Wolfram syndrome and those with certain types of respiratory chain diseases suggests mitochondrial DNA (mtDNA) involvement in Wolfram syndrome patients. We report a Tunisian patient with clinical features of moderate Wolfram syndrome including diabetes, dilated cardiomyopathy and neurological complications. The results showed the presence of the mitochondrial ND1 m.3337G>A mutation in almost homoplasmic form in 3 tested tissues of the proband (blood leukocytes, buccal mucosa and skeletal muscle). In addition, the long-range PCR amplifications revealed the presence of multiple deletions of the mitochondrial DNA extracted from the patient's skeletal muscle removing several tRNA and protein-coding genes. Our study reported a Tunisian patient with clinical features of moderate Wolfram syndrome associated with cardiomyopathy, in whom we detected the ND1 m.3337G>A mutation with mitochondrial multiple deletions.

  2. The mitochondrial ND1 m.3337G>A mutation associated to multiple mitochondrial DNA deletions in a patient with Wolfram syndrome and cardiomyopathy

    International Nuclear Information System (INIS)

    Highlights: → We reported a patient with Wolfram syndrome and dilated cardiomyopathy. → We detected the ND1 mitochondrial m.3337G>A mutation in 3 tested tissues (blood leukocytes, buccal mucosa and skeletal muscle). → Long-range PCR amplification revealed the presence of multiple mitochondrial deletions in the skeletal muscle. → The deletions remove several tRNA and protein-coding genes. -- Abstract: Wolfram syndrome (WFS) is a rare hereditary disorder also known as DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). It is a heterogeneous disease and full characterization of all clinical and biological features of this disorder is difficult. The wide spectrum of clinical expression, affecting several organs and tissues, and the similarity in phenotype between patients with Wolfram syndrome and those with certain types of respiratory chain diseases suggests mitochondrial DNA (mtDNA) involvement in Wolfram syndrome patients. We report a Tunisian patient with clinical features of moderate Wolfram syndrome including diabetes, dilated cardiomyopathy and neurological complications. The results showed the presence of the mitochondrial ND1 m.3337G>A mutation in almost homoplasmic form in 3 tested tissues of the proband (blood leukocytes, buccal mucosa and skeletal muscle). In addition, the long-range PCR amplifications revealed the presence of multiple deletions of the mitochondrial DNA extracted from the patient's skeletal muscle removing several tRNA and protein-coding genes. Our study reported a Tunisian patient with clinical features of moderate Wolfram syndrome associated with cardiomyopathy, in whom we detected the ND1 m.3337G>A mutation with mitochondrial multiple deletions.

  3. Comparison of Boiling and Robotics Automation Method in DNA Extraction for Metagenomic Sequencing of Human Oral Microbes

    Science.gov (United States)

    Shinozaki, Natsuko; Ye, Bin; Tsuboi, Akito; Nagasaki, Masao; Yamashita, Riu

    2016-01-01

    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no “gold standard” for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study. PMID:27104353

  4. Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing and resistance mutations in the plasma DNA of patients with advanced non-small cell lung cancer during treatment with erlotinib

    DEFF Research Database (Denmark)

    Sorensen, Boe S; Wu, Lin; Wei, Wen; Tsai, Julie; Weber, Britta; Nexo, Ebba; Meldgaard, Peter

    2014-01-01

    was tested in plasma DNA from patients with advanced NSCLC with allele-specific polymerase chain reaction assays. Blood samples from 23 patients with adenocarcinoma of NSCLC that carried tyrosine kinase inhibitor-sensitizing EGFR mutations were taken immediately before treatment with erlotinib......BACKGROUND: The feasibility of monitoring epidermal growth factor receptor (EGFR) mutations in plasma DNA from patients with advanced non-small cell lung cancer (NSCLC) during treatment with erlotinib and its relation to disease progression was investigated. METHODS: The amount of EGFR-mutant DNA....... Additional blood samples were taken at timed intervals until erlotinib treatment was withdrawn. RESULTS: The amount of plasma DNA with sensitizing EGFR mutations was found to be reduced after the first cycle of erlotinib treatment in 22 of 23 patients (96%). No patients presented with the resistant T790M...

  5. Postmortem Genetic Testing for Conventional Autopsy–Negative Sudden Unexplained Death: An Evaluation of Different DNA Extraction Protocols and the Feasibility of Mutational Analysis From Archival Paraffin-Embedded Heart Tissue

    OpenAIRE

    Carturan, Elisa; Tester, David J.; Brost, Brian C.; Basso, Cristina; Thiene, Gaetano; Ackerman, Michael J.

    2008-01-01

    One third of autopsy-negative sudden unexplained deaths (SUDs) can be attributed to a cardiac channelopathy. Typically, paraffin-embedded tissue (PET) is the only source of DNA available for genetic analyses. We examined different DNA extraction procedures, involving 2 deparaffinization methods, 2 digestion methods, 4 laboratory-based purification methods, and 5 commercial kits. Mutational analysis involving 25 RYR2 exons was performed on PET DNA from 35 SUD cases to evaluate the feasibility ...

  6. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients

    OpenAIRE

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-01-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fi...

  7. Sequence of DNA flanking the exons of the HEXA gene, and identification of mutations in Tay-Sachs disease.

    OpenAIRE

    Triggs-Raine, B L; Akerman, B R; Clarke, J T; Gravel, R A

    1991-01-01

    The rapid identification of mutations causing Tay-Sachs disease requires the capacity to readily screen the regions of the HEXA gene most likely to be affected by mutation. We have sequenced the portions of the introns flanking each of the 14 HEXA exons in order to specify oligonucleotide primers for the PCR-dependent amplification of each exon and splice-junction sequence. The amplified products were analyzed, by electrophoresis in nondenaturing polyacrylamide gels, for the presence of eithe...

  8. Genome-wide SNP scan of pooled DNA reveals nonsense mutation in FGF20 in the scaleless line of featherless chickens

    Directory of Open Access Journals (Sweden)

    Wells Kirsty L

    2012-06-01

    Full Text Available Abstract Background Scaleless (sc/sc chickens carry a single recessive mutation that causes a lack of almost all body feathers, as well as foot scales and spurs, due to a failure of skin patterning during embryogenesis. This spontaneous mutant line, first described in the 1950s, has been used extensively to explore the tissue interactions involved in ectodermal appendage formation in embryonic skin. Moreover, the trait is potentially useful in tropical agriculture due to the ability of featherless chickens to tolerate heat, which is at present a major constraint to efficient poultry meat production in hot climates. In the interests of enhancing our understanding of feather placode development, and to provide the poultry industry with a strategy to breed heat-tolerant meat-type chickens (broilers, we mapped and identified the sc mutation. Results Through a cost-effective and labour-efficient SNP array mapping approach using DNA from sc/sc and sc/+ blood sample pools, we map the sc trait to chromosome 4 and show that a nonsense mutation in FGF20 is completely associated with the sc/sc phenotype. This mutation, common to all sc/sc individuals and absent from wild type, is predicted to lead to loss of a highly conserved region of the FGF20 protein important for FGF signalling. In situ hybridisation and quantitative RT-PCR studies reveal that FGF20 is epidermally expressed during the early stages of feather placode patterning. In addition, we describe a dCAPS genotyping assay based on the mutation, developed to facilitate discrimination between wild type and sc alleles. Conclusions This work represents the first loss of function genetic evidence supporting a role for FGF ligand signalling in feather development, and suggests FGF20 as a novel central player in the development of vertebrate skin appendages, including hair follicles and exocrine glands. In addition, this is to our knowledge the first report describing the use of the chicken SNP array to

  9. Analysis of adaptive mutations selected during the consecutive passages of hepatitis E virus produced from an infectious cDNA clone.

    Science.gov (United States)

    Nagashima, Shigeo; Kobayashi, Tominari; Tanaka, Toshinori; Tanggis; Jirintai, Suljid; Takahashi, Masaharu; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2016-09-01

    To characterize the genomic mutations of hepatitis E virus (HEV) during consecutive passages associated with adaptation to growth in cell culture, a cloned genotype 3 HEV [pJE03-1760F/wt, starting virus (SV)] was passaged 10 times in A549 cells, and the entire genomic sequence of the passage 10 (P10) progeny was determined. Compared to SV, P10 virus possessed two non-synonymous (T2808C and A5054G) and four synonymous mutations (C1213T, T2557C, C3118T and C4435T) in the ORF1. Full-length infectious cDNA clones with a single, double (T2808C and A5054G), or all six mutations, identical to P10, were constructed, and their replication capacity was compared. Four (C1213T, T2557C, T2808C and A5054G) of the six viruses with a single mutation grew more efficiently than SV. The P10 virus propagated more rapidly and grew more efficiently than SV and T2808C+A5054G and reached a higher viral load (95.1- and 8.5-fold, respectively) at 20days post-inoculation. An immunofluorescence analysis revealed that a high percentage (>80%) of cells inoculated with the P10 virus expressed ORF2 proteins, while relatively low percentages (nearly 30% or 5%) inoculated with T2808C+A5054G or SV, respectively, expressed ORF2 proteins. We found that not only non-synonymous but also synonymous HEV mutations are independently associated with increased virus production. PMID:27485920

  10. Comparison of Xpert MTB/RIF Assay and GenoType MTBDRplus DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis

    Science.gov (United States)

    Rahman, Arfatur; Sahrin, Mahfuza; Afrin, Sadia; Earley, Keith; Ahmed, Shahriar; Rahman, S. M. Mazidur; Banu, Sayera

    2016-01-01

    Background GeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (rpoB) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding. Methods We analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing. Results The agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases. Conclusion Although the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis. PMID:27054344

  11. A new sieving matrix for DNA sequencing, genotyping and mutation detection and high-throughput genotyping with a 96-capillary array system

    Energy Technology Data Exchange (ETDEWEB)

    Gao, David

    1999-11-08

    Capillary electrophoresis has been widely accepted as a fast separation technique in DNA analysis. In this dissertation, a new sieving matrix is described for DNA analysis, especially DNA sequencing, genetic typing and mutation detection. A high-throughput 96 capillary array electrophoresis system was also demonstrated for simultaneous multiple genotyping. The authors first evaluated the influence of different capillary coatings on the performance of DNA sequencing. A bare capillary was compared with a DB-wax, an FC-coated and a polyvinylpyrrolidone dynamically coated capillary with PEO as sieving matrix. It was found that covalently-coated capillaries had no better performance than bare capil