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Sample records for accurate dna copy

  1. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material

    PubMed Central

    Dong, Lianhua; Meng, Ying; Sui, Zhiwei; Wang, Jing; Wu, Liqing; Fu, Boqiang

    2015-01-01

    Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments. PMID:26302947

  2. Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

    PubMed Central

    Whale, Alexandra S.; Cowen, Simon; Foy, Carole A.; Huggett, Jim F.

    2013-01-01

    Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA. PMID:23472156

  3. Hacking DNA copy number for circuit engineering.

    PubMed

    Wu, Feilun; You, Lingchong

    2017-07-27

    DNA copy number represents an essential parameter in the dynamics of synthetic gene circuits but typically is not explicitly considered. A new study demonstrates how dynamic control of DNA copy number can serve as an effective strategy to program robust oscillations in gene expression circuits.

  4. Single copy DNA homology in sea stars.

    PubMed

    Smith, M J; Nicholson, R; Stuerzl, M; Lui, A

    1982-01-01

    The sequence homology in the single copy DNA of sea stars has been measured. Labeled single copy DNA from Pisaster ochraceus was reannealed with excess genomic DNA from P. brevispinus, Evasterias troschelii, Pycnopodia helianthoides, Solaster stimpsoni, and Dermasterias imbricata. Reassociation reactions were performed under two criteria of salt and temperature. The extent of reassociation and thermal denaturation characteristics of hybrid single copy DNA molecules follow classical taxonomic lines. P. brevispinus DNA contains essentially all of the sequences present in P. ochraceus single copy tracer while Evasterias and Pycnopodia DNAs contain 52% and 46% of such sequences respectively. Reciprocal reassociation reactions with labeled Evasterias single copy DNA confirm the amount and fidelity of the sequence homology. There is a small definite reaction of uncertain homology between P. ochraceus single copy DNA and Solaster or Dermasterias DNA. Similarly Solaster DNA contains sequences homologous to approximately 18% of Dermasterias unique DNA. The thermal denaturation temperatures of heteroduplexes indicate that the genera Pisaster and Evasterias diverged shortly after the divergence of the subfamilies Pycnopodiinae and Asteriinae. The two Pisaster species diverged more recently, probably in the most recent quarter of the interval since the separation of the genera Pisaster and Evasterias.

  5. Systematic biases in DNA copy number originate from isolation procedures

    PubMed Central

    2013-01-01

    Background The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal. Results While GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing. Conclusion These results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses. PMID:23618369

  6. DNA replication stress restricts ribosomal DNA copy number

    PubMed Central

    Salim, Devika; Bradford, William D.; Freeland, Amy; Cady, Gillian; Wang, Jianmin

    2017-01-01

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100–200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how “normal” copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen a yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a “normal” rDNA copy number. PMID:28915237

  7. DNA replication stress restricts ribosomal DNA copy number.

    PubMed

    Salim, Devika; Bradford, William D; Freeland, Amy; Cady, Gillian; Wang, Jianmin; Pruitt, Steven C; Gerton, Jennifer L

    2017-09-15

    Ribosomal RNAs (rRNAs) in budding yeast are encoded by ~100-200 repeats of a 9.1kb sequence arranged in tandem on chromosome XII, the ribosomal DNA (rDNA) locus. Copy number of rDNA repeat units in eukaryotic cells is maintained far in excess of the requirement for ribosome biogenesis. Despite the importance of the repeats for both ribosomal and non-ribosomal functions, it is currently not known how "normal" copy number is determined or maintained. To identify essential genes involved in the maintenance of rDNA copy number, we developed a droplet digital PCR based assay to measure rDNA copy number in yeast and used it to screen the yeast conditional temperature-sensitive mutant collection of essential genes. Our screen revealed that low rDNA copy number is associated with compromised DNA replication. Further, subculturing yeast under two separate conditions of DNA replication stress selected for a contraction of the rDNA array independent of the replication fork blocking protein, Fob1. Interestingly, cells with a contracted array grew better than their counterparts with normal copy number under conditions of DNA replication stress. Our data indicate that DNA replication stresses select for a smaller rDNA array. We speculate that this liberates scarce replication factors for use by the rest of the genome, which in turn helps cells complete DNA replication and continue to propagate. Interestingly, tumors from mini chromosome maintenance 2 (MCM2)-deficient mice also show a loss of rDNA repeats. Our data suggest that a reduction in rDNA copy number may indicate a history of DNA replication stress, and that rDNA array size could serve as a diagnostic marker for replication stress. Taken together, these data begin to suggest the selective pressures that combine to yield a "normal" rDNA copy number.

  8. Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats

    PubMed Central

    Armour, John A. L.; Palla, Raquel; Zeeuwen, Patrick L. J. M.; den Heijer, Martin; Schalkwijk, Joost; Hollox, Edward J.

    2007-01-01

    Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies. PMID:17175532

  9. Most Cancers Caused by Random DNA Copying Errors

    MedlinePlus

    ... fullstory_164252.html Most Cancers Caused by Random DNA Copying Errors While habits, environment can be key ... factors, genes inherited from parents, or simply random DNA copying errors. From their calculations, the researchers now ...

  10. PCR-Based Detection of DNA Copy Number Variation.

    PubMed

    Mehrotra, Meenakshi

    2016-01-01

    Copy number variations are important polymorphisms that can influence gene expression within and close to the rearranged region, and results in phenotypic variation. Techniques that detect abnormalities in DNA copy number are therefore useful for studying the associations between DNA aberrations and disease phenotype and for locating critical genes. PCR-based detection of copy number of target gene using TaqMan copy number assay offers a reliable method to measure copy number variation in human genome.

  11. Accurate measurement of transgene copy number in crop plants using droplet digital PCR.

    PubMed

    Collier, Ray; Dasgupta, Kasturi; Xing, Yan-Ping; Hernandez, Bryan Tarape; Shao, Min; Rohozinski, Dominica; Kovak, Emma; Lin, Jeanie; de Oliveira, Maria Luiza P; Stover, Ed; McCue, Kent F; Harmon, Frank G; Blechl, Ann; Thomson, James G; Thilmony, Roger

    2017-02-23

    Genetic transformation is a powerful means for the improvement of crop plants, but requires labor and resource intensive methods. An efficient method for identifying single copy transgene insertion events from a population of independent transgenic lines is desirable. Currently transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (qPCR) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time-consuming and often requires a large amount of genomic DNA and radioactively labeled probes. Alternatively, qPCR requires less DNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one and two copy events in transgenic plants with large genomes. To address this need, we developed a droplet digital PCR (dPCR)-based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato, and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence-specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species. This article is protected by copyright. All rights reserved.

  12. Getting DNA copy numbers without control samples

    PubMed Central

    2012-01-01

    Background The selection of the reference to scale the data in a copy number analysis has paramount importance to achieve accurate estimates. Usually this reference is generated using control samples included in the study. However, these control samples are not always available and in these cases, an artificial reference must be created. A proper generation of this signal is crucial in terms of both noise and bias. We propose NSA (Normality Search Algorithm), a scaling method that works with and without control samples. It is based on the assumption that genomic regions enriched in SNPs with identical copy numbers in both alleles are likely to be normal. These normal regions are predicted for each sample individually and used to calculate the final reference signal. NSA can be applied to any CN data regardless the microarray technology and preprocessing method. It also finds an optimal weighting of the samples minimizing possible batch effects. Results Five human datasets (a subset of HapMap samples, Glioblastoma Multiforme (GBM), Ovarian, Prostate and Lung Cancer experiments) have been analyzed. It is shown that using only tumoral samples, NSA is able to remove the bias in the copy number estimation, to reduce the noise and therefore, to increase the ability to detect copy number aberrations (CNAs). These improvements allow NSA to also detect recurrent aberrations more accurately than other state of the art methods. Conclusions NSA provides a robust and accurate reference for scaling probe signals data to CN values without the need of control samples. It minimizes the problems of bias, noise and batch effects in the estimation of CNs. Therefore, NSA scaling approach helps to better detect recurrent CNAs than current methods. The automatic selection of references makes it useful to perform bulk analysis of many GEO or ArrayExpress experiments without the need of developing a parser to find the normal samples or possible batches within the data. The method is

  13. Getting DNA copy numbers without control samples.

    PubMed

    Ortiz-Estevez, Maria; Aramburu, Ander; Rubio, Angel

    2012-08-16

    The selection of the reference to scale the data in a copy number analysis has paramount importance to achieve accurate estimates. Usually this reference is generated using control samples included in the study. However, these control samples are not always available and in these cases, an artificial reference must be created. A proper generation of this signal is crucial in terms of both noise and bias.We propose NSA (Normality Search Algorithm), a scaling method that works with and without control samples. It is based on the assumption that genomic regions enriched in SNPs with identical copy numbers in both alleles are likely to be normal. These normal regions are predicted for each sample individually and used to calculate the final reference signal. NSA can be applied to any CN data regardless the microarray technology and preprocessing method. It also finds an optimal weighting of the samples minimizing possible batch effects. Five human datasets (a subset of HapMap samples, Glioblastoma Multiforme (GBM), Ovarian, Prostate and Lung Cancer experiments) have been analyzed. It is shown that using only tumoral samples, NSA is able to remove the bias in the copy number estimation, to reduce the noise and therefore, to increase the ability to detect copy number aberrations (CNAs). These improvements allow NSA to also detect recurrent aberrations more accurately than other state of the art methods. NSA provides a robust and accurate reference for scaling probe signals data to CN values without the need of control samples. It minimizes the problems of bias, noise and batch effects in the estimation of CNs. Therefore, NSA scaling approach helps to better detect recurrent CNAs than current methods. The automatic selection of references makes it useful to perform bulk analysis of many GEO or ArrayExpress experiments without the need of developing a parser to find the normal samples or possible batches within the data. The method is available in the open-source R package

  14. Ribosomal DNA copy number loss and sequence variation in cancer

    PubMed Central

    Xu, Baoshan; Li, Hua; Perry, John M.; Singh, Vijay Pratap; Yu, Zulin; Zakari, Musinu; Li, Linheng

    2017-01-01

    Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments. PMID:28640831

  15. Ribosomal DNA copy number loss and sequence variation in cancer.

    PubMed

    Xu, Baoshan; Li, Hua; Perry, John M; Singh, Vijay Pratap; Unruh, Jay; Yu, Zulin; Zakari, Musinu; McDowell, William; Li, Linheng; Gerton, Jennifer L

    2017-06-01

    Ribosomal DNA is one of the most variable regions in the human genome with respect to copy number. Despite the importance of rDNA for cellular function, we know virtually nothing about what governs its copy number, stability, and sequence in the mammalian genome due to challenges associated with mapping and analysis. We applied computational and droplet digital PCR approaches to measure rDNA copy number in normal and cancer states in human and mouse genomes. We find that copy number and sequence can change in cancer genomes. Counterintuitively, human cancer genomes show a loss of copies, accompanied by global copy number co-variation. The sequence can also be more variable in the cancer genome. Cancer genomes with lower copies have mutational evidence of mTOR hyperactivity. The PTEN phosphatase is a tumor suppressor that is critical for genome stability and a negative regulator of the mTOR kinase pathway. Surprisingly, but consistent with the human cancer genomes, hematopoietic cancer stem cells from a Pten-/- mouse model for leukemia have lower rDNA copy number than normal tissue, despite increased proliferation, rRNA production, and protein synthesis. Loss of copies occurs early and is associated with hypersensitivity to DNA damage. Therefore, copy loss is a recurrent feature in cancers associated with mTOR activation. Ribosomal DNA copy number may be a simple and useful indicator of whether a cancer will be sensitive to DNA damaging treatments.

  16. Mitochondrial DNA Copy Number Is Associated with Breast Cancer Risk

    PubMed Central

    Thyagarajan, Bharat; Wang, Renwei; Nelson, Heather; Barcelo, Helene; Koh, Woon-Puay; Yuan, Jian-Min

    2013-01-01

    Mitochondrial DNA (mtDNA) copy number in peripheral blood is associated with increased risk of several cancers. However, data from prospective studies on mtDNA copy number and breast cancer risk are lacking. We evaluated the association between mtDNA copy number in peripheral blood and breast cancer risk in a nested case-control study of 183 breast cancer cases with pre-diagnostic blood samples and 529 individually matched controls among participants of the Singapore Chinese Health Study. The mtDNA copy number was measured using real time PCR. Conditional logistic regression analyses showed that there was an overall positive association between mtDNA copy number and breast cancer risk (Ptrend = 0.01). The elevated risk for higher mtDNA copy numbers was primarily seen for women with <3 years between blood draw and cancer diagnosis; ORs (95% CIs) for 2nd, 3rd, 4th, and 5th quintile of mtDNA copy number were 1.52 (0.61, 3.82), 2.52 (1.03, 6.12), 3.12 (1.31, 7.43), and 3.06 (1.25, 7.47), respectively, compared with the 1st quintile (Ptrend = 0.004). There was no association between mtDNA copy number and breast cancer risk among women who donated a blood sample ≥3 years before breast cancer diagnosis (Ptrend = 0.41). This study supports a prospective association between increased mtDNA copy number and breast cancer risk that is dependent on the time interval between blood collection and breast cancer diagnosis. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in breast cancer risk. PMID:23776581

  17. No association between mitochondrial DNA copy number and colorectal adenomas.

    PubMed

    Thyagarajan, Bharat; Guan, Weihua; Fedirko, Veronika; Barcelo, Helene; Tu, Huakang; Gross, Myron; Goodman, Michael; Bostick, Roberd M

    2016-08-01

    Despite previously reported associations between peripheral blood mtDNA copy number and colorectal cancer, it remains unclear whether altered mtDNA copy number in peripheral blood is a risk factor for colorectal cancer or a biomarker for undiagnosed colorectal cancer. Though colorectal adenomas are well-recognized precursor lesions to colorectal cancer, no study has evaluated an association between mtDNA copy number and colorectal adenoma risk. Hence, we investigated an association between peripheral blood mtDNA copy number and incident, sporadic colorectal adenoma in 412 colorectal adenoma cases and 526 cancer-free controls pooled from three colonoscopy-based case-control studies that used identical methods for case ascertainment, risk factor determination, and biospecimen collection. We also evaluated associations between relative mtDNA copy number and markers of oxidative stress, including circulating F2 -isoprostanes, carotenoids, and fluorescent oxidation products. We measured mtDNA copy number using a quantitative real time polymerase chain reaction (PCR). We used unconditional logistic regression to analyze the association between mtDNA copy number and colorectal adenoma risk after multivariable adjustment. We found no association between logarithmically transformed relative mtDNA copy number, analyzed as a continuous variable, and colorectal adenoma risk (odds ratio = 1.02, 95%CI: 0.82-1.27; P = 0.86). There were no statistically significant associations between relative mtDNA copy number and other markers of oxidative stress. Our findings, taken together with those from previous studies, suggest that relative mtDNA copy number in peripheral blood may more likely be a marker of early colorectal cancer than of risk for the disease or of in vivo oxidative stress. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  18. Mitochondrial DNA Copy Number in Sleep Duration Discordant Monozygotic Twins.

    PubMed

    Wrede, Joanna E; Mengel-From, Jonas; Buchwald, Dedra; Vitiello, Michael V; Bamshad, Michael; Noonan, Carolyn; Christiansen, Lene; Christensen, Kaare; Watson, Nathaniel F

    2015-10-01

    Mitochondrial DNA (mtDNA) copy number is an important component of mitochondrial function and varies with age, disease, and environmental factors. We aimed to determine whether mtDNA copy number varies with habitual differences in sleep duration within pairs of monozygotic twins. Academic clinical research center. 15 sleep duration discordant monozygotic twin pairs (30 twins, 80% female; mean age 42.1 years [SD 15.0]). Sleep duration was phenotyped with wrist actigraphy. Each twin pair included a "normal" (7-9 h/24) and "short" (< 7 h/24) sleeping twin. Fasting peripheral blood leukocyte DNA was assessed for mtDNA copy number via the n-fold difference between qPCR measured mtDNA and nuclear DNA creating an mtDNA measure without absolute units. We used generalized estimating equation linear regression models accounting for the correlated data structure to assess within-pair effects of sleep duration on mtDNA copy number. Mean within-pair sleep duration difference per 24 hours was 94.3 minutes (SD 62.6 min). We found reduced sleep duration (β = 0.06; 95% CI 0.004, 0.12; P < 0.05) and sleep efficiency (β = 0.51; 95% CI 0.06, 0.95; P < 0.05) were significantly associated with reduced mtDNA copy number within twin pairs. Thus every 1-minute decrease in actigraphy-defined sleep duration was associated with a decrease in mtDNA copy number of 0.06. Likewise, a 1% decrease in actigraphy-defined sleep efficiency was associated with a decrease in mtDNA copy number of 0.51. Reduced sleep duration and sleep efficiency were associated with reduced mitochondrial DNA copy number in sleep duration discordant monozygotic twins offering a potential mechanism whereby short sleep impairs health and longevity through mitochondrial stress. © 2015 Associated Professional Sleep Societies, LLC.

  19. Mitochondrial DNA copy number in peripheral blood and melanoma risk.

    PubMed

    Shen, Jie; Gopalakrishnan, Vancheswaran; Lee, Jeffrey E; Fang, Shenying; Zhao, Hua

    2015-01-01

    Mitochondrial DNA (mtDNA) copy number in peripheral blood has been suggested as risk modifier in various types of cancer. However, its influence on melanoma risk is unclear. We evaluated the association between mtDNA copy number in peripheral blood and melanoma risk in 500 melanoma cases and 500 healthy controls from an ongoing melanoma study. The mtDNA copy number was measured using real-time polymerase chain reaction. Overall, mean mtDNA copy number was significantly higher in cases than in controls (1.15 vs 0.99, P<0.001). Increased mtDNA copy number was associated with a 1.45-fold increased risk of melanoma (95% confidence interval: 1.12-1.97). Significant joint effects between mtDNA copy number and variables related to pigmentation and history of sunlight exposure were observed. This study supports an association between increased mtDNA copy number and melanoma risk that is independent on the known melanoma risk factors (pigmentation and history of sunlight exposure).

  20. Number matters: control of mammalian mitochondrial DNA copy number.

    PubMed

    Clay Montier, Laura L; Deng, Janice J; Bai, Yidong

    2009-03-01

    Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA (mtDNA) depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has advanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.

  1. Mitochondrial DNA Copy Number in Sleep Duration Discordant Monozygotic Twins

    PubMed Central

    Wrede, Joanna E.; Mengel-From, Jonas; Buchwald, Dedra; Vitiello, Michael V.; Bamshad, Michael; Noonan, Carolyn; Christiansen, Lene; Christensen, Kaare; Watson, Nathaniel F.

    2015-01-01

    Study Objectives: Mitochondrial DNA (mtDNA) copy number is an important component of mitochondrial function and varies with age, disease, and environmental factors. We aimed to determine whether mtDNA copy number varies with habitual differences in sleep duration within pairs of monozygotic twins. Setting: Academic clinical research center. Participants: 15 sleep duration discordant monozygotic twin pairs (30 twins, 80% female; mean age 42.1 years [SD 15.0]). Design: Sleep duration was phenotyped with wrist actigraphy. Each twin pair included a “normal” (7–9 h/24) and “short” (< 7 h/24) sleeping twin. Fasting peripheral blood leukocyte DNA was assessed for mtDNA copy number via the n-fold difference between qPCR measured mtDNA and nuclear DNA creating an mtDNA measure without absolute units. We used generalized estimating equation linear regression models accounting for the correlated data structure to assess within-pair effects of sleep duration on mtDNA copy number. Measurements and Results: Mean within-pair sleep duration difference per 24 hours was 94.3 minutes (SD 62.6 min). We found reduced sleep duration (β = 0.06; 95% CI 0.004, 0.12; P < 0.05) and sleep efficiency (β = 0.51; 95% CI 0.06, 0.95; P < 0.05) were significantly associated with reduced mtDNA copy number within twin pairs. Thus every 1-minute decrease in actigraphy-defined sleep duration was associated with a decrease in mtDNA copy number of 0.06. Likewise, a 1% decrease in actigraphy-defined sleep efficiency was associated with a decrease in mtDNA copy number of 0.51. Conclusions: Reduced sleep duration and sleep efficiency were associated with reduced mitochondrial DNA copy number in sleep duration discordant monozygotic twins offering a potential mechanism whereby short sleep impairs health and longevity through mitochondrial stress. Citation: Wrede JE, Mengel-From J, Buchwald D, Vitiello MV, Bamshad M, Noonan C, Christiansen L, Christensen K, Watson NF. Mitochondrial DNA copy number

  2. RECONSTRUCTING DNA COPY NUMBER BY PENALIZED ESTIMATION AND IMPUTATION.

    PubMed

    Zhang, Zhongyang; Lange, Kenneth; Ophoff, Roel; Sabatti, Chiara

    2010-12-01

    Recent advances in genomics have underscored the surprising ubiquity of DNA copy number variation (CNV). Fortunately, modern genotyping platforms also detect CNVs with fairly high reliability. Hidden Markov models and algorithms have played a dominant role in the interpretation of CNV data. Here we explore CNV reconstruction via estimation with a fused-lasso penalty as suggested by Tibshirani and Wang [Biostatistics 9 (2008) 18-29]. We mount a fresh attack on this difficult optimization problem by the following: (a) changing the penalty terms slightly by substituting a smooth approximation to the absolute value function, (b) designing and implementing a new MM (majorization-minimization) algorithm, and (c) applying a fast version of Newton's method to jointly update all model parameters. Together these changes enable us to minimize the fused-lasso criterion in a highly effective way.We also reframe the reconstruction problem in terms of imputation via discrete optimization. This approach is easier and more accurate than parameter estimation because it relies on the fact that only a handful of possible copy number states exist at each SNP. The dynamic programming framework has the added bonus of exploiting information that the current fused-lasso approach ignores. The accuracy of our imputations is comparable to that of hidden Markov models at a substantially lower computational cost.

  3. Accurate quantification of supercoiled DNA by digital PCR.

    PubMed

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-04-11

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry.

  4. Accurate quantification of supercoiled DNA by digital PCR

    PubMed Central

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  5. Mitochondrial DNA copy number variation across human cancers

    PubMed Central

    Reznik, Ed; Miller, Martin L; Şenbabaoğlu, Yasin; Riaz, Nadeem; Sarungbam, Judy; Tickoo, Satish K; Al-Ahmadie, Hikmat A; Lee, William; Seshan, Venkatraman E; Hakimi, A Ari; Sander, Chris

    2016-01-01

    Mutations, deletions, and changes in copy number of mitochondrial DNA (mtDNA), are observed throughout cancers. Here, we survey mtDNA copy number variation across 22 tumor types profiled by The Cancer Genome Atlas project. We observe a tendency for some cancers, especially of the bladder, breast, and kidney, to be depleted of mtDNA, relative to matched normal tissue. Analysis of genetic context reveals an association between incidence of several somatic alterations, including IDH1 mutations in gliomas, and mtDNA content. In some but not all cancer types, mtDNA content is correlated with the expression of respiratory genes, and anti-correlated to the expression of immune response and cell-cycle genes. In tandem with immunohistochemical evidence, we find that some tumors may compensate for mtDNA depletion to sustain levels of respiratory proteins. Our results highlight the extent of mtDNA copy number variation in tumors and point to related therapeutic opportunities. DOI: http://dx.doi.org/10.7554/eLife.10769.001 PMID:26901439

  6. Reconstructing DNA copy number by joint segmentation of multiple sequences

    PubMed Central

    2012-01-01

    Background Variations in DNA copy number carry information on the modalities of genome evolution and mis-regulation of DNA replication in cancer cells. Their study can help localize tumor suppressor genes, distinguish different populations of cancerous cells, and identify genomic variations responsible for disease phenotypes. A number of different high throughput technologies can be used to identify copy number variable sites, and the literature documents multiple effective algorithms. We focus here on the specific problem of detecting regions where variation in copy number is relatively common in the sample at hand. This problem encompasses the cases of copy number polymorphisms, related samples, technical replicates, and cancerous sub-populations from the same individual. Results We present a segmentation method named generalized fused lasso (GFL) to reconstruct copy number variant regions. GFL is based on penalized estimation and is capable of processing multiple signals jointly. Our approach is computationally very attractive and leads to sensitivity and specificity levels comparable to those of state-of-the-art specialized methodologies. We illustrate its applicability with simulated and real data sets. Conclusions The flexibility of our framework makes it applicable to data obtained with a wide range of technology. Its versatility and speed make GFL particularly useful in the initial screening stages of large data sets. PMID:22897923

  7. DNA Copy Number Control Through Inhibition of Replication Fork Progression

    PubMed Central

    Nordman, Jared T.; Kozhevnikova, Elena N.; Verrijzer, C. Peter; Pindyurin, Alexey V.; Andreyeva, Evgeniya N.; Shloma, Victor V.; Zhimulev, Igor F.; Orr-Weaver, Terry L.

    2014-01-01

    Summary Proper control of DNA replication is essential to ensure faithful transmission of genetic material and to prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict cell cycle regulation. Importantly, DNA replication is highly influenced by developmental cues. In Drosophila, specific regions of the genome are repressed for DNA replication during differentiation by the SNF2 domain-containing protein SUUR through an unknown mechanism. We demonstrate that SUUR is recruited to active replication forks and mediates repression of DNA replication by directly inhibiting replication fork progression instead of functioning as a replication fork barrier. Mass-spec identification of SUUR associated proteins identified the replicative helicase member CDC45 as a SUUR-associated protein, supporting a role for SUUR directly at replication forks. Our results reveal that control of eukaryotic DNA copy number can occur through inhibition of replication fork progression. PMID:25437540

  8. Rates of single-copy DNA evolution in herons.

    PubMed

    Sheldon, F H

    1987-01-01

    DNA-DNA hybridization was used to discover the extent of single-copy DNA similarity among 13 species of herons and one ibis. Genetic distances among taxa were summarized as Tm values in a folded matrix. From this matrix, trees with the same branching pattern were constructed by least squares under one of two assumptions: (1) that sister branches are equal in length and (2) that sister branches are not necessarily equal in length. The residual sums of squares of these trees were compared by F-test to see whether the branches of the tree built under assumption (2) fit the matrix data significantly better than those of the tree built under assumption (1). By this method the existence of different rates of DNA evolution in different heron lineages was established. Bittern single-copy DNA has evolved at a rate approximately 25% faster, and boat-billed heron (Cochearius) and rufescent tiger heron (Tigrisoma lineatum) DNA has evolved approximately 19% slower, than that of day and night herons. It appears that the differences in rates of DNA evolution may increase proportionally with genealogical distance.

  9. Genome-wide copy number analysis using copy number inferring tool (CNIT) and DNA pooling.

    PubMed

    Lin, Chien-hsing; Huang, Mei-chu; Li, Ling-hui; Wu, Jer-yuarn; Chen, Yuan-tsong; Fann, Cathy S J

    2008-08-01

    Copy number variation (CNV) has become an important genomic structure element in the human population, and some CNVs are related to specific traits and diseases. Moreover, analysis of human genomes has been potentiated by the use of high-resolution microarrays that assess single nucleotide polymorphisms (SNPs). Although many programs have been designed to analyze data from Affymetrix SNP microarrays, they all have high false-positive rates (FPRs) in copy number (CN) analyses. Copy number analysis tool (CNAT) 4.0 is a recently developed program that offers improved CN estimation, but small amplifications and deletions are lost when using the smoothing procedure. Here, we propose a copy number inferring tool (CNIT) algorithm for the 100K SNP microarray to investigate CNVs at 29.6-kb resolution. CNIT estimated SNP allelic and total CN with reliable P values based on intensity data. In addition, the hidden Markov model (HMM) method was applied to predict regions having altered CN by considering contiguous SNPs. Based on a CN analysis of 23 unrelated Taiwanese and 30 HapMap Centre d'Etude du Polymorphisme Humain (CEPH) trios, CNIT showed higher accuracy and power than other programs. The FPRs and false-negative rates (FNRs) of CNIT were 0.1% and 0.16%, respectively. CNIT also showed better sensitivity for detecting small amplifications and deletions. Furthermore, DNA pooling of 10 and 30 normal unrelated individuals were applied to the 100K SNP microarray, respectively, and 12 common CN-variable regions were identified, suggesting that DNA pooling can be applied to discover common CNVs.

  10. Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants

    PubMed Central

    Morley, Stewart A.; Nielsen, Brent L.

    2016-01-01

    Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles. PMID:26870072

  11. Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants.

    PubMed

    Morley, Stewart A; Nielsen, Brent L

    2016-01-01

    Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles.

  12. Molecular Poltergeists: Mitochondrial DNA Copies (numts) in Sequenced Nuclear Genomes

    PubMed Central

    Hazkani-Covo, Einat; Zeller, Raymond M.; Martin, William

    2010-01-01

    The natural transfer of DNA from mitochondria to the nucleus generates nuclear copies of mitochondrial DNA (numts) and is an ongoing evolutionary process, as genome sequences attest. In humans, five different numts cause genetic disease and a dozen human loci are polymorphic for the presence of numts, underscoring the rapid rate at which mitochondrial sequences reach the nucleus over evolutionary time. In the laboratory and in nature, numts enter the nuclear DNA via non-homolgous end joining (NHEJ) at double-strand breaks (DSBs). The frequency of numt insertions among 85 sequenced eukaryotic genomes reveal that numt content is strongly correlated with genome size, suggesting that the numt insertion rate might be limited by DSB frequency. Polymorphic numts in humans link maternally inherited mitochondrial genotypes to nuclear DNA haplotypes during the past, offering new opportunities to associate nuclear markers with mitochondrial markers back in time. PMID:20168995

  13. Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device

    PubMed Central

    Dube, Simant; Qin, Jian; Ramakrishnan, Ramesh

    2008-01-01

    Copy Number Variations (CNVs) of regions of the human genome have been associated with multiple diseases. We present an algorithm which is mathematically sound and computationally efficient to accurately analyze CNV in a DNA sample utilizing a nanofluidic device, known as the digital array. This numerical algorithm is utilized to compute copy number variation and the associated statistical confidence interval and is based on results from probability theory and statistics. We also provide formulas which can be used as close approximations. PMID:18682853

  14. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage

    PubMed Central

    Gonzalez-Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi

    2016-01-01

    Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  15. Mitochondrial DNA Copy Number and Exposure to Polycyclic Aromatic Hydrocarbons

    PubMed Central

    Pavanello, Sofia; Dioni, Laura; Hoxha, Mirjam; Fedeli, Ugo; Mielzynska-Švach, Danuta; Baccarelli, Andrea A.

    2013-01-01

    Background Increased mitochondrial DNA copy number (mtDNAcn) is a biological response to mtDNA damage and dysfunction predictive of lung cancer risk. Polycyclic aromatic hydrocarbons (PAHs) are established lung carcinogens and may cause mitochondrial toxicity. Whether PAH exposure and PAH-related nuclear DNA (nDNA) genotoxic effects are linked with increased mtDNAcn has never been evaluated. Methods We investigated the effect of chronic exposure to PAHs on mtDNAcn in peripheral blood lymphocytes (PBLs) of 46 Polish male non-current smoking cokeoven workers and 44 matched controls, who were part of a group of 94 study individuals examined in our previous work. Subjects PAH exposure and genetic alterations were characterized through measures of internal dose (urinary 1-pyrenol), target dose [anti-benzo[a]pyrene diolepoxide (anti-BPDE)-DNA adduct], genetic instability (micronuclei, MN and telomere length [TL]) and DNA methylation [p53 promoter] in PBLs. mtDNAcn (MT/S) was measured using a validated real-time PCR method. Results Workers with PAH exposure above the median value (>3 µmol 1-pyrenol/mol creatinine) showed higher mtDNAcn [geometric means (GM) of 1.06 (unadjusted) and 1.07 (age-adjusted)] compared to controls [GM 0.89 (unadjusted); 0.89 (age-adjusted)] (p=0.029 and 0.016), as well as higher levels of genetic and chromosomal [i.e. anti-BPDE-DNA adducts (p<0.001), MN (p<0.001) and TL (p=0.053)] and epigenetic [i.e., p53 gene-specific promoter methylation (p<0.001)] alterations in the nDNA. In the whole study population, unadjusted and age-adjusted mtDNAcn was positively correlated with 1-pyrenol (p=0.043 and 0.032) and anti-BPDE-DNA adducts (p=0.046 and 0.049). Conclusions PAH exposure and PAH-related nDNA genotoxicity are associated with increased mtDNAcn. Impact The present study is suggestive of potential roles of mtDNAcn in PAH-induced carcinogenesis. PMID:23885040

  16. Loss of the antioxidant enzyme CuZnSOD (Sod1) mimics an age-related increase in absolute mitochondrial DNA copy number in the skeletal muscle.

    PubMed

    Masser, Dustin R; Clark, Nicholas W; Van Remmen, Holly; Freeman, Willard M

    2016-08-01

    Mitochondria contain multiple copies of the circular mitochondrial genome (mtDNA) that encodes ribosomal RNAs and proteins locally translated for oxidative phosphorylation. Loss of mtDNA integrity, both altered copy number and increased mutations, is implicated in cellular dysfunction with aging. Published data on mtDNA copy number and aging is discordant which may be due to methodological limitations for quantifying mtDNA copy number. Existing quantitative PCR (qPCR) mtDNA copy number quantification methods provide only relative abundances and are problematic to normalize to different template input amounts and across tissues/sample types. As well, existing methods cannot quantify mtDNA copy number in subcellular isolates, such as isolated mitochondria and neuronal synaptic terminals, which lack nuclear genomic DNA for normalization. We have developed and validated a novel absolute mtDNA copy number quantitation method that uses chip-based digital polymerase chain reaction (dPCR) to count the number of copies of mtDNA and used this novel method to assess the literature discrepancy in which there is no clear consensus whether mtDNA numbers change with aging in skeletal muscle. Skeletal muscle in old mice was found to have increased absolute mtDNA numbers compared to young controls. Furthermore, young Sod1 (-/-) mice were assessed and show an age-mimicking increase in skeletal muscle mtDNA. These findings reproduce a number of previous studies that demonstrate age-related increases in mtDNA. This simple and cost effective dPCR approach should enable precise and accurate mtDNA copy number quantitation in mitochondrial studies, eliminating contradictory studies of mitochondrial DNA content with aging.

  17. Reconstructing labroid evolution with single-copy nuclear DNA.

    PubMed Central

    Streelman, J T; Karl, S A

    1997-01-01

    Fifteen per cent of all living fishes are united in a single suborder (Labroidei) and display a dazzling array of behavioural and ecological traits. The labroids are considered monophyletic and members share a pharyngeal jaw apparatus (PJA) modified for crushing and processing prey. Outside of the explicitly functional PJA, there is no corroborative evidence for a monophyletic Labroidei. Here, we report the first molecular phylogenetic analysis of the suborder. Contrary to morphology-based phylogenies, our single-copy nuclear DNA data do not support labroid families as a natural group. Our data indicate that pharyngognathy has evolved independently among labroid families and that characters of the PJA are not reliable markers of perciform evolution. This work 'crushes' conventional views of fish phylogeny and should engender novel concepts of piscine life history evolution. PMID:9263469

  18. Lower mitochondrial DNA copy number in peripheral blood leukocytes increases the risk of endometrial cancer.

    PubMed

    Sun, Yuhui; Zhang, Liren; Ho, Simon S; Wu, Xifeng; Gu, Jian

    2016-06-01

    Mitochondria are the primary source of energy generation in human cells. Low mitochondrial DNA (mtDNA) copy number in peripheral blood leukocytes (PBLs) has been associated with obesity and increased risks of several cancers. Since obesity is a significant risk factor for endometrial cancer, we hypothesize that low mtDNA copy number in PBLs is associated with an increased susceptibility to endometrial cancer. Using a Caucasian case-control study, we measured mtDNA copy number in PBLs from 139 endometrial cancer patients and 139 age-matched controls and determined the association of mtDNA copy number with the risk of endometrial cancer using multivariate logistic regression analysis. The normalized mtDNA copy number was significantly lower in endometrial cancer cases (median, 0.84; range, 0.24-2.00) than in controls (median, 1.06; range, 0.64-1.96) (P < 0.001). Dichotomized into high and low groups based on the median mtDNA copy number value in the controls, individuals with low mtDNA copy number had a significantly increased risk of endometrial cancer (adjusted OR, 5.59; 95%CI, 3.05-10.25; P < 0.001) compared to those with high mtDNA copy number. There was a significant dose-response association in tertile analysis. In addition, there was a significant joint effect between lower mtDNA copy number and never smoking, hypertension, diabetes, and obesity in elevating the risk of endometrial cancer. Low mtDNA copy number in PBLs is significantly associated with an increased risk of endometrial cancer in Caucasians. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  19. Increased leukocyte mitochondrial DNA copy number is associated with oral premalignant lesions: an epidemiology study.

    PubMed

    He, Yonggang; Gong, Yilei; Gu, Jian; Lee, J Jack; Lippman, Scott M; Wu, Xifeng

    2014-08-01

    Although changes in the mitochondrial DNA (mtDNA) copy number in peripheral blood leukocytes (PBLs) have been linked to increased susceptibility to several cancers, the relationship between the mtDNA copy number in PBLs and the risk of cancer precursors has not been investigated. In this study, we measured the relative mtDNA copy number in PBLs of 143 patients with histologically confirmed oral premalignant lesions (OPLs) and of 357 healthy controls that were frequency-matched to patients according to age, sex and race. OPL patients had a significantly higher mtDNA copy number than the controls (1.36 ± 0.74 versus 1.11 ± 0.32; P < 0.001). In analyses stratified by sex, race, alcohol consumption and smoking status, the mtDNA copy number was higher in the OPL patients than in the controls in all the strata. Using the median mtDNA copy number in the control group as a cutoff, we found that individuals with a high mtDNA copy number had significantly higher risk of having OPLs than individuals with a low mtDNA copy number (adjusted odds ratio, 1.93; 95% confidence interval, 1.23-3.05, P = 0.004). Analysis of the joint effect of alcohol consumption and smoking revealed even greater risk for OPLs. Our results suggest that high mtDNA copy number in PBLs is significantly associated with having OPLs. To our knowledge, this is the first epidemiologic study to show that the mtDNA copy number may indicate the risk of cancer precursors.

  20. Accurate measure of transgene copy number in crop plants using droplet digital PCR

    USDA-ARS?s Scientific Manuscript database

    Genetic transformation is a powerful means for the improvement of crop plants, but requires labor- and resource-intensive methods. An efficient method for identifying single-copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy numb...

  1. Mitochondrial DNA copy number variation as a potential predictor of renal cell carcinoma.

    PubMed

    Elsayed, Eman T; Hashad, Mohamed M; Elgohary, Iman E

    2017-07-24

    Peripheral blood mitochondrial DNA (mtDNA) copy number alteration has been suggested as a risk factor for several types of cancer. The aim of the present study was to assess the role of peripheral blood mtDNA copy number variation as a noninvasive biomarker in the prediction and early detection of renal cell carcinoma (RCC) in a cohort of Egyptian patients. Quantitative real-time polymerase chain reaction (qPCR) was used to measure peripheral blood mtDNA copy numbers in 57 patients with newly diagnosed, early-stage localized RCC and 60 age- and sex-matched healthy individuals as a control group. Median mtDNA copy number was significantly higher in RCC cases than in controls (166 vs. 91, p<0.001). Increased mtDNA copy number was associated with an 18-fold increased risk of RCC (95% confidence interval: 5.065-63.9). On receiver operating characteristic curve analysis, it was found that mtDNA could distinguish between RCC patients and healthy controls, with 86% sensitivity, 80% specificity, 80.3% positive predictive value and 85.7% negative predictive value at a cutoff value of 108.5. Our results showed that increased peripheral blood mtDNA copy number was associated with increased risk of RCC. Therefore, RCC might be considered as part of a range of potential tumors in cases with elevated blood mtDNA copy number.

  2. DNA Copy Number Variations Characterize Benign and Malignant Thyroid Tumors

    PubMed Central

    Liu, Yan; Cope, Leslie; Sun, Wenyue; Wang, Yongchun; Prasad, Nijaguna; Sangenario, Lauren; Talbot, Kristen; Somervell, Helina; Westra, William; Bishop, Justin; Califano, Joseph; Zeiger, Martha

    2013-01-01

    Context: Fine-needle aspiration (FNA) is the best diagnostic tool for preoperative evaluation of thyroid nodules but is often inconclusive as a guide for surgical management. Objective: Our hypothesis was that thyroid tumor subtypes may show characteristic DNA copy number variation (CNV) patterns, which may further improve the preoperative classification. Design: Our study cohorts included benign follicular adenomas (FAs), classic papillary thyroid carcinomas (PTCs), and follicular variant PTCs (FVPTCs), the three subtypes most commonly associated with inconclusive preoperative cytopathology. Setting: Tissue and FNA samples were obtained at an academic tertiary referral center. Patients: Cases were identified that underwent partial or complete thyroidectomy for malignant or indeterminate thyroid lesions between 2000 and 2008 and had adequate snap-frozen tissue. Interventions: Pairs of tumor tissue and matching normal thyroid tissue-derived DNA were compared using 550K single-nucleotide polymorphism arrays. Main Outcome Measure: Statistically significant differences in CNV patterns between tumor subtypes were identified. Results: Segmental amplifications in chromosomes (Ch) 7 and 12 were more common in FAs than in PTCs or FVPTCs. Additionally, a subset of FAs and FVPTCs showed deletions in Ch22. We identified the 5 CNV-associated genes best at discriminating between FAs and PTCs/FVPTCs, which correctly classified 90% of cases. These 5 Ch12 genes were validated by quantitative genomic PCR and gene expression array analyses on the same patient cohort. The 5-gene signature was then successfully validated against an independent test cohort of benign and malignant tumor samples. Finally, we performed a feasibility study on matched FA-derived intraoperative FNA samples and were able to correctly identify FAs harboring the Ch12 amplification signature, whereas FAs without amplification showed a normal Ch12 signature. Conclusions: Thyroid tumor subtypes possess

  3. DNA Copy Number Signature to Predict Recurrence in Early-Stage Ovarian Cancer

    DTIC Science & Technology

    2015-08-01

    AWARD NUMBER: W81XWH-14-1-0194 TITLE: DNA Copy Number Signature to Predict Recurrence in Early-Stage Ovarian Cancer PRINCIPAL INVESTIGATOR...SUBTITLE 5a. CONTRACT NUMBER DNA Copy Number Signature to Predict Recurrence in Early Stage Ovarian Cancer 5b. GRANT NUMBER W81XWH-14-1-0194 5c...tasks Major Task 1: Obtain DNA samples from consortium specimens • Subtask 1 Pathological review of 592 early-stage high-grade ovarian cancer specimens

  4. Modulation of Mitochondrial DNA Copy Number to Induce Hepatocytic Differentiation of Human Amniotic Epithelial Cells.

    PubMed

    Vaghjiani, Vijesh; Cain, Jason E; Lee, William; Vaithilingam, Vijayaganapathy; Tuch, Bernard E; St John, Justin C

    2017-09-05

    Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.

  5. Comparison of methods for accurate quantification of DNA mass concentration with traceability to the international system of units.

    PubMed

    Bhat, Somanath; Curach, Natalie; Mostyn, Thomas; Bains, Gursharan Singh; Griffiths, Kate R; Emslie, Kerry R

    2010-09-01

    Accurate estimation of total DNA concentration (mass concentration, e.g., ng/muL) that is traceable to the International System of Units (SI) is a crucial starting point for improving reproducible measurements in many applications involving nucleic acid testing and requires a DNA reference material which has been certified for its total DNA concentration. In this study, the concentrations of six different lambda DNA preparations were determined using different measurement platforms: UV Absorbance at 260 nm (A(260)) with and without prior sodium hydroxide (NaOH) treatment of the DNA, PicoGreen assay, and digital polymerase chain reaction (dPCR). DNA concentration estimates by A(260) with and without prior NaOH treatment were significantly different for five of the six samples tested. There were no significant differences in concentration estimates based on A(260) with prior NaOH treatment, PicoGreen analysis, and dPCR for two of the three samples tested using dPCR. Since the measurand in dPCR is amount (copy number) concentration (copies/muL), the results suggest that accurate estimation of DNA mass concentration based on copy number concentration is achievable provided the DNA is fully characterized and in the double-stranded form or amplification is designed to be initiated from only one of the two complementary strands.

  6. Mitochondrial DNA copy number in whole blood and glioma risk: A case control study.

    PubMed

    Shen, Jie; Song, Renduo; Lu, Zhimin; Zhao, Hua

    2016-12-01

    Alterations in mitochondrial DNA (mtDNA) copy number are observed in human gliomas. However, whether variations in mtDNA copy number in whole blood play any role in glioma carcinogenesis is still largely unknown. In current study with 395 glioma patients and 425 healthy controls, we intended to investigate the association between mtDNA copy number in whole blood and glioma risk. Overall, we found that levels of mtDNA copy number were significantly higher in glioma cases than healthy controls (mean: 1.48 vs. 1.32, P < 0.01). In both cases and controls, levels of mtDNA copy number were inversely correlated with age (P < 0.01, respectively). And in cases, newly diagnosed, glioblastoma (GBM), and high grade glioma patients had significantly lower mtDNA copy number than their counterparts (P = 0.02, P < 0.01, and P = 0.04, respectively). In the multivariate analysis, elevated mtDNA copy number levels were associated with a 1.63-fold increased risk of glioma (adjusted odds ratio (OR) = 1.63, 95% confidence interval (CI) = 1.23-2.14). In further quartile analysis, study subjects who had highest levels of mtNDA copy number had 1.75-fold increased risk of gliomas (adjOR = 1.75, 95%CI = 1.18-2.61). In brief, our findings support the role of mtDNA copy number in the glioma carcinogenesis. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Telomere length is correlated with mitochondrial DNA copy number in intestinal, but not diffuse, gastric cancer.

    PubMed

    Jung, Soo-Jung; Cho, Ji-Hyoung; Park, Won-Jin; Heo, Yu-Ran; Lee, Jae-Ho

    2017-07-01

    A positive correlation between telomere length and mitochondrial DNA (mtDNA) copy number has previously been observed in healthy individuals, and in patients with psychiatric disorders. In the present study, telomere length and mtDNA copy number were evaluated in gastric cancer (GC) tissue samples. DNA was extracted from 109 GC samples (including 82 intestinal, and 27 diffuse cases), and the telomere length and mtDNA copy number were analyzed using a quantitative-polymerase chain reaction assay. The relative telomere length and mtDNA copy number in tumor tissue, as compared with in normal tissue, (mean ± standard deviation) in all GC samples were 11.48±1.14 and 14.86±1.35, respectively. Telomere length and mtDNA copy number were not identified as exhibiting clinical or prognostic value for GC. However, positive correlations between telomere length and mitochondrial DNA copy number were identified in GC (r=0.408, P<0.001) and in the adjacent normal mucosa (r=0.363; P<0.001). When stratified by Lauren classification, the correlation was identified in intestinal type GC samples (r=0.461; P<0.001), but not in diffuse type GC samples (r=0.225; P=0.260). This result indicated that loss of the correlation of telomeres and mitochondrial function may induce the initiation or progression of GC pathogenesis.

  8. Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs

    PubMed Central

    Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

    2015-01-01

    Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors. PMID:25837486

  9. Accurate measurement of transgene copy number in crop plants using droplet digital PCR

    USDA-ARS?s Scientific Manuscript database

    Technical abstract: Genetic transformation is a powerful means for the improvement of crop plants, but requires labor and resource intensive methods. An efficient method for identifying single copy transgene insertion events from a population of independent transgenic lines is desirable. Currently ...

  10. Mitochondrial DNA copy number, but not haplogroup is associated with keratoconus in Han Chinese population.

    PubMed

    Hao, Xiao-Dan; Chen, Peng; Wang, Ye; Li, Su-Xia; Xie, Li-Xin

    2015-03-01

    Oxidative stress may play a role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is closely related to mitochondrion function, and variations may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. To test whether mtDNA background and copy number confer genetic susceptibility to KC in the Han Chinese population, we performed this association study. We analyzed mtDNA sequence variations in 210 KC patients and 309 matched individuals from China, and classified each subject by haplogroup. Mitochondrial DNA copy number was measured in a subset of these subjects (193 patients and 103 controls). Comparison of matrilineal components of the cases and control populations revealed no significant difference. However, measurement of mtDNA copy number showed that KC patients had significantly lower mtDNA copy numbers than controls (P = 0.0002), even when age, gender, and mtDNA background were considered. Our results suggest that mtDNA copy number, but not haplogroup, is associated with keratoconus, and may contribute to its pathogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

    PubMed Central

    Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F.

    2016-01-01

    Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion. PMID:27031233

  12. Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

    PubMed

    Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F

    2016-01-01

    Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

  13. Porcine oocyte mtDNA copy number is high or low depending on the donor.

    PubMed

    Pedersen, Hanne Skovsgaard; Løvendahl, Peter; Larsen, Knud; Madsen, Lone Bruhn; Callesen, Henrik

    2016-08-01

    Oocyte capacity is relevant in understanding decreasing female fertility and in the use of assisted reproductive technologies in human and farm animals. Mitochondria are important to the development of a functionally good oocyte and the oocyte mtDNA copy number has been introduced as a useful parameter for prediction of oocyte competence. The aim of this study was to investigate: (i) if the oocyte donor has an influence on its oocyte's mtDNA copy number; and (ii) the relation between oocyte size and mtDNA copy number using pre- and postpubertal pig oocytes. Cumulus-oocyte complexes were collected from individual donor pigs. The oocytes were allocated into different size-groups, snap-frozen and single-oocyte mtDNA copy number was estimated by quantitative real-time PCR using the genes ND1 and COX1. Results showed that mean mtDNA copy number in oocytes from any individual donor could be categorized as either 'high' (≥100,000) or 'low' (<100,000) with no difference in threshold between pre- and postpubertal oocytes. No linear correlation was detected between oocyte size and mtDNA copy number within pre- and postpubertal oocytes. This study demonstrates the importance of the oocyte donor in relation to oocyte mtDNA copy number, irrespectively of the donor's puberty status and the oocyte's growth stage. Observations from this study facilitate both further investigations of the importance of mtDNA copy number and the unravelling of relations between different mitochondrial parameters and oocyte competence.

  14. A prospective study of mitochondrial DNA copy number and the risk of prostate cancer.

    PubMed

    Moore, Amy; Lan, Qing; Hofmann, Jonathan N; Liu, Chin-San; Cheng, Wen-Ling; Lin, Ta-Tsung; Berndt, Sonja I

    2017-06-01

    Evidence suggests that mitochondrial DNA (mtDNA) copy number increases in response to DNA damage. Increased mtDNA copy number has been observed in prostate cancer (PCa) cells, suggesting a role in PCa development, but this association has not yet been investigated prospectively. We conducted a nested case-control study (793 cases and 790 controls) of men randomized to the screening arm of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) to evaluate the association between pre-diagnosis mtDNA copy number, measured in peripheral blood leukocytes, and the risk of PCa. We used logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI) and polytomous logistic regression to analyze differences in associations by non-aggressive (Stage I/II AND Gleason grade < 8) or aggressive (Stage III/IV OR Gleason grade ≥ 8) PCa. Although mtDNA copy number was not significantly associated with PCa risk overall (OR 1.23, 95% CI 0.97-1.55, p = 0.089), increasing mtDNA copy number was associated with an increased risk of non-aggressive PCa (OR 1.29, 95% CI 1.01-1.65, p = 0.044) compared to controls. No association was observed with aggressive PCa (OR 1.02, 95% CI 0.64-1.63, p = 0.933). Higher mtDNA copy number was also associated with increased PSA levels among controls (p = 0.014). These results suggest that alterations in mtDNA copy number may reflect disruption of the normal prostate glandular architecture seen in early-stage disease, as opposed to reflecting the large number of tumor cells seen with advanced PCa.

  15. The relationship between mitochondrial DNA copy number and stallion sperm function.

    PubMed

    Darr, Christa R; Moraes, Luis E; Connon, Richard E; Love, Charles C; Teague, Sheila; Varner, Dickson D; Meyers, Stuart A

    2017-05-01

    Mitochondrial DNA (mtDNA) copy number has been utilized as a measure of sperm quality in several species including mice, dogs, and humans, and has been suggested as a potential biomarker of fertility in stallion sperm. The results of the present study extend this recent discovery using sperm samples from American Quarter Horse stallions of varying age. By determining copy number of three mitochondrial genes, cytochrome b (CYTB), NADH dehydrogenase 1 (ND1) and NADH dehydrogenase 4 (ND4), instead of a single gene, we demonstrate an improved understanding of mtDNA fate in stallion sperm mitochondria following spermatogenesis. Sperm samples from 37 stallions ranging from 3 to 24 years old were collected at four breeding ranches in north and central Texas during the 2015 breeding season. Samples were analyzed for sperm motion characteristics, nuclear DNA denaturability and mtDNA copy number. Mitochondrial DNA content in individual sperm was determined by real-time qPCR and normalized with a single copy nuclear gene, Beta actin. Exploratory correlation analysis revealed that total motility was negatively correlated with CYTB copy number and sperm chromatin structure. Stallion age did not have a significant effect on copy number for any of the genes. Copy number differences existed between the three genes with CYTB having the greatest number of copies (20.6 ± 1.2 copies, range: 6.0 to 41.1) followed by ND4 (15.5 ± 0.8 copies, range: 6.7 to 27.8) and finally ND1 (12.0 ± 1.0 copies, range: 0.4 to 26.6) (P < 0.05). Varying copy number across mitochondrial genes is likely to be a result of mtDNA fragmentation and degradation since downregulation of sperm mtDNA occurs during spermatogenesis and may be important for normal sperm function. Beta regression analysis suggested that for every unit increase in mtDNA copy number of CYTB, there was a 4% decrease in the odds of sperm movement (P = 0.001). Influential analysis suggested that results are robust and not highly

  16. High copy number of mitochondrial DNA predicts poor prognosis in patients with advanced stage colon cancer.

    PubMed

    Wang, Yun; He, Shuixiang; Zhu, Xingmei; Qiao, Wei; Zhang, Juan

    2016-12-23

    The aim of this investigation was to determine whether alterations in mitochondrial DNA (mtDNA) copy number in colon cancer were associated with clinicopathological parameters and postsurgical outcome. By quantitative real-time PCR assay, the mtDNA copy number was detected in a cohort of colon cancer and matched adjacent colon tissues (n = 162). The majority of patients had higher mtDNA content in colon cancer tissues than matched adjacent colon tissues. Moreover, high mtDNA content in tumor tissues was associated with larger tumor size, higher serum CEA level, advanced TNM stage, vascular emboli, and liver metastases. Further survival curve analysis showed that high mtDNA content was related to the worst survival in patients with colon cancer at advanced TNM stage. High mtDNA content is a potential effective factor of poor prognosis in patients with advanced stage colon cancer.

  17. Association between Leukocyte Mitochondrial DNA Copy Number and Regular Exercise in Postmenopausal Women.

    PubMed

    Chang, Yu Kyung; Kim, Da Eun; Cho, Soo Hyun; Kim, Jung-Ha

    2016-11-01

    Previous studies suggest that habitual exercise can improve skeletal mitochondrial function; however, to date, the association between exercise and mitochondrial function in peripheral leukocytes has not been reported. The aim of this study was to evaluate the relationship between regular exercise and mitochondrial function by measuring leukocyte mitochondrial DNA (mtDNA) copy number in postmenopausal women. This cross-sectional study included 144 relatively healthy, non-diabetic, non-smoking, postmenopausal women. Clinical parameters, including anthropometric measurements and cardio-metabolic parameters, were assessed. Regular exercise was defined as at least 150 minutes per week of moderate-intensity activity, or an equivalent combination of moderate and vigorous-intensity activity, over a duration of at least 6 months. Leukocyte mtDNA copy numbers were measured using real-time polymerase chain reaction assays, and these were normalized to the β-globin copy number to give the relative mtDNA copy number. The mtDNA copy number of peripheral leukocytes was significantly greater in the exercise group (1.33±0.02) than in the no exercise group (1.05±0.02, P<0.01). Stepwise multiple regression analysis showed that regular exercise was independently associated with mtDNA copy number (β=0.25, P<0.01) after adjusting for the variables age, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure, homeostasis model assessment of insulin resistance value, and levels of high-density lipoprotein cholesterol, triglycerides, and homocysteine. Regular exercise is associated with greater leukocyte mtDNA copy number in postmenopausal women.

  18. Association between Leukocyte Mitochondrial DNA Copy Number and Regular Exercise in Postmenopausal Women

    PubMed Central

    Chang, Yu Kyung; Kim, Da Eun; Cho, Soo Hyun

    2016-01-01

    Background Previous studies suggest that habitual exercise can improve skeletal mitochondrial function; however, to date, the association between exercise and mitochondrial function in peripheral leukocytes has not been reported. The aim of this study was to evaluate the relationship between regular exercise and mitochondrial function by measuring leukocyte mitochondrial DNA (mtDNA) copy number in postmenopausal women. Methods This cross-sectional study included 144 relatively healthy, non-diabetic, non-smoking, postmenopausal women. Clinical parameters, including anthropometric measurements and cardio-metabolic parameters, were assessed. Regular exercise was defined as at least 150 minutes per week of moderate-intensity activity, or an equivalent combination of moderate and vigorous-intensity activity, over a duration of at least 6 months. Leukocyte mtDNA copy numbers were measured using real-time polymerase chain reaction assays, and these were normalized to the β-globin copy number to give the relative mtDNA copy number. Results The mtDNA copy number of peripheral leukocytes was significantly greater in the exercise group (1.33±0.02) than in the no exercise group (1.05±0.02, P<0.01). Stepwise multiple regression analysis showed that regular exercise was independently associated with mtDNA copy number (β=0.25, P<0.01) after adjusting for the variables age, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure, homeostasis model assessment of insulin resistance value, and levels of high-density lipoprotein cholesterol, triglycerides, and homocysteine. Conclusion Regular exercise is associated with greater leukocyte mtDNA copy number in postmenopausal women. PMID:27900071

  19. Integration of DNA Copy Number Alterations and Transcriptional Expression Analysis in Human Gastric Cancer

    PubMed Central

    Coral, Ho; Yuen, Siu Tsan; Chu, Kent Man; Law, Simon; Zhang, Lianhai; Ji, Jiafu; Leung, Suet Yi; Chen, Xin

    2012-01-01

    Background Genomic instability with frequent DNA copy number alterations is one of the key hallmarks of carcinogenesis. The chromosomal regions with frequent DNA copy number gain and loss in human gastric cancer are still poorly defined. It remains unknown how the DNA copy number variations contributes to the changes of gene expression profiles, especially on the global level. Principal Findings We analyzed DNA copy number alterations in 64 human gastric cancer samples and 8 gastric cancer cell lines using bacterial artificial chromosome (BAC) arrays based comparative genomic hybridization (aCGH). Statistical analysis was applied to correlate previously published gene expression data obtained from cDNA microarrays with corresponding DNA copy number variation data to identify candidate oncogenes and tumor suppressor genes. We found that gastric cancer samples showed recurrent DNA copy number variations, including gains at 5p, 8q, 20p, 20q, and losses at 4q, 9p, 18q, 21q. The most frequent regions of amplification were 20q12 (7/72), 20q12–20q13.1 (12/72), 20q13.1–20q13.2 (11/72) and 20q13.2–20q13.3 (6/72). The most frequent deleted region was 9p21 (8/72). Correlating gene expression array data with aCGH identified 321 candidate oncogenes, which were overexpressed and showed frequent DNA copy number gains; and 12 candidate tumor suppressor genes which were down-regulated and showed frequent DNA copy number losses in human gastric cancers. Three networks of significantly expressed genes in gastric cancer samples were identified by ingenuity pathway analysis. Conclusions This study provides insight into DNA copy number variations and their contribution to altered gene expression profiles during human gastric cancer development. It provides novel candidate driver oncogenes or tumor suppressor genes for human gastric cancer, useful pathway maps for the future understanding of the molecular pathogenesis of this malignancy, and the construction of new therapeutic

  20. Ribosomal DNA and Stellate gene copy number variation on the Y chromosome of Drosophila melanogaster.

    PubMed

    Lyckegaard, E M; Clark, A G

    1989-03-01

    Multigene families on the Y chromosome face an unusual array of evolutionary forces. Both ribosomal DNA and Stellate, the two families examined here, have multiple copies of similar sequences on the X and Y chromosomes. Although the rate of sequence divergence on the Y chromosome depends on rates of mutation, gene conversion and exchange with the X chromosome, as well as purifying selection, the regulation of gene copy number may also depend on other pleiotropic functions, such as maintenance of chromosome pairing. Gene copy numbers were estimated for a series of 34 Y chromosome replacement lines using densitometric measurements of slot blots of genomic DNA from adult Drosophila melanogaster. Scans of autoradiographs of the same blots probed with the cloned alcohol dehydrogenase gene, a single copy gene, served as internal standards. Copy numbers span a 6-fold range for ribosomal DNA and a 3-fold range for Stellate DNA. Despite this magnitude of variation, there was no association between copy number and segregation variation of the sex chromosomes.

  1. rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants.

    PubMed

    Kwan, Elizabeth X; Wang, Xiaobin S; Amemiya, Haley M; Brewer, Bonita J; Raghuraman, M K

    2016-09-08

    The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae. Copyright © 2016 Kwan et al.

  2. rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants

    PubMed Central

    Kwan, Elizabeth X.; Wang, Xiaobin S.; Amemiya, Haley M.; Brewer, Bonita J.; Raghuraman, M. K.

    2016-01-01

    The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae. PMID:27449518

  3. Rapid detection of chromosome 18 copy number in buccal smears using DNA probes and FISH

    SciTech Connect

    Harris, C.; Nunez, M.; Giraldez, R.

    1994-09-01

    Rapid diagnosis of trisomy 18 in newborns is often critical to clinical management decisions that must be made in a minimum of time. DNA probes combined with FISH can be used to accurately to determine the copy number of chromosome 18 in interphase cells. We have used the D18Z1 alpha satellite DNA probe to determine signal frequency in normal, previously karyotyped subjects, 12 females and 6 males. We also present one clinical case of trisomy 18, confirmed by karyotype, for comparison to the results obtained from normal subjects. Buccal smears, unlike cytogenetic preparations from peripheral blood, are quite resistant to penetration of probes and detection reagents resulting in higher levels of false monosomy. We have studied 19 individuals and have obtained consistent FISH results, ranging from 64 to 90% disomy. False monosomy rates ranged from 10 to 36%, while false trisomy or tetrasomy was less than 1% in all samples. High rates of false monosomy make this test questionable for detection of low order mosaicism for monosomy, but the extremely low false hyperploidy rate suggests that this is a dependable procedure for detection of trisomy 18, enabling the use of buccal epithelium which can be collected easily from even premature and tiny infants.

  4. The potential role for use of mitochondrial DNA copy number as predictive biomarker in presbycusis

    PubMed Central

    Falah, Masoumeh; Houshmand, Massoud; Najafi, Mohammad; Balali, Maryam; Mahmoudian, Saeid; Asghari, Alimohamad; Emamdjomeh, Hessamaldin; Farhadi, Mohammad

    2016-01-01

    Objectives Age-related hearing impairment, or presbycusis, is the most common communication disorder and neurodegenerative disease in the elderly. Its prevalence is expected to increase, due to the trend of growth of the elderly population. The current diagnostic test for detection of presbycusis is implemented after there has been a change in hearing sensitivity. Identification of a pre-diagnostic biomarker would raise the possibility of preserving hearing sensitivity before damage occurs. Mitochondrial dysfunction, including the production of reactive oxygen species and induction of expression of apoptotic genes, participates in the progression of presbycusis. Mitochondrial DNA sequence variation has a critical role in presbycusis. However, the nature of the relationship between mitochondrial DNA copy number, an important biomarker in many other diseases, and presbycusis is undetermined. Methods Fifty-four subjects with presbycusis and 29 healthy controls were selected after ear, nose, throat examination and pure-tone audiometry. DNA was extracted from peripheral blood samples. The copy number of mitochondrial DNA relative to the nuclear genome was measured by quantitative real-time polymerase chain reaction. Results Subjects with presbycusis had a lower median mitochondrial DNA copy number than healthy subjects and the difference was statistically significant (P=0.007). Mitochondrial DNA copy number was also significantly associated with degree of hearing impairment (P=0.025) and audiogram configuration (P=0.022). Conclusion The findings of this study suggest that lower mitochondrial DNA copy number is responsible for presbycusis through alteration of mitochondrial function. Moreover, the significant association of mitochondrial DNA copy number in peripheral blood samples with the degree of hearing impairment and audiogram configuration has potential for use as a standard test for presbycusis, providing the possibility of the development of an easy

  5. Loss of the Association between Telomere Length and Mitochondrial DNA Copy Number Contribute to Colorectal Carcinogenesis.

    PubMed

    Lee, Hyunsu; Cho, Ji-Hyoung; Park, Won-Jin; Jung, Soo-Jung; Choi, In-Jang; Lee, Jae-Ho

    2017-05-09

    Positive association between telomere length and mitochondrial DNA (mtDNA) copy number were introduced in healthy and patients with psychiatric disorder. Based on frequent genetic changes of telomere and mitochondria in colorectal carcinomas (CRC), we studied their clinical characteristics and their association in colorectal carcinogenesis. DNA was extracted from 109 CRCs, 64 colorectal tubular adenomas (TAs), and 28 serrated polyps (SPs), and then, telomere length and mtDNA copy number were analyzed in these legions by using a real-time PCR assay. Telomere length and mtDNA copy number (mean ± S.D) in CRCs was 1.87 ± 1.52 and 1.61 ± 1.37, respectively. In TAs and SPs, relative mtDNA copy number was 0.92 ± 0.71 and 1.84 ± 1.06, respectively, shoing statistical difference (p = 0.017). However, telomere length was similar in these precancerous legions. Telomere length and mtDNA copy number did not show clinical and prognostic values in CRCs, however, positive correlation between telomere length and mitochondrial DNA copy number were found in CRC (r = 0.408, p < 0.001). However, this association was not shown in precancerous lesions (r = -0.031, p = 0.765). This result suggests that loss of co-regulation between telomeres and mitochondrial function may induce the initiation or play a role as trigger factor of colorectal carcinogenesis.

  6. DNA barcode data accurately assign higher spider taxa.

    PubMed

    Coddington, Jonathan A; Agnarsson, Ingi; Cheng, Ren-Chung; Čandek, Klemen; Driskell, Amy; Frick, Holger; Gregorič, Matjaž; Kostanjšek, Rok; Kropf, Christian; Kweskin, Matthew; Lokovšek, Tjaša; Pipan, Miha; Vidergar, Nina; Kuntner, Matjaž

    2016-01-01

    The use of unique DNA sequences as a method for taxonomic identification is no longer fundamentally controversial, even though debate continues on the best markers, methods, and technology to use. Although both existing databanks such as GenBank and BOLD, as well as reference taxonomies, are imperfect, in best case scenarios "barcodes" (whether single or multiple, organelle or nuclear, loci) clearly are an increasingly fast and inexpensive method of identification, especially as compared to manual identification of unknowns by increasingly rare expert taxonomists. Because most species on Earth are undescribed, a complete reference database at the species level is impractical in the near term. The question therefore arises whether unidentified species can, using DNA barcodes, be accurately assigned to more inclusive groups such as genera and families-taxonomic ranks of putatively monophyletic groups for which the global inventory is more complete and stable. We used a carefully chosen test library of CO1 sequences from 49 families, 313 genera, and 816 species of spiders to assess the accuracy of genus and family-level assignment. We used BLAST queries of each sequence against the entire library and got the top ten hits. The percent sequence identity was reported from these hits (PIdent, range 75-100%). Accurate assignment of higher taxa (PIdent above which errors totaled less than 5%) occurred for genera at PIdent values >95 and families at PIdent values ≥ 91, suggesting these as heuristic thresholds for accurate generic and familial identifications in spiders. Accuracy of identification increases with numbers of species/genus and genera/family in the library; above five genera per family and fifteen species per genus all higher taxon assignments were correct. We propose that using percent sequence identity between conventional barcode sequences may be a feasible and reasonably accurate method to identify animals to family/genus. However, the quality of the

  7. DNA barcode data accurately assign higher spider taxa

    PubMed Central

    Coddington, Jonathan A.; Agnarsson, Ingi; Cheng, Ren-Chung; Čandek, Klemen; Driskell, Amy; Frick, Holger; Gregorič, Matjaž; Kostanjšek, Rok; Kropf, Christian; Kweskin, Matthew; Lokovšek, Tjaša; Pipan, Miha; Vidergar, Nina

    2016-01-01

    The use of unique DNA sequences as a method for taxonomic identification is no longer fundamentally controversial, even though debate continues on the best markers, methods, and technology to use. Although both existing databanks such as GenBank and BOLD, as well as reference taxonomies, are imperfect, in best case scenarios “barcodes” (whether single or multiple, organelle or nuclear, loci) clearly are an increasingly fast and inexpensive method of identification, especially as compared to manual identification of unknowns by increasingly rare expert taxonomists. Because most species on Earth are undescribed, a complete reference database at the species level is impractical in the near term. The question therefore arises whether unidentified species can, using DNA barcodes, be accurately assigned to more inclusive groups such as genera and families—taxonomic ranks of putatively monophyletic groups for which the global inventory is more complete and stable. We used a carefully chosen test library of CO1 sequences from 49 families, 313 genera, and 816 species of spiders to assess the accuracy of genus and family-level assignment. We used BLAST queries of each sequence against the entire library and got the top ten hits. The percent sequence identity was reported from these hits (PIdent, range 75–100%). Accurate assignment of higher taxa (PIdent above which errors totaled less than 5%) occurred for genera at PIdent values >95 and families at PIdent values ≥ 91, suggesting these as heuristic thresholds for accurate generic and familial identifications in spiders. Accuracy of identification increases with numbers of species/genus and genera/family in the library; above five genera per family and fifteen species per genus all higher taxon assignments were correct. We propose that using percent sequence identity between conventional barcode sequences may be a feasible and reasonably accurate method to identify animals to family/genus. However, the quality of

  8. Cardiometabolic phenotypes and mitochondrial DNA copy number in two cohorts of UK women.

    PubMed

    Guyatt, Anna L; Burrows, Kimberley; Guthrie, Philip A I; Ring, Sue; McArdle, Wendy; Day, Ian N M; Ascione, Raimondo; Lawlor, Debbie A; Gaunt, Tom R; Rodriguez, Santiago

    2017-08-15

    The mitochondrial genome is present at variable copy number between individuals. Mitochondria are vulnerable to oxidative stress, and their dysfunction may be associated with cardiovascular disease. The association of mitochondrial DNA copy number with cardiometabolic risk factors (lipids, glycaemic traits, inflammatory markers, anthropometry and blood pressure) was assessed in two independent cohorts of European origin women, one in whom outcomes were measured at mean (SD) age 30 (4.3) years (N=2278) and the second at 69.4 (5.5) years (N=2872). Mitochondrial DNA copy number was assayed by quantitative polymerase chain reaction. Associations were adjusted for smoking, sociodemographic status, laboratory factors and white cell traits. Out of a total of 12 outcomes assessed in both cohorts, mitochondrial DNA copy number showed little or no association with the majority (point estimates were close to zero and nearly all p-values were >0.01). The strongest evidence was for an inverse association in the older cohort with insulin (standardised beta [95%CI]: -0.06, [-0.098, -0.022], p=0.002), but this association did not replicate in the younger cohort. Our findings do not provide support for variation in mitochondrial DNA copy number having an important impact on cardio-metabolic risk factors in European origin women. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Global Genetic Determinants of Mitochondrial DNA Copy Number

    PubMed Central

    Zhang, Hengshan; Singh, Keshav K.

    2014-01-01

    Many human diseases including development of cancer is associated with depletion of mitochondrial DNA (mtDNA) content. These diseases are collectively described as mitochondrial DNA depletion syndrome (MDS). High similarity between yeast and human mitochondria allows genomic study of the budding yeast to be used to identify human disease genes. In this study, we systematically screened the pre-existing respiratory-deficient Saccharomyces cerevisiae yeast strains using fluorescent microscopy and identified 102 nuclear genes whose deletions result in a complete mtDNA loss, of which 52 are not reported previously. Strikingly, these genes mainly encode protein products involved in mitochondrial protein biosynthesis process (54.9%). The rest of these genes either encode protein products associated with nucleic acid metabolism (14.7%), oxidative phosphorylation (3.9%), or other protein products (13.7%) responsible for bud-site selection, mitochondrial intermembrane space protein import, assembly of cytochrome-c oxidase, vacuolar protein sorting, protein-nucleus import, calcium-mediated signaling, heme biosynthesis and iron homeostasis. Thirteen (12.7%) of the genes encode proteins of unknown function. We identified human orthologs of these genes, conducted the interaction between the gene products and linked them to human mitochondrial disorders and other pathologies. In addition, we screened for genes whose defects affect the nuclear genome integrity. Our data provide a systematic view of the nuclear genes involved in maintenance of mitochondrial DNA. Together, our studies i) provide a global view of the genes regulating mtDNA content; ii) provide compelling new evidence toward understanding novel mechanism involved in mitochondrial genome maintenance and iii) provide useful clues in understanding human diseases in which mitochondrial defect and in particular depletion of mitochondrial genome plays a critical role. PMID:25170845

  10. Genome-wide DNA copy number analysis in pancreatic cancer using high-density single nucleotide polymorphism arrays

    PubMed Central

    Harada, T; Chelala, C; Bhakta, V; Chaplin, T; Caulee, K; Baril, P; Young, BD; Lemoine, NR

    2008-01-01

    To identify genomic abnormalities characteristic of pancreatic ductal adenocarcinoma (PDAC) in vivo, a panel of 27 microdissected PDAC specimens were analysed using high-density microarrays representing ∼116 000 single nucleotide polymorphism (SNP) loci. We detected frequent gains of 1q, 2, 3, 5, 7p, 8q, 11, 14q and 17q (≥78% of cases), and losses of 1p, 3p, 6, 9p, 13q, 14q, 17p and 18q (≥44%). Although the results were comparable with those from array CGH, regions of those genetic changes were defined more accurately by SNP arrays. Integrating the Ensembl public data, we have generated ‘gene’ copy number indices that facilitate the search for novel candidates involved in pancreatic carcinogenesis. Copy numbers in a subset of the genes were validated using quantitative real-time PCR. The SKAP2/SCAP2 gene (7p15.2), which belongs to the src family kinases, was most frequently (63%) amplified in our sample set and its recurrent overexpression (67%) was confirmed by reverse transcription–PCR. Furthermore, fluorescence in situ hybridization and in situ RNA hybridization analyses for this gene have demonstrated a significant correlation between DNA copy number and mRNA expression level in an independent sample set (P<0.001). These findings indicate that the dysregulation of SKAP2/SCAP2, which is mostly caused by its increased gene copy number, is likely to be associated with the development of PDAC. PMID:17952125

  11. Quantitation of DNA copy number in individual mitochondrial particles by capillary electrophoresis.

    PubMed

    Navratil, Marian; Poe, Bobby G; Arriaga, Edgar A

    2007-10-15

    Here, we present a direct method for determining mitochondrial DNA (mtDNA) copy numbers in individual mitochondrial particles, isolated from cultured cells, by means of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. We demonstrate that this method can detect a single molecule of PicoGreen-stained mtDNA in intact DsRed2-labeled mitochondrial particles isolated from human osteosarcoma 143B cells. This ultimate limit of mtDNA detection made it possible to confirm that an individual mitochondrial nucleoid, the genetic unit of mitochondrial inheritance, can contain a single copy of mtDNA. The validation of this approach was achieved via monitoring chemically induced mtDNA depletion and comparing the CE-LIF results to those obtained by quantitative microscopy imaging and multiplex real-time PCR analysis. Owing to its sensitivity, the CE-LIF method may become a powerful tool for investigating the copy number and organization of mtDNA in mitochondrial disease and aging, and in molecular biology techniques requiring manipulation and quantitation of DNA molecules such as plasmids.

  12. Human TOP3: a single-copy gene encoding DNA topoisomerase III.

    PubMed Central

    Hanai, R; Caron, P R; Wang, J C

    1996-01-01

    A human cDNA encoding a protein homologous to the Escherichia coli DNA topoisomerase I subfamily of enzymes has been identified through cloning and sequencing. Expressing the cloned human cDNA in yeast (delta)top1 cells lacking endogenous DNA topoisomerase I yielded an activity in cell extracts that specifically reduces the number of supercoils in a highly negatively supercoiled DNA. On the basis of these results, the human gene containing the cDNA sequence has been denoted TOP3, and the protein it encodes has been denoted DNA topoisomerase III. Screening of a panel of human-rodent somatic hybrids and fluorescence in situ hybridization of cloned TOP3 genomic DNA to metaphase chromosomes indicate that human TOP3 is a single-copy gene located at chromosome 17p11.2-12. Images Fig. 2 PMID:8622991

  13. Allele-specific copy number profiling by next-generation DNA sequencing.

    PubMed

    Chen, Hao; Bell, John M; Zavala, Nicolas A; Ji, Hanlee P; Zhang, Nancy R

    2015-02-27

    The progression and clonal development of tumors often involve amplifications and deletions of genomic DNA. Estimation of allele-specific copy number, which quantifies the number of copies of each allele at each variant loci rather than the total number of chromosome copies, is an important step in the characterization of tumor genomes and the inference of their clonal history. We describe a new method, falcon, for finding somatic allele-specific copy number changes by next generation sequencing of tumors with matched normals. falcon is based on a change-point model on a bivariate mixed Binomial process, which explicitly models the copy numbers of the two chromosome haplotypes and corrects for local allele-specific coverage biases. By using the Binomial distribution rather than a normal approximation, falcon more effectively pools evidence from sites with low coverage. A modified Bayesian information criterion is used to guide model selection for determining the number of copy number events. Falcon is evaluated on in silico spike-in data and applied to the analysis of a pre-malignant colon tumor sample and late-stage colorectal adenocarcinoma from the same individual. The allele-specific copy number estimates obtained by falcon allows us to draw detailed conclusions regarding the clonal history of the individual's colon cancer.

  14. Copy number variations in urine cell free DNA as biomarkers in advanced prostate cancer.

    PubMed

    Xia, Yun; Huang, Chiang-Ching; Dittmar, Rachel; Du, Meijun; Wang, Yuan; Liu, Hongyan; Shenoy, Niraj; Wang, Liang; Kohli, Manish

    2016-06-14

    Genetic profiling of urine cell free DNA (cfDNA) has not been evaluated in advanced prostate cancer. We performed whole genome sequencing of urine cfDNAs to identify tumor-associated copy number variations in urine before and after initiating androgen deprivation therapy in HSPC stage and docetaxel chemotherapy in CRPC stage. A log2 ratio-based copy number analysis detected common genomic abnormalities in prostate cancer including AR amplification in 5/10 CRPC patients. Other abnormalities identified included TMPRSS2-ERG fusion, PTEN gene deletion, NOTCH1 locus amplification along with genomic amplifications at 8q24.3, 9q34.3, 11p15.5 and 14q11.2, and deletions at 4q35.2, 5q31.3, 7q36.3, 12q24.33, and 16p11.2. By comparing copy number between pre- and post-treatment, we found significant copy number changes in 34 genomic loci. To estimate the somatic tumor DNA fraction in urine cfDNAs, we developed a Urine Genomic Abnormality (UGA) score algorithm that summed the top ten most significant segments with copy number changes. The UGA scores correlated with tumor burden and the change in UGA score after stage-specific therapies reflected disease progression status and overall survival. The study demonstrates the potential clinical utility of urine cfDNAs in predicting treatment response and monitoring disease progression.

  15. DNA copy number changes define spatial patterns of heterogeneity in colorectal cancer

    PubMed Central

    Mamlouk, Soulafa; Childs, Liam Harold; Aust, Daniela; Heim, Daniel; Melching, Friederike; Oliveira, Cristiano; Wolf, Thomas; Durek, Pawel; Schumacher, Dirk; Bläker, Hendrik; von Winterfeld, Moritz; Gastl, Bastian; Möhr, Kerstin; Menne, Andrea; Zeugner, Silke; Redmer, Torben; Lenze, Dido; Tierling, Sascha; Möbs, Markus; Weichert, Wilko; Folprecht, Gunnar; Blanc, Eric; Beule, Dieter; Schäfer, Reinhold; Morkel, Markus; Klauschen, Frederick; Leser, Ulf; Sers, Christine

    2017-01-01

    Genetic heterogeneity between and within tumours is a major factor determining cancer progression and therapy response. Here we examined DNA sequence and DNA copy-number heterogeneity in colorectal cancer (CRC) by targeted high-depth sequencing of 100 most frequently altered genes. In 97 samples, with primary tumours and matched metastases from 27 patients, we observe inter-tumour concordance for coding mutations; in contrast, gene copy numbers are highly discordant between primary tumours and metastases as validated by fluorescent in situ hybridization. To further investigate intra-tumour heterogeneity, we dissected a single tumour into 68 spatially defined samples and sequenced them separately. We identify evenly distributed coding mutations in APC and TP53 in all tumour areas, yet highly variable gene copy numbers in numerous genes. 3D morpho-molecular reconstruction reveals two clusters with divergent copy number aberrations along the proximal–distal axis indicating that DNA copy number variations are a major source of tumour heterogeneity in CRC. PMID:28120820

  16. Automated design of paralogue ratio test assays for the accurate and rapid typing of copy number variation

    PubMed Central

    Veal, Colin D.; Xu, Hang; Reekie, Katherine; Free, Robert; Hardwick, Robert J.; McVey, David; Brookes, Anthony J.; Hollox, Edward J.; Talbot, Christopher J.

    2013-01-01

    Motivation: Genomic copy number variation (CNV) can influence susceptibility to common diseases. High-throughput measurement of gene copy number on large numbers of samples is a challenging, yet critical, stage in confirming observations from sequencing or array Comparative Genome Hybridization (CGH). The paralogue ratio test (PRT) is a simple, cost-effective method of accurately determining copy number by quantifying the amplification ratio between a target and reference amplicon. PRT has been successfully applied to several studies analyzing common CNV. However, its use has not been widespread because of difficulties in assay design. Results: We present PRTPrimer (www.prtprimer.org) software for automated PRT assay design. In addition to stand-alone software, the web site includes a database of pre-designed assays for the human genome at an average spacing of 6 kb and a web interface for custom assay design. Other reference genomes can also be analyzed through local installation of the software. The usefulness of PRTPrimer was tested within known CNV, and showed reproducible quantification. This software and database provide assays that can rapidly genotype CNV, cost-effectively, on a large number of samples and will enable the widespread adoption of PRT. Availability: PRTPrimer is available in two forms: a Perl script (version 5.14 and higher) that can be run from the command line on Linux systems and as a service on the PRTPrimer web site (www.prtprimer.org). Contact: cjt14@le.ac.uk Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:23742985

  17. Comparison of dna-copying fidelity during repair and semiconservative synthesis by radioactive precursor distribution analysis

    SciTech Connect

    Nemirovskii, L.E.; Vasil'ev, V.K.

    1986-04-01

    The authors compare the fidelity of DNA copying during semiconservative and reparative synthesis under normal conditions and during cortisol-induced activation of free-radical processes, by examining the distribution of radioactivity among DNA pyrimidine isopliths. Radioactivity of nucleotide material in the isopliths was measured by counting in appropriate zones of the chromatograms in toluene scintillator. The investigation shows that injury to DNA of different organs, both directly and as a result of faulty repair, leads to shortening of the pyrimidine isopliths, i.e., to changes in the primary structure of DNA. These data help to explain the simultaneously cytostatic, carcinostatic, and mutagenic action of irradiation, cortisol and hydroxyurea.

  18. Detection of DNA copy number alterations in cancer by array comparative genomic hybridization.

    PubMed

    Michels, Evi; De Preter, Katleen; Van Roy, Nadine; Speleman, Frank

    2007-09-01

    Over the past few years, various reliable platforms for high-resolution detection of DNA copy number changes have become widely available. Together with optimized protocols for labeling and hybridization and algorithms for data analysis and representation, this has lead to a rapid increase in the application of this technology in the study of copy number variation in the human genome in normal cells and copy number imbalances in genetic diseases, including cancer. In this review, we briefly discuss specific technical issues relevant for array comparative genomic hybridization analysis in cancer tissues. We specifically focus on recent successes of array comparative genomic hybridization technology in the progress of our understanding of oncogenesis in a variety of cancer types. A third section highlights the potential of sensitive genome-wide detection of patterns of DNA imbalances or molecular portraits for class discovery and therapeutic stratification.

  19. Sequential Model Selection based Segmentation to Detect DNA Copy Number Variation

    PubMed Central

    Hu, Jianhua; Zhang, Liwen; Wang, Huixia Judy

    2016-01-01

    Summary Array-based CGH experiments are designed to detect genomic aberrations or regions of DNA copy-number variation that are associated with an outcome, typically a state of disease. Most of the existing statistical methods target on detecting DNA copy number variations in a single sample or array. We focus on the detection of group effect variation, through simultaneous study of multiple samples from multiple groups. Rather than using direct segmentation or smoothing techniques, as commonly seen in existing detection methods, we develop a sequential model selection procedure that is guided by a modified Bayesian information criterion. This approach improves detection accuracy by accumulatively utilizing information across contiguous clones, and has computational advantage over the existing popular detection methods. Our empirical investigation suggests that the performance of the proposed method is superior to that of the existing detection methods, in particular, in detecting small segments or separating neighboring segments with differential degrees of copy-number variation. PMID:26954760

  20. Optimal design of oligonucleotide microarrays for measurement of DNA copy-number.

    PubMed

    Sharp, Andrew J; Itsara, Andy; Cheng, Ze; Alkan, Can; Schwartz, Stuart; Eichler, Evan E

    2007-11-15

    Copy-number variants (CNVs) occur frequently within the human genome, and may be associated with many human phenotypes. If disease association studies of CNVs are to be performed routinely, it is essential that the copy-number status be accurately genotyped. We systematically assessed the dynamic range response of an oligonucleotide microarray platform to accurately predict copy-number in a set of seven patients who had previously been shown to carry between 1 and 6 copies of an approximately 4 Mb region of 15q12.2-q13.1. We identify probe uniqueness, probe length, uniformity of probe melting temperature, overlap with SNPs and common repeats (particularly Alu elements) and guanine homopolymer content as parameters that significantly affect probe performance. Further, we prove the influence of these criteria on array performance by using these parameters to prospectively filter data from a second array design covering an independent genomic region and observing significant improvements in data quality. The informed selection of probes which have superior performance characteristics allows the prospective design of oligonucleotide arrays which show increased sensitivity and specificity compared with current designs. Although based on the analysis of data from comparative genomic hybridization experiments, we anticipate that our results are relevant to the design of improved oligonucleotide arrays for high-throughput copy-number genotyping of complex regions of the human genome.

  1. DiNAMIC: a method to identify recurrent DNA copy number aberrations in tumors

    PubMed Central

    Walter, Vonn; Nobel, Andrew B.; Wright, Fred A.

    2011-01-01

    Motivation: DNA copy number gains and losses are commonly found in tumor tissue, and some of these aberrations play a role in tumor genesis and development. Although high resolution DNA copy number data can be obtained using array-based techniques, no single method is widely used to distinguish between recurrent and sporadic copy number aberrations. Results: Here we introduce Discovering Copy Number Aberrations Manifested In Cancer (DiNAMIC), a novel method for assessing the statistical significance of recurrent copy number aberrations. In contrast to competing procedures, the testing procedure underlying DiNAMIC is carefully motivated, and employs a novel cyclic permutation scheme. Extensive simulation studies show that DiNAMIC controls false positive discoveries in a variety of realistic scenarios. We use DiNAMIC to analyze two publicly available tumor datasets, and our results show that DiNAMIC detects multiple loci that have biological relevance. Availability: Source code implemented in R, as well as text files containing examples and sample datasets are available at http://www.bios.unc.edu/research/genomic_software/DiNAMIC. Contact: vwalter@email.unc.edu; fwright@bios.unc.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21183584

  2. Quantification of Fewer than Ten Copies of a DNA Biomarker without Amplification or Labeling.

    PubMed

    Lee, Yoonhee; Kim, Youngkyu; Lee, Donggyu; Roy, Dhruvajyoti; Park, Joon Won

    2016-06-08

    Polymerase chain reaction (PCR) is a highly sensitive diagnosis technique for detection of nucleic acids and for monitoring residual disease; however, PCR can be unreliable for samples containing very few target molecules. Here, we describe a quantification method, using force-distance (FD) curve based atomic force microscopy (AFM) to detect a target DNA bound to small (1.4-1.9 μm diameter) probe DNA spots, allowing mapping of entire spots to nanometer resolution. Using a synthetic BCR-ABL fusion gene sequence target, we examined samples containing between one and 10 target copies. A high degree of correlation (r(2) = 0.994) between numbers of target copies and detected probe clusters was observed, and the approach could detect the BCR-ABL biomarker when only a single copy was present, although multiple screens were required. Our results clearly demonstrate that FD curve-based imaging is suitable for quantitative analysis of fewer than 10 copies of DNA biomarkers without amplification, modification, or labeling.

  3. Mitochondrial DNA detection and copy number determination in the spermatozoa of the sea urchin Arbacia lixula.

    PubMed

    De Giorgi, C; D'Alessandro, A; Saccone, C

    1992-02-14

    The Polymerase Chain reaction technique has been used in order to detect and amplify a specific region of mtDNA, in a total DNA preparation extracted from the sperm of the sea urchin Arbacia lixula. The amplified fragment is the D-loop region which hybridizes with the homologous region extracted from the egg mtDNA. The results demonstrate that mtDNA is present in sperm cell, and, since the replication origin is present it is potentially able to replicate in the zygote. Furthermore, the technique used allowed us to estimate mtDNA copy number in sea urchin sperm, which has never been done before. Our results are that sea urchin sperm cell contains between 4 and 28 mtDNA molecules.

  4. High interindividual restriction fragment length and copy number of polymorphism of a TVRI family in moderate human DNA repeats

    SciTech Connect

    Rogaev, E.I.; Shapiro, Yu.A.

    1987-06-01

    The authors describe the selection of cloned human DNA sequences, with a copy number not exceeding 1000 copies per diploid genome, and their testing for interindividual restriction fragment lengths and copy number of polymorphism (RFLCP). As a result of the investigation a DNA clone was found (TVRI-6), about 2.8 kilobase-pairs in size, for which an unusually high level of interindividual RFLCP was discovered. The TVRI-6 sequence was obtained from a bank of Pst I restriction fragments of human placental nuclear DNA cloned in pBR 322. The bank was analyzed by hybridization of colonies with phosphorus 32-labelled human nuclear DNA.

  5. Assessing Mitochondrial DNA Variation and Copy Number in Lymphocytes of ~2,000 Sardinians Using Tailored Sequencing Analysis Tools

    PubMed Central

    Ding, Jun; Sidore, Carlo; Butler, Thomas J.; Wing, Mary Kate; Qian, Yong; Meirelles, Osorio; Busonero, Fabio; Tsoi, Lam C.; Maschio, Andrea; Angius, Andrea; Kang, Hyun Min; Nagaraja, Ramaiah; Cucca, Francesco; Abecasis, Gonçalo R.; Schlessinger, David

    2015-01-01

    DNA sequencing identifies common and rare genetic variants for association studies, but studies typically focus on variants in nuclear DNA and ignore the mitochondrial genome. In fact, analyzing variants in mitochondrial DNA (mtDNA) sequences presents special problems, which we resolve here with a general solution for the analysis of mtDNA in next-generation sequencing studies. The new program package comprises 1) an algorithm designed to identify mtDNA variants (i.e., homoplasmies and heteroplasmies), incorporating sequencing error rates at each base in a likelihood calculation and allowing allele fractions at a variant site to differ across individuals; and 2) an estimation of mtDNA copy number in a cell directly from whole-genome sequencing data. We also apply the methods to DNA sequence from lymphocytes of ~2,000 SardiNIA Project participants. As expected, mothers and offspring share all homoplasmies but a lesser proportion of heteroplasmies. Both homoplasmies and heteroplasmies show 5-fold higher transition/transversion ratios than variants in nuclear DNA. Also, heteroplasmy increases with age, though on average only ~1 heteroplasmy reaches the 4% level between ages 20 and 90. In addition, we find that mtDNA copy number averages ~110 copies/lymphocyte and is ~54% heritable, implying substantial genetic regulation of the level of mtDNA. Copy numbers also decrease modestly but significantly with age, and females on average have significantly more copies than males. The mtDNA copy numbers are significantly associated with waist circumference (p-value = 0.0031) and waist-hip ratio (p-value = 2.4×10-5), but not with body mass index, indicating an association with central fat distribution. To our knowledge, this is the largest population analysis to date of mtDNA dynamics, revealing the age-imposed increase in heteroplasmy, the relatively high heritability of copy number, and the association of copy number with metabolic traits. PMID:26172475

  6. Cell-free DNA copy number variations in plasma from colorectal cancer patients.

    PubMed

    Li, Jian; Dittmar, Rachel L; Xia, Shu; Zhang, Huijuan; Du, Meijun; Huang, Chiang-Ching; Druliner, Brooke R; Boardman, Lisa; Wang, Liang

    2017-08-01

    To evaluate the clinical utility of cell-free DNA (cfDNA), we performed whole-genome sequencing to systematically examine plasma cfDNA copy number variations (CNVs) in a cohort of patients with colorectal cancer (CRC, n = 80), polyps (n = 20), and healthy controls (n = 35). We initially compared cfDNA yield in 20 paired serum-plasma samples and observed significantly higher cfDNA concentration in serum (median = 81.20 ng, range 7.18-500 ng·mL(-1) ) than in plasma (median = 5.09 ng, range 3.76-62.8 ng·mL(-1) ) (P < 0.0001). However, tumor-derived cfDNA content was significantly lower in serum than in matched plasma samples tested. With ~10 million reads per sample, the sequencing-based copy number analysis showed common CNVs in multiple chromosomal regions, including amplifications on 1q, 8q, and 5q and deletions on 1p, 4q, 8p, 17p, 18q, and 22q. Copy number changes were also evident in genes critical to the cell cycle, DNA repair, and WNT signaling pathways. To evaluate whether cumulative copy number changes were associated with tumor stages, we calculated plasma genomic abnormality in colon cancer (PGA-C) score by summing the most significant CNVs. The PGA-C score showed predictive performance with an area under the curve from 0.54 to 0.84 for CRC stages I-IV. Locus-specific copy number analysis identified nine genomic regions where CNVs were significantly associated with survival in stage III-IV CRC patients. A multivariate model using six of nine genomic regions demonstrated a significant association of high-risk score with shorter survival (HR = 5.33, 95% CI = 6.76-94.44, P < 0.0001). Our study demonstrates the importance of using plasma (rather than serum) to test tumor-related genomic variations. Plasma cfDNA-based tests can capture tumor-specific genetic changes and may provide a measurable classifier for assessing clinical outcomes in advanced CRC patients. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  7. Association between Salivary Mitochondrial DNA Copy Number and Chronic Fatigue according to Combined Symptoms in Korean Adults.

    PubMed

    Shin, Jinyoung; Kim, Kyong Chol; Lee, Duk Chul; Lee, Hye Ree; Shim, Jae Yong

    2017-07-01

    We examined the association between salivary mitochondrial DNA (mtDNA) copy number and chronic fatigue combined with depression and insomnia. This cross-sectional study included 58 healthy adults with moderate to severe fatigue (Brief Fatigue Inventory [BFI] ≥4) for longer than 6 months. Subjects were classified as those without combined symptoms, with either depression (Beck Depression Inventory [BDI] ≥13) or insomnia (Pittsburgh Sleep Quality Index [PSQI] ≥5), or with both depression and insomnia. Salivary mtDNA copy number was measured by real-time quantitative polymerase chain reaction. The association was evaluated using a general linear model. About 76% of participants had either depression or insomnia as additional symptoms. These subjects were predominately female, drank more alcohol, and exercised less than those without combined symptoms (P<0.05). The group with both depression and insomnia exhibited significantly higher BFI and lower mtDNA copy number than those without combined symptoms (P<0.05). After adjusting for confounding factors, significant negative associations between mtDNA copy number and usual fatigue were found in the group without combined symptoms, whereas the negative associations in the group with combined symptoms were attenuated. BDI and PSQI were not associated with mtDNA copy number. Chronic fatigue is negatively associated with salivary mtDNA copy number. Salivary mtDNA copy number may be a biological marker of fatigue with or without combined symptoms, indicating that a separate approach is necessary.

  8. Alterations of Mitochondrial DNA Copy Number and Telomere Length with Early Adversity and Psychopathology

    PubMed Central

    Tyrka, Audrey R.; Parade, Stephanie H.; Price, Lawrence H.; Kao, Hung-Teh; Porton, Barbara; Philip, Noah S.; Welch, Emma S.; Carpenter, Linda L.

    2015-01-01

    Background Telomere shortening and alterations of mitochondrial biogenesis are involved in cellular aging. Childhood adversity is associated with telomere shortening, and several investigations have shown short telomeres in psychiatric disorders. Recent studies have examined whether mitochondria might be involved in neuropsychiatric conditions; findings are limited and no prior work has examined this in relation to stress exposure. Methods Two-hundred and ninety healthy adults provided information on childhood parental loss and maltreatment and completed diagnostic interviews. Participants were categorized into four groups based upon the presence or absence of childhood adversity and the presence or absence of lifetime psychopathology (depressive, anxiety, and substance use disorders). Telomere length and mtDNA copy number were measured from leukocyte DNA by qPCR. Results Childhood adversity and lifetime psychopathology were each associated with shorter telomeres (p < .01) and higher mtDNA copy numbers (p < .001). Significantly higher mtDNA copy numbers and shorter telomeres were seen in individuals with major depression, depressive disorders, and anxiety disorders, as well as those with parental loss and childhood maltreatment. A history of substance disorders was also associated with significantly higher mtDNA copy numbers. Conclusion This study provides the first evidence of an alteration of mitochondrial biogenesis with early life stress and with anxiety and substance use disorders. We replicate prior work on telomere length and psychopathology, and show that this effect is not secondary to medication use or comorbid medical illness. Finally, we show that early life stress and psychopathology are each associated with these markers of cellular aging. PMID:25749099

  9. Mutation of the little finger domain in human DNA polymerase η alters fidelity when copying undamaged DNA.

    PubMed

    Beardslee, Renee A; Suarez, Samuel C; Toffton, Shannon M; McCulloch, Scott D

    2013-10-01

    DNA polymerase η (pol η) synthesizes past cyclobutane pyrimidine dimer and possibly 7,8-dihydro-8-oxoguanine (8-oxoG) lesions during DNA replication. Loss of pol η is associated with an increase in mutation rate, demonstrating its indispensable role in mutation suppression. It has been recently reported that β-strand 12 (amino acids 316-324) of the little finger region correctly positions the template strand with the catalytic core of the enzyme. The authors hypothesized that modification of β-strand 12 residues would disrupt correct enzyme-DNA alignment and alter pol η's activity and fidelity. To investigate this, the authors purified proteins containing the catalytic core of the polymerase, incorporated single amino acid changes to select β-strand 12 residues, and evaluated DNA synthesis activity for each pol η. Lesion bypass efficiencies and replication fidelities when copying DNA-containing cis-syn cyclobutane thymine-thymine dimer and 8-oxoG lesions were determined and compared with the corresponding values for the wild-type polymerase. The results confirm the importance of the β-strand in polymerase function and show that fidelity is most often altered when undamaged DNA is copied. Additionally, it is shown that DNA-protein contacts distal to the active site can significantly affect the fidelity of synthesis.

  10. Association between Mitochondrial DNA Copy Number in Peripheral Blood and Incident CKD in the Atherosclerosis Risk in Communities Study.

    PubMed

    Tin, Adrienne; Grams, Morgan E; Ashar, Foram N; Lane, John A; Rosenberg, Avi Z; Grove, Megan L; Boerwinkle, Eric; Selvin, Elizabeth; Coresh, Josef; Pankratz, Nathan; Arking, Dan E

    2016-08-01

    Mitochondrial dysfunction in kidney cells has been implicated in the pathogenesis of CKD. Mitochondrial DNA (mtDNA) copy number is a surrogate measure of mitochondrial function, and higher mtDNA copy number in peripheral blood has been associated with lower risk of two important risk factors for CKD progression, diabetes and microalbuminuria. We evaluated whether mtDNA copy number in peripheral blood associates with incident CKD in a population-based cohort of middle-aged adults. We estimated mtDNA copy number using 25 high-quality mitochondrial single nucleotide polymorphisms from the Affymetrix 6.0 array. Among 9058 participants, those with higher mtDNA copy number had a lower rate of prevalent diabetes and lower C-reactive protein levels and white blood cell counts. Baseline eGFR did not differ significantly by mtDNA copy number. Over a median follow-up of 19.6 years, 1490 participants developed CKD. Higher mtDNA copy number associated with lower risk of incident CKD (highest versus lowest quartile: hazard ratio 0.65; 95% confidence interval, 0.56 to 0.75; P<0.001) after adjusting for age, sex, and race. After adjusting for additional risk factors of CKD, including prevalent diabetes, hypertension, C-reactive protein level, and white blood cell count, this association remained significant (highest versus lowest quartile: hazard ratio 0.75; 95% confidence interval, 0.64 to 0.87; P<0.001). In conclusion, higher mtDNA copy number associated with lower incidence of CKD independent of traditional risk factors and inflammation biomarker levels in this cohort. Further research on modifiable factors influencing mtDNA copy number may lead to improvement in the prevention and treatment of CKD. Copyright © 2016 by the American Society of Nephrology.

  11. Discovering subgroups of patients from DNA copy number data using NMF on compacted matrices.

    PubMed

    de Campos, Cassio P; Rancoita, Paola M V; Kwee, Ivo; Zucca, Emanuele; Zaffalon, Marco; Bertoni, Francesco

    2013-01-01

    In the study of complex genetic diseases, the identification of subgroups of patients sharing similar genetic characteristics represents a challenging task, for example, to improve treatment decision. One type of genetic lesion, frequently investigated in such disorders, is the change of the DNA copy number (CN) at specific genomic traits. Non-negative Matrix Factorization (NMF) is a standard technique to reduce the dimensionality of a data set and to cluster data samples, while keeping its most relevant information in meaningful components. Thus, it can be used to discover subgroups of patients from CN profiles. It is however computationally impractical for very high dimensional data, such as CN microarray data. Deciding the most suitable number of subgroups is also a challenging problem. The aim of this work is to derive a procedure to compact high dimensional data, in order to improve NMF applicability without compromising the quality of the clustering. This is particularly important for analyzing high-resolution microarray data. Many commonly used quality measures, as well as our own measures, are employed to decide the number of subgroups and to assess the quality of the results. Our measures are based on the idea of identifying robust subgroups, inspired by biologically/clinically relevance instead of simply aiming at well-separated clusters. We evaluate our procedure using four real independent data sets. In these data sets, our method was able to find accurate subgroups with individual molecular and clinical features and outperformed the standard NMF in terms of accuracy in the factorization fitness function. Hence, it can be useful for the discovery of subgroups of patients with similar CN profiles in the study of heterogeneous diseases.

  12. Mitochondrial DNA copy numbers in pyramidal neurons are decreased and mitochondrial biogenesis transcriptome signaling is disrupted in Alzheimer's disease hippocampi.

    PubMed

    Rice, Ann C; Keeney, Paula M; Algarzae, Norah K; Ladd, Amy C; Thomas, Ravindar R; Bennett, James P

    2014-01-01

    Alzheimer's disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aβ1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aβ25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD.

  13. Copy Number of the Transposon, Pokey, in rDNA Is Positively Correlated with rDNA Copy Number in Daphnia obtusa

    PubMed Central

    LeRiche, Kaitlynn; Eagle, Shannon H. C.; Crease, Teresa J.

    2014-01-01

    Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species. PMID:25490398

  14. Mutation of the Little Finger Domain in Human DNA Polymerase η Alters Fidelity When Copying Undamaged DNA

    PubMed Central

    Beardslee, Renee A.; Suarez, Samuel C.; Toffton, Shannon M.; McCulloch, Scott D.

    2014-01-01

    DNA polymerase η (pol η) synthesizes past cyclobutane pyrimidine dimer and possibly 7,8-dihydro-8-oxoguanine (8-oxoG) lesions during DNA replication. Loss of pol η is associated with an increase in mutation rate, demonstrating its indispensable role in mutation suppression. It has been recently reported that β-strand 12 (amino acids 316–324) of the little finger region correctly positions the template strand with the catalytic core of the enzyme. The authors hypothesized that modification of β-strand 12 residues would disrupt correct enzyme–DNA alignment and alter pol η’s activity and fidelity. To investigate this, the authors purified proteins containing the catalytic core of the polymerase, incorporated single amino acid changes to select β-strand 12 residues, and evaluated DNA synthesis activity for each pol η. Lesion bypass efficiencies and replication fidelities when copying DNA-containing cis-syn cyclobutane thymine-thymine dimer and 8-oxoG lesions were determined and compared with the corresponding values for the wild-type polymerase. The results confirm the importance of the β-strand in polymerase function and show that fidelity is most often altered when undamaged DNA is copied. Additionally, it is shown that DNA–protein contacts distal to the active site can significantly affect the fidelity of synthesis. PMID:23913529

  15. A novel satellite DNA sequence in the Peromyscus genome (PMSat): Evolution via copy number fluctuation.

    PubMed

    Louzada, Sandra; Vieira-da-Silva, Ana; Mendes-da-Silva, Ana; Kubickova, Svatava; Rubes, Jiri; Adega, Filomena; Chaves, Raquel

    2015-11-01

    Satellite DNAs (satDNA) are tandemly arrayed repeated sequences largely present in eukaryotic genomes, which play important roles in genome evolution and function, and therefore, their analysis is vital. Here, we describe the isolation of a novel satellite DNA family (PMSat) from the rodent Peromyscus eremicus (Cricetidae, Rodentia), which is located in pericentromeric regions and exhibits a typical satellite DNA genome organization. Orthologous PMSat sequences were isolated and characterized from three species belonging to Cricetidae: Cricetus cricetus, Phodopus sungorus and Microtus arvalis. In these species, PMSat is highly conserved, with the absence of fixed species-specific mutations. Strikingly, different numbers of copies of this sequence were found among the species, suggesting evolution by copy number fluctuation. Repeat units of PMSat were also found in the Peromyscus maniculatus bairdii BioProject, but our results suggest that these repeat units are from genome regions outside the pericentromere. The remarkably high evolutionary sequence conservation along with the preservation of a few numbers of copies of this sequence in the analyzed genomes may suggest functional significance but a different sequence nature/organization. Our data highlight that repeats are difficult to analyze due to the limited tools available to dissect genomes and the fact that assemblies do not cover regions of constitutive heterochromatin.

  16. Mitochondrial DNA Copy Number and Risk of Oral Cancer: A Report from Northeast India

    PubMed Central

    Mondal, Rosy; Ghosh, Sankar Kumar; Choudhury, Javed Hussain; Seram, Anil; Sinha, Kavita; Hussain, Marine; Laskar, Ruhina Shirin; Rabha, Bijuli; Dey, Pradip; Ganguli, Sabitri; NathChoudhury, Monisha; Talukdar, Fazlur Rahman; Chaudhuri, Biswadeep; Dhar, Bishal

    2013-01-01

    Background Oral squamous cell carcinoma (OSCC) is the sixth most common cancer globally. Tobacco consumption and HPV infection, both are the major risk factor for the development of oral cancer and causes mitochondrial dysfunction. Genetic polymorphisms in xenobiotic-metabolizing enzymes modify the effect of environmental exposures, thereby playing a significant role in gene–environment interactions and hence contributing to the individual susceptibility to cancer. Here, we have investigated the association of tobacco - betel quid chewing, HPV infection, GSTM1-GSTT1 null genotypes, and tumour stages with mitochondrial DNA (mtDNA) content variation in oral cancer patients. Methodology/Principal Findings The study comprised of 124 cases of OSCC and 140 control subjects to PCR based detection was done for high-risk HPV using a consensus primer and multiplex PCR was done for detection of GSTM1-GSTT1 polymorphism. A comparative ΔCt method was used for determination of mtDNA content. The risk of OSCC increased with the ceased mtDNA copy number (Ptrend = 0.003). The association between mtDNA copy number and OSCC risk was evident among tobacco – betel quid chewers rather than tobacco – betel quid non chewers; the interaction between mtDNA copy number and tobacco – betel quid was significant (P = 0.0005). Significant difference was observed between GSTM1 - GSTT1 null genotypes (P = 0.04, P = 0.001 respectively) and HPV infection (P<0.001) with mtDNA content variation in cases and controls. Positive correlation was found with decrease in mtDNA content with the increase in tumour stages (P<0.001). We are reporting for the first time the association of HPV infection and GSTM1-GSTT1 null genotypes with mtDNA content in OSCC. Conclusion Our results indicate that the mtDNA content in tumour tissues changes with tumour stage and tobacco-betel quid chewing habits while low levels of mtDNA content suggests invasive thereby serving as a biomarker in detection

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

    PubMed

    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.

  18. Development and validation of InnoQuant(®) HY, a system for quantitation and quality assessment of total human and male DNA using high copy targets.

    PubMed

    Loftus, Andrew; Murphy, Gina; Brown, Hiromi; Montgomery, Anne; Tabak, Jonathan; Baus, James; Carroll, Marion; Green, André; Sikka, Suresh; Sinha, Sudhir

    2017-07-01

    The development and validation of InnoQuant(®) HY, a real-time PCR system containing four DNA targets-two RE autosomal targets of different sizes, male specific targets, and an internal positive control target-are described herein. The ratio of the two autosomal targets provides a Degradation Index, or a quantitative value of a sample's degradation state. The male specific targets are multi-copy targets located on the Y chromosome, which provides information about a sample's male DNA composition. The experimental results demonstrate InnoQuant HY as a robust qPCR method producing accurate DNA quantitation results even at low dynamic ranges, with reproducibility among population groups. The system is human specific with low level higher primate cross reactivity and is able to consistently and reproducibly detect sub-picogram concentrations of human and human male DNA. The use of high copy number Alu and SVA (>1000 copies per genome) retrotransposable elements as the two autosomal targets significantly enhances both sensitivity and reproducibility of determination of DNA quantitation as well as DNA degradation in forensic samples. The inclusion of a sensitive multi-copy Y-chromosome specific target provides accurate quantitation of DNA from a male in challenging male-female mixtures (i.e. sexual assault samples). Even in the presence of a large excess of DNA from a female, accurate quantitation was achieved with a male to female ratio of 1:128,000. Population database studies reveal an average Short/Y target ratio of the quantification values across all four populations tested was 1.124±0.282, exhibiting the system's reproducibility across multiple populations. The results from InnoQuant HY provide a tool equipping a forensic analyst with crucial data about a sample's DNA quantitation, male:female ratio, degradation state, and the presence or absence of PCR inhibitors. With the information gained from the InnoQuant HY kit, a more streamlined and efficient workflow

  19. Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes

    PubMed Central

    Gibbons, John G.; Branco, Alan T.; Godinho, Susana A.; Yu, Shoukai; Lemos, Bernardo

    2015-01-01

    Tandemly repeated ribosomal DNA (rDNA) arrays are among the most evolutionary dynamic loci of eukaryotic genomes. The loci code for essential cellular components, yet exhibit extensive copy number (CN) variation within and between species. CN might be partly determined by the requirement of dosage balance between the 5S and 45S rDNA arrays. The arrays are nonhomologous, physically unlinked in mammals, and encode functionally interdependent RNA components of the ribosome. Here we show that the 5S and 45S rDNA arrays exhibit concerted CN variation (cCNV). Despite 5S and 45S rDNA elements residing on different chromosomes and lacking sequence similarity, cCNV between these loci is strong, evolutionarily conserved in humans and mice, and manifested across individual genotypes in natural populations and pedigrees. Finally, we observe that bisphenol A induces rapid and parallel modulation of 5S and 45S rDNA CN. Our observations reveal a novel mode of genome variation, indicate that natural selection contributed to the evolution and conservation of cCNV, and support the hypothesis that 5S CN is partly determined by the requirement of dosage balance with the 45S rDNA array. We suggest that human disease variation might be traced to disrupted rDNA dosage balance in the genome. PMID:25583482

  20. [Research on potential interaction between mitochondrial DNA copy number and related factors on risk of hypertension in coal miners].

    PubMed

    Guo, J Y; Lei, L J; Qiao, N; Fan, G Q; Sun, C M; Huang, J J; Wang, T

    2017-01-10

    Objective: To investigate the effects of mitochondrial DNA (mtDNA) copy number in peripheral blood and related factors on the risk of hypertension in coal miners. Methods: A case-control study was conducted in 378 coal miners with hypertension and 325 healthy coal miners recruited from Datong Coal Mine Group. A standard questionnaire was used to collect their general information, such as demographic characteristics, habits and occupational history. Fluorescence quantitative PCR was performed to detect the copy number of mtDNA. Logistic regression model was applied for identifying the related risk factors of hypertension and analyzing the interaction between mtDNA copy number and risk factors. Results: The prevalence of hypertension of high mtDNA copy number was lower than mtDNA copy numberin 0-5.67 group, but the difference was not statistically significant (P=0.414). Alcohol drinking (OR=1.80, 95% CI: 1.26-2.56), family history of hypertension (OR=1.74, 95% CI: 1.20- 2.50), work shifts (OR=0.69, 95% CI: 0.48-0.99), education level (P=0.012) and family monthly income level (P=0.001) were related to the prevalence of hypertension. There were potential interactions between mtDNA copy number and alcohol drinking, family monthly income level, family history of hypertension, respectively. Alcohol drinking was a risk factor for hypertension [1.77 (1.25-2.50)]. Potential interactions between mtDNA copy number and alcohol drinking reduced the risk of hypertension (OR=1.20, 95% CI: 1.07-1.35). Family history of hypertension was a risk factor for hypertension [1.81(1.26-2.59)]. Potential interactions between mtDNA copy number and family history of hypertension reduced the risk of hypertension (OR=1.24, 95%CI: 1.09-1.41). Family monthly income level was a protect factor for hypertension [0.55(0.46-0.66)]. Potential interactions between mtDNA copy number and family monthly income level increased the protection role of hypertension (OR=0.90, 95% CI: 0.86-0.94). Conclusion: mtDNA

  1. A High-Resolution Map of Segmental DNA Copy Number Variation in the Mouse Genome

    PubMed Central

    Graubert, Timothy A; Selzer, Rebecca R; Richmond, Todd A; Eis, Peggy S; Shannon, William D; Li, Xia; McLeod, Howard L; Cheverud, James M; Ley, Timothy J

    2007-01-01

    Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits. PMID:17206864

  2. DNA copy number aberrations associated with lymphovascular invasion in upper urinary tract urothelial carcinoma.

    PubMed

    Misumi, Taku; Yamamoto, Yoshiaki; Miyachika, Yoshihiro; Eguchi, Satoshi; Chochi, Yasuyo; Nakao, Motonao; Nagao, Kazuhiro; Hara, Takahiko; Sakano, Shigeru; Furuya, Tomoko; Oga, Atsunori; Kawauchi, Shigeto; Sasaki, Kohsuke; Matsuyama, Hideyasu

    2012-06-01

    Recent studies have reported that lymphovascular invasion (LVI) is a predictor of patient prognosis in upper urinary tract urothelial carcinoma (UUTUC). DNA copy number aberrations (DCNAs) identified by array-based comparative genomic hybridization (aCGH) had not previously been examined in UUTUC. We therefore examined DCNAs in UUTUC and compared them with DCNAs in LVI. We applied aCGH technology using DNA chips spotted with 4,030 BAC clones to 32 UUTUC patients. Frequent copy number gains were detected on chromosomal regions 8p23.1 and 20q13.12, whereas frequent copy number losses were detected on chromosomal regions 13q21.1, 17p13.1, 6q16.3, and 17p11.2. DCNAs occurred more frequently in tumors with LVI than in those without it (P = 0.0002), and this parameter was more closely associated with LVI than with the tumor grade or pT stage. Disease-specific survival rate was higher in tumors without LVI than in those with it (P = 0.0120); however, tumor grade and stage were not significant prognostic factors of patient outcome. These data support our hypothesis that tumors with LVI have more genetic alterations in terms of total numbers of DCNAs than those without, and provide proof that aggressive adjuvant therapy should be considered for UUTUC patients with LVI.

  3. How accurate are accurate force-fields for B-DNA?

    PubMed Central

    Ivani, Ivan; Hospital, Adam; Portella, Guillem

    2017-01-01

    Abstract Last generation of force-fields are raising expectations on the quality of molecular dynamics (MD) simulations of DNA, as well as to the belief that theoretical models can substitute experimental ones in several cases. However these claims are based on limited benchmarks, where MD simulations have shown the ability to reproduce already existing ‘experimental models’, which in turn, have an unclear accuracy to represent DNA conformation in solution. In this work we explore the ability of different force-fields to predict the structure of two new B-DNA dodecamers, determined herein by means of 1H nuclear magnetic resonance (NMR). The study allowed us to check directly for experimental NMR observables on duplexes previously not solved, and also to assess the reliability of ‘experimental structures’. We observed that technical details in the annealing procedures can induce non-negligible local changes in the final structures. We also found that while not all theoretical simulations are equally reliable, those obtained using last generation of AMBER force-fields (BSC1 and BSC0OL15) show predictive power in the multi-microsecond timescale and can be safely used to reproduce global structure of DNA duplexes and fine sequence-dependent details. PMID:28088759

  4. An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control.

    PubMed

    Inuzuka, M; Wada, Y

    1988-02-08

    Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.

  5. DNA is hypomethylated at repetitive and single copy loci in patients with ICF syndrome

    SciTech Connect

    Schuffenhauer, S.; Buchholz, B.; Neitinger, T.

    1994-09-01

    ICF syndrome (immunodeficiency, centromeric heterochromatin instability, facial anomaly) is a very rare genetic disorder, reported in only 12 cases. Chromosomal rearrangements occur predominantly in the heterochromatic regions of HC 1 and 16 and include stretching, whole arm deletions and multibranched configurations. The molecular defect of these abnormalities is not known. Similar abnormalities have been found in cell cultures treated with viruses or 5-acacytidine, agents which cause DNA hypomethylation. Because heterochromatic DNA is known to be highly methylated, we hypothesise that DNA hypomethylation and subsequent disturbance of heterochromatin structure may play a role in the chromosomal rearrangements of ICF syndrome. Methylation studies in DNA from peripheral lymphocytes of two non-related ICF patients revealed hypomethylation of satellite-2 DNA localized in the heterochromatic regions of HC 1 and 16. DNA hypomethylation was also found at single copy loci, e.g. D15S63, D15S9, H19 and DXS255. Differences between the two patients suggest a random distribution of DNA hypomethylation. While a causal relationship between the molecular and cytogenetic abnormalities is likely, the postulated relationship between hypomethylation and the clinical symptoms in ICF syndrome remains to be elucidated.

  6. Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma †

    PubMed Central

    Lin, Chen-Sung; Lee, Hui-Ting; Lee, Ming-Huei; Pan, Siao-Cian; Ke, Chen-Yeh; Chiu, Allen Wen-Hsiang; Wei, Yau-Huei

    2016-01-01

    We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XFe-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0

  7. Role of Mitochondrial DNA Copy Number Alteration in Human Renal Cell Carcinoma.

    PubMed

    Lin, Chen-Sung; Lee, Hui-Ting; Lee, Ming-Huei; Pan, Siao-Cian; Ke, Chen-Yeh; Chiu, Allen Wen-Hsiang; Wei, Yau-Huei

    2016-05-25

    We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively. Protein or mRNA expression levels of TFAM; mtDNA-encoded NADH dehydrogenase subunit 1 (ND1), ND6 and cytochrome c oxidase subunit 2 (COX-2); nuclear DNA (nDNA)-encoded succinate dehydrogenase subunit A (SDHA); v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic enzymes including hexokinase II (HK-II), glucose 6-phosphate isomerase (GPI), phosphofructokinase (PFK), and lactate dehydrogenase subunit A (LDHA); and hypoxia-inducible factors the HIF-1α and HIF-2α, pyruvate dehydrogenase kinase 1 (PDK1), and pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XF(e)-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and vimentin expression. Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower mtDNA copy number (p = 0.034), lower mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower protein levels of TFAM and COX-2 than did the NT clone. By contrast, the protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and vimentin; trans-well migration activity (p = 0

  8. Two Drosophila retrotransposon gypsy subfamilies differ in ability to produce new DNA copies via reverse transcription in Drosophila cultured cells.

    PubMed Central

    Lyubomirskaya, N V; Avedisov, S N; Surkov, S A; Ilyin, Y V

    1993-01-01

    Plasmid DNA constructs containing 5' end truncated retrotransposon gypsy were introduced into Drosophila cultured cells. Appearance of new complete DNA copies with reconstructed via reverse transcription 5'LTR were detected by PCR after transient expression and by Southern blot analysis of genome DNA of stably transformed cells. Two gypsy subfamilies supposed to be different in transpositional activity were analyzed in terms of their ability to produce new DNA copies via reverse transcription in D. hydei cultured cells. It was demonstrated that both gypsy variants undergo retrotransposition but with different efficiency. Images PMID:7688116

  9. The effects of mitochondrial DNA deletion and copy number variations on different exercise intensities in highly trained swimmers.

    PubMed

    Baykara, O; Sahin, S K; Akbas, F; Guven, M; Onaran, I

    2016-10-31

    It has been suggested that heavy exercise might increase oxidative stress, causing mitochondrial DNA (mtDNA) mutations as well as DNA mutations and changes in the mtDNA copy number in cells. mtDNA4977 deletion is one of the most common deletions seen on mitochondria. We hypothesize association between exercise induced oxidative stress and mtDNA damage in peripheral blood lymphocytes (PBLs) of highly trained swimmers. Therefore we studied the mtDNA4977 deletion level, mtDNA copy number and their relationship with cellular ATP and oxidative stress status in PBLs of swimmers. 8 highly trained and 8 normal trained swimmers and 8 non-athlete subjects were included in the study. The mtDNA4977 deletion and amount of mtDNA were measured using RT-PCR method whereas dichlorohydrofluoroscein (DCF) assay method was used to assess cellular oxidative stress and ATP levels were measured using bioluminescence method. Even though an increase in mtDNA4977 deletion was found in all study groups, the difference was not statistically significant (p=0.98). The mtDNA copy numbers were found to be surprisingly high in highly trained swimmers compared to normal trained swimmers and non-athlete subjects by 4.03 fold (p= 0.0002) and 5.58 fold (p=0.0003), respectively. No significant differences were found between groups by means of intracellular ATP levels (p=0.406) and oxidative stress (p=0.430).  No correlation was found between mtDNA copy number and intracellular ATP content of the PBLs (p=0.703). Our results suggest that heavy training does not have a specific effect on mtDNA4977 deletion but it may be affecting mitochondrial copy numbers which may act as a compensatory mechanism related to ATP levels in blood.

  10. Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse

    PubMed Central

    Krzeminski, Patryk; Corchete, Luis A.; García, Juan L.; López-Corral, Lucía; Fermiñán, Encarna; García, Eva M.; Martín, Ana A.; Hernández-Rivas, Jesús M.; García-Sanz, Ramón; Miguel, Jesús F. San; Gutiérrez, Norma C.

    2016-01-01

    Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse. PMID:27811368

  11. Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse.

    PubMed

    Krzeminski, Patryk; Corchete, Luis A; García, Juan L; López-Corral, Lucía; Fermiñán, Encarna; García, Eva M; Martín, Ana A; Hernández-Rivas, Jesús M; García-Sanz, Ramón; San Miguel, Jesús F; Gutiérrez, Norma C

    2016-12-06

    Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.

  12. QUANTITATIVE SCREENING OF SINGLE COPIES OF HUMAN PAPILLOMA VIRAL DNA WITHOUT AMPLIFICATION

    PubMed Central

    Li, Jiangwei; Lee, Ji-Young; Yeung, Edward S.

    2008-01-01

    We describe a novel quantitative viral screening method based on single-molecule detection that does not require amplification. DNA of human papilloma virus (HPV), the major etiological agent of cervical cancer, served as the screening target in this study. Eight 100-nucleotide (nt) single-stranded (ss)-DNA probes were designed complementary to the E6-E7 gene of HPV-16 DNA. The probes were covalently stained with Alexa Fluor 532 and hybridized to the target in solution. The individual hybridized molecules were imaged with an intensified charge-coupled device (ICCD) in two ways. In the single-color mode, target molecules were detected via fluorescence from hybridized probes only. This system could detect HPV-16 DNA in the presence of human genomic DNA down to 0.7 copy/cell, and had a linear dynamic range of over six orders of magnitude. In the dual-color mode, we employed fluorescence resonance energy transfer (FRET) and added YOYO-3 dye as the acceptor. The two colors from Alexa Fluor 532 and YOYO-3 were dispersed by a transmission grating located in front of the ICCD. With this reinforced criteria for identifying the hybridized molecules, zero false-positive count was achieved. We also showed that DNA extracts from Pap test specimens did not interfere with the measurements. PMID:16970325

  13. Classification of human cancers based on DNA copy number amplification modeling

    PubMed Central

    Myllykangas, Samuel; Tikka, Jarkko; Böhling, Tom; Knuutila, Sakari; Hollmén, Jaakko

    2008-01-01

    Background DNA amplifications alter gene dosage in cancer genomes by multiplying the gene copy number. Amplifications are quintessential in a considerable number of advanced cancers of various anatomical locations. The aims of this study were to classify human cancers based on their amplification patterns, explore the biological and clinical fundamentals behind their amplification-pattern based classification, and understand the characteristics in human genomic architecture that associate with amplification mechanisms. Methods We applied a machine learning approach to model DNA copy number amplifications using a data set of binary amplification records at chromosome sub-band resolution from 4400 cases that represent 82 cancer types. Amplification data was fused with background data: clinical, histological and biological classifications, and cytogenetic annotations. Statistical hypothesis testing was used to mine associations between the data sets. Results Probabilistic clustering of each chromosome identified 111 amplification models and divided the cancer cases into clusters. The distribution of classification terms in the amplification-model based clustering of cancer cases revealed cancer classes that were associated with specific DNA copy number amplification models. Amplification patterns – finite or bounded descriptions of the ranges of the amplifications in the chromosome – were extracted from the clustered data and expressed according to the original cytogenetic nomenclature. This was achieved by maximal frequent itemset mining using the cluster-specific data sets. The boundaries of amplification patterns were shown to be enriched with fragile sites, telomeres, centromeres, and light chromosome bands. Conclusions Our results demonstrate that amplifications are non-random chromosomal changes and specifically selected in tumor tissue microenvironment. Furthermore, statistical evidence showed that specific chromosomal features co-localize with

  14. Association of telomere length and mitochondrial DNA copy number in a community sample of healthy adults.

    PubMed

    Tyrka, Audrey R; Carpenter, Linda L; Kao, Hung-Teh; Porton, Barbara; Philip, Noah S; Ridout, Samuel J; Ridout, Kathryn K; Price, Lawrence H

    2015-06-01

    Cellular aging plays a role in longevity and senescence, and has been implicated in medical and psychiatric conditions, including heart disease, cancer, major depression and posttraumatic stress disorder. Telomere shortening and mitochondrial dysfunction are thought to be central to the cellular aging process. The present study examined the association between mitochondrial DNA (mtDNA) copy number and telomere length in a sample of medically healthy adults. Participants (total n=392) were divided into 4 groups based on the presence or absence of early life adversity and lifetime psychopathology: No Adversity/No Disorder, n=136; Adversity/No Disorder, n=91; No Adversity/Disorder, n=46; Adversity/Disorder, n=119. Telomere length and mtDNA copy number were measured using quantitative polymerase chain reaction. There was a positive correlation between mtDNA and telomere length in the entire sample (r=0.120, p<0.001) and in each of the four groups of participants (No Adversity/No Disorder, r=0.291, p=0.001; Adversity/No Disorder r=0.279, p=0.007; No Adversity/Disorder r=0.449, p=0.002; Adversity/Disorder, r=0.558, p<0.001). These correlations remained significant when controlling for age, smoking, and body mass index and establish an association between mtDNA and telomere length in a large group of women and men both with and without early adversity and psychopathology, suggesting co-regulation of telomeres and mitochondrial function. The mechanisms underlying this association may be important in the pathophysiology of age-related medical conditions, such as heart disease and cancer, as well as for stress-associated psychiatric disorders.

  15. Familial longevity study reveals a significant association of mitochondrial DNA copy number between centenarians and their offspring.

    PubMed

    He, Yong-Han; Chen, Xiao-Qiong; Yan, Dong-Jing; Xiao, Fu-Hui; Lin, Rong; Liao, Xiao-Ping; Liu, Yao-Wen; Pu, Shao-Yan; Yu, Qin; Sun, Hong-Peng; Jiang, Jian-Jun; Cai, Wang-Wei; Kong, Qing-Peng

    2016-11-01

    Reduced mitochondrial function is an important cause of aging and age-related diseases. We previously revealed a relatively higher level of mitochondrial DNA (mtDNA) content in centenarians. However, it is still unknown whether such an mtDNA content pattern of centenarians could be passed on to their offspring and how it was regulated. To address these issues, we recruited 60 longevity families consisting of 206 family members (cohort 1) and explored their mtDNA copy number. The results showed that the first generation of the offspring (F1 offspring) had a higher level of mtDNA copy number than their spouses (p < 0.05) independent of a gender effect. In addition, we found a positive association of mtDNA copy number in centenarians with that in F1 offspring (r = 0.54, p = 0.0008) but not with that in F1 spouses. These results were replicated in another independent cohort consisting of 153 subjects (cohort 2). RNA sequencing analysis suggests that the single-stranded DNA-binding protein 4 was significantly associated with mtDNA copy number and was highly expressed in centenarians as well as F1 offspring versus the F1 spouses, thus likely regulates the mtDNA copy number in the long-lived family members. In conclusion, our results suggest that the pattern of high mtDNA copy number is likely inheritable, which may act as a favorable factor to familial longevity through assuring adequate energy supply. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. From DNA Copy Number to Gene Expression: Local aberrations, Trisomies and Monosomies

    NASA Astrophysics Data System (ADS)

    Shay, Tal

    The goal of my PhD research was to study the effect of DNA copy number changes on gene expression. DNA copy number aberrations may be local, encompassing several genes, or on the level of an entire chromosome, such as trisomy and monosomy. The main dataset I studied was of Glioblastoma, obtained in the framework of a collaboration, but I worked also with public datasets of cancer and Down's Syndrome. The molecular basis of expression changes in Glioblastoma. Glioblastoma is the most common and aggressive type of primary brain tumors in adults. In collaboration with Prof. Hegi (CHUV, Switzerland), we analyzed a rich Glioblastoma dataset including clinical information, DNA copy number (array CGH) and expression profiles. We explored the correlation between DNA copy number and gene expression at the level of chromosomal arms and local genomic aberrations. We detected known amplification and over expression of oncogenes, as well as deletion and down-regulation of tumor suppressor genes. We exploited that information to map alterations of pathways that are known to be disrupted in Glioblastoma, and tried to characterize samples that have no known alteration in any of the studied pathways. Identifying local DNA aberrations of biological significance. Many types of tumors exhibit chromosomal losses or gains and local amplifications and deletions. A region that is aberrant in many tumors, or whose copy number change is stronger, is more likely to be clinically relevant, and not just a by-product of genetic instability. We developed a novel method that defines and prioritizes aberrations by formalizing these intuitions. The method scores each aberration by the fraction of patients harboring it, its length and its amplitude, and assesses the significance of the score by comparing it to a null distribution obtained by permutations. This approach detects genetic locations that are significantly aberrant, generating a 'genomic aberration profile' for each sample. The 'genomic

  17. Adenoviral vector DNA for accurate genome editing with engineered nucleases.

    PubMed

    Holkers, Maarten; Maggio, Ignazio; Henriques, Sara F D; Janssen, Josephine M; Cathomen, Toni; Gonçalves, Manuel A F V

    2014-10-01

    Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.

  18. Characterization of an inducible promoter in different DNA copy number conditions

    PubMed Central

    2012-01-01

    Background The bottom-up programming of living organisms to implement novel user-defined biological capabilities is one of the main goals of synthetic biology. Currently, a predominant problem connected with the construction of even simple synthetic biological systems is the unpredictability of the genetic circuitry when assembled and incorporated in living cells. Copy number, transcriptional/translational demand and toxicity of the DNA-encoded functions are some of the major factors which may lead to cell overburdening and thus to nonlinear effects on system output. It is important to disclose the linearity working boundaries of engineered biological systems when dealing with such phenomena. Results The output of an N-3-oxohexanoyl-L-homoserine lactone (HSL)-inducible RFP-expressing device was studied in Escherichia coli in different copy number contexts, ranging from 1 copy per cell (integrated in the genome) to hundreds (via multicopy plasmids). The system is composed by a luxR constitutive expression cassette and a RFP gene regulated by the luxI promoter, which is activated by the HSL-LuxR complex. System output, in terms of promoter activity as a function of HSL concentration, was assessed relative to the one of a reference promoter in identical conditions by using the Relative Promoter Units (RPU) approach. Nonlinear effects were observed in the maximum activity, which is identical in single and low copy conditions, while it decreases for higher copy number conditions. In order to properly compare the luxI promoter strength among all the conditions, a mathematical modeling approach was used to relate the promoter activity to the estimated HSL-LuxR complex concentration, which is the actual activator of transcription. During model fitting, a correlation between the copy number and the dissociation constant of HSL-LuxR complex and luxI promoter was observed. Conclusions Even in a simple inducible system, nonlinear effects are observed and non-trivial data

  19. Characterization of an inducible promoter in different DNA copy number conditions.

    PubMed

    Zucca, Susanna; Pasotti, Lorenzo; Mazzini, Giuliano; De Angelis, Maria Gabriella Cusella; Magni, Paolo

    2012-03-28

    The bottom-up programming of living organisms to implement novel user-defined biological capabilities is one of the main goals of synthetic biology. Currently, a predominant problem connected with the construction of even simple synthetic biological systems is the unpredictability of the genetic circuitry when assembled and incorporated in living cells. Copy number, transcriptional/translational demand and toxicity of the DNA-encoded functions are some of the major factors which may lead to cell overburdening and thus to nonlinear effects on system output. It is important to disclose the linearity working boundaries of engineered biological systems when dealing with such phenomena. The output of an N-3-oxohexanoyl-L-homoserine lactone (HSL)-inducible RFP-expressing device was studied in Escherichia coli in different copy number contexts, ranging from 1 copy per cell (integrated in the genome) to hundreds (via multicopy plasmids). The system is composed by a luxR constitutive expression cassette and a RFP gene regulated by the luxI promoter, which is activated by the HSL-LuxR complex. System output, in terms of promoter activity as a function of HSL concentration, was assessed relative to the one of a reference promoter in identical conditions by using the Relative Promoter Units (RPU) approach. Nonlinear effects were observed in the maximum activity, which is identical in single and low copy conditions, while it decreases for higher copy number conditions. In order to properly compare the luxI promoter strength among all the conditions, a mathematical modeling approach was used to relate the promoter activity to the estimated HSL-LuxR complex concentration, which is the actual activator of transcription. During model fitting, a correlation between the copy number and the dissociation constant of HSL-LuxR complex and luxI promoter was observed. Even in a simple inducible system, nonlinear effects are observed and non-trivial data processing is necessary to fully

  20. Copy Number Variation of Mitochondrial DNA Genes in Pneumocystis jirovecii According to the Fungal Load in BAL Specimens

    PubMed Central

    Valero, Clara; Buitrago, María José; Gits-Muselli, Maud; Benazra, Marion; Sturny-Leclère, Aude; Hamane, Samia; Guigue, Nicolas; Bretagne, Stéphane; Alanio, Alexandre

    2016-01-01

    Pneumocystis jirovecii is an unculturable fungus and the causative agent of Pneumocystis pneumonia, a life-threatening opportunistic infection. Although molecular diagnosis is often based on the detection of mtLSU rRNA mitochondrial gene, the number of copies of mitochondrial genes had not been investigated. We developed and optimized six real-time PCR assays in order to determine the copy number of four mitochondrial genes (mtSSU rRNA, mtLSU rRNA, NAD1, and CYTB) in comparison to nuclear genome (DHPS and HSP70) and tested 84 bronchoalveolar fluids of patients at different stages of the infection. Unexpectedly, we found that copy number of mitochondrial genes varied from gene to gene with mtSSU rRNA gene being more represented (37 copies) than NAD1 (23 copies), mtLSU rRNA (15 copies) and CYTB (6 copies) genes compared to nuclear genome. Hierarchical clustering analysis (HCA) allowed us to define five major clusters, significantly associated with fungal load (p = 0.029), in which copy number of mitochondrial genes was significantly different among them. More importantly, copy number of mtLSU rRNA, NAD1, and CYTB but not mtSSU rRNA differed according to P. jirovecii physiological state with a decreased number of copies when the fungal load is low. This suggests the existence of a mixture of various subspecies of mtDNA that can harbor different amplification rates. Overall, we revealed here an unexpected variability of P. jirovecii mtDNA copy number that fluctuates according to P. jirovecii’s physiological state, except for mtSSU that is the most stable and the most present mitochondrial gene. PMID:27672381

  1. Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB

    PubMed Central

    Sugiman-Marangos, Seiji N.; Weiss, Yoni M.; Junop, Murray S.

    2016-01-01

    Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing. PMID:27044084

  2. DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies.

    PubMed Central

    Knuutila, S.; Björkqvist, A. M.; Autio, K.; Tarkkanen, M.; Wolf, M.; Monni, O.; Szymanska, J.; Larramendy, M. L.; Tapper, J.; Pere, H.; El-Rifai, W.; Hemmer, S.; Wasenius, V. M.; Vidgren, V.; Zhu, Y.

    1998-01-01

    This review summarizes reports of recurrent DNA sequence copy number amplifications in human neoplasms detected by comparative genomic hybridization. Some of the chromosomal areas with recurrent DNA copy number amplifications (amplicons) of 1p22-p31, 1p32-p36, 1q, 2p13-p16, 2p23-p25, 2q31-q33, 3q, 5p, 6p12-pter, 7p12-p13, 7q11.2, 7q21-q22, 8p11-p12, 8q, 11q13-q14, 12p, 12q13-q21, 13q14, 13q22-qter, 14q13-q21, 15q24-qter, 17p11.2-p12, 17q12-q21, 17q22-qter, 18q, 19p13.2-pter, 19cen-q13.3, 20p11.2-p12, 20q, Xp11.2-p21, and Xp11-q13 and genes therein are presented in more detail. The paper with more than 150 references and two tables can be accessed from our web site http://www.helsinki.fi/lglvwww/CMG.html. The data will be updated biannually until the year 2001. PMID:9588877

  3. Accurate phylogenetic classification of DNA fragments based onsequence composition

    SciTech Connect

    McHardy, Alice C.; Garcia Martin, Hector; Tsirigos, Aristotelis; Hugenholtz, Philip; Rigoutsos, Isidore

    2006-05-01

    Metagenome studies have retrieved vast amounts of sequenceout of a variety of environments, leading to novel discoveries and greatinsights into the uncultured microbial world. Except for very simplecommunities, diversity makes sequence assembly and analysis a verychallenging problem. To understand the structure a 5 nd function ofmicrobial communities, a taxonomic characterization of the obtainedsequence fragments is highly desirable, yet currently limited mostly tothose sequences that contain phylogenetic marker genes. We show that forclades at the rank of domain down to genus, sequence composition allowsthe very accurate phylogenetic 10 characterization of genomic sequence.We developed a composition-based classifier, PhyloPythia, for de novophylogenetic sequence characterization and have trained it on adata setof 340 genomes. By extensive evaluation experiments we show that themethodis accurate across all taxonomic ranks considered, even forsequences that originate fromnovel organisms and are as short as 1kb.Application to two metagenome datasets 15 obtained from samples ofphosphorus-removing sludge showed that the method allows the accurateclassification at genus level of most sequence fragments from thedominant populations, while at the same time correctly characterizingeven larger parts of the samples at higher taxonomic levels.

  4. Accurate phylogenetic classification of variable-length DNA fragments.

    PubMed

    McHardy, Alice Carolyn; Martín, Héctor García; Tsirigos, Aristotelis; Hugenholtz, Philip; Rigoutsos, Isidore

    2007-01-01

    Metagenome studies have retrieved vast amounts of sequence data from a variety of environments leading to new discoveries and insights into the uncultured microbial world. Except for very simple communities, the encountered diversity has made fragment assembly and the subsequent analysis a challenging problem. A taxonomic characterization of metagenomic fragments is required for a deeper understanding of shotgun-sequenced microbial communities, but success has mostly been limited to sequences containing phylogenetic marker genes. Here we present PhyloPythia, a composition-based classifier that combines higher-level generic clades from a set of 340 completed genomes with sample-derived population models. Extensive analyses on synthetic and real metagenome data sets showed that PhyloPythia allows the accurate classification of most sequence fragments across all considered taxonomic ranks, even for unknown organisms. The method requires no more than 100 kb of training sequence for the creation of accurate models of sample-specific populations and can assign fragments >or=1 kb with high specificity.

  5. Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants

    PubMed Central

    Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A.

    2016-01-01

    Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes. PMID:27622766

  6. Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants.

    PubMed

    Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A

    2016-01-01

    Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes.

  7. Elevated Mitochondrial DNA Copy Number in Peripheral Blood and Tissue Predict the Opposite Outcome of Cancer: A Meta-Analysis

    PubMed Central

    Chen, Nan; Wen, Shu; Sun, Xiaoru; Fang, Qian; Huang, Lin; Liu, Shuai; Li, Wanling; Qiu, Meng

    2016-01-01

    Previous studies have suggested that mitochondrial DNA (mtDNA) copy number was associated with cancer risk. However, no solid conclusion revealed the potential predictive value of mtDNA copy number for cancer prognosis. The present meta-analysis was performed to clarify the problem. Hence, we performed a systematic search in PubMed, EmBase, Web of Science databases independently and a total of eighteen studies comprising 3961 cases satisfied the criteria and finally enrolled. Our results didn’t show the association between them but significant heterogeneity in overall analysis (OS: HR = 0.923, 95% CI: 0.653–1.306, p = 0.652; DFS: HR = 0.997, 95% CI: 0.599–1.659, p = 0.99). However, subgroup analysis stratified by sample came to the opposite conclusion. High level mitochondrial DNA copy number in peripheral blood predicted a poor cancer prognosis (OS: HR = 1.624, 95% CI: 1.211–2.177, p = 0.001; DFS: HR = 1.582, 95% CI: 1.026–2.439, p = 0.038) while patients with high level mitochondrial DNA copy number in tumor tissue exhibited better outcomes (OS: HR = 0.604 95% CI: 0.406–0.899, p = 0.013; DFS: HR = 0.593, 95% CI: 0.411–0.857, p = 0.005). These findings were further proved in detailed analyses in blood or tissue subgroup. In conclusion, our study suggested the elevated mtDNA copy number in peripheral blood predicted a poor cancer prognosis while the better outcome was presented among patients with elevated mtDNA copy number in tumor tissue. PMID:27857175

  8. Mitochondrial DNA copy number and biogenesis in different tissues of early- and late-lactating dairy cows.

    PubMed

    Laubenthal, L; Hoelker, M; Frahm, J; Dänicke, S; Gerlach, K; Südekum, K-H; Sauerwein, H; Häussler, S

    2016-02-01

    Energy balance in dairy cows changes during the course of lactation due to alterations in voluntary feed intake and energy required for milk synthesis. To adapt to the demands of lactation, energy metabolism needs to be regulated and coordinated in key organs such as adipose tissue (AT), liver, and mammary gland. Mitochondria are the main sites of energy production in mammalian cells and their number varies depending on age, organ, and physiological condition. The copy number of the mitochondrial genome, the mitochondrial DNA (mtDNA), reflects the abundance of mitochondria within a cell and is regulated by transcriptional and translational factors. Environmental, physiological, and energetic conditions change during lactation and we thus hypothesized that these changes may influence the mtDNA copy number and the abundance of genes regulating mitochondrial biogenesis. Therefore, we aimed to provide an overview of mitochondrial biogenesis in liver, subcutaneous (sc)AT, mammary gland, and peripheral blood cells during early and late lactation in dairy cows. German Holstein cows (n=21) were fed according to their requirements, and biopsies from scAT, liver, mammary gland, and blood were collected in early and late lactation and assayed for relative mtDNA copy numbers and the mRNA abundance of genes regulating mitochondrial biogenesis, such as nuclear-respiratory factor 1 and 2 (NRF-1, NRF-2), mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC-1α). The number of mtDNA copies increased from early to late lactation in all tissues, whereas that in peripheral blood cells was greater in early compared with late lactation. Moreover, mitochondrial activity enzymes (i.e., citrate synthase and cytochrome c oxidase) increased from early to late lactation in scAT. Comparing the number of mtDNA copies between tissues and blood in dairy cows, the highest mtDNA content was observed in liver. The mRNA abundance of

  9. An evaluation of new and established methods to determine T‐DNA copy number and homozygosity in transgenic plants.

    PubMed Central

    Głowacka, Katarzyna; Kromdijk, Johannes; Leonelli, Lauriebeth; Niyogi, Krishna K.; Clemente, Tom E.

    2016-01-01

    Abstract Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL‐)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T‐DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL‐PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T‐DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T‐DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided. PMID:26670088

  10. An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants.

    PubMed

    Głowacka, Katarzyna; Kromdijk, Johannes; Leonelli, Lauriebeth; Niyogi, Krishna K; Clemente, Tom E; Long, Stephen P

    2016-04-01

    Stable transformation of plants is a powerful tool for hypothesis testing. A rapid and reliable evaluation method of the transgenic allele for copy number and homozygosity is vital in analysing these transformations. Here the suitability of Southern blot analysis, thermal asymmetric interlaced (TAIL-)PCR, quantitative (q)PCR and digital droplet (dd)PCR to estimate T-DNA copy number, locus complexity and homozygosity were compared in transgenic tobacco. Southern blot analysis and ddPCR on three generations of transgenic offspring with contrasting zygosity and copy number were entirely consistent, whereas TAIL-PCR often underestimated copy number. qPCR deviated considerably from the Southern blot results and had lower precision and higher variability than ddPCR. Comparison of segregation analyses and ddPCR of T1 progeny from 26 T0 plants showed that at least 19% of the lines carried multiple T-DNA insertions per locus, which can lead to unstable transgene expression. Segregation analyses failed to detect these multiple copies, presumably because of their close linkage. This shows the importance of routine T-DNA copy number estimation. Based on our results, ddPCR is the most suitable method, because it is as reliable as Southern blot analysis yet much faster. A protocol for this application of ddPCR to large plant genomes is provided.

  11. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    PubMed

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis.

  12. Micro-Scale Genomic DNA Copy Number Aberrations as Another Means of Mutagenesis in Breast Cancer

    PubMed Central

    Chao, Hann-Hsiang; He, Xiaping; Parker, Joel S.; Zhao, Wei; Perou, Charles M.

    2012-01-01

    Introduction In breast cancer, the basal-like subtype has high levels of genomic instability relative to other breast cancer subtypes with many basal-like-specific regions of aberration. There is evidence that this genomic instability extends to smaller scale genomic aberrations, as shown by a previously described micro-deletion event in the PTEN gene in the Basal-like SUM149 breast cancer cell line. Methods We sought to identify if small regions of genomic DNA copy number changes exist by using a high density, gene-centric Comparative Genomic Hybridizations (CGH) array on cell lines and primary tumors. A custom tiling array for CGH (244,000 probes, 200 bp tiling resolution) was created to identify small regions of genomic change, which was focused on previously identified basal-like-specific, and general cancer genes. Tumor genomic DNA from 94 patients and 2 breast cancer cell lines was labeled and hybridized to these arrays. Aberrations were called using SWITCHdna and the smallest 25% of SWITCHdna-defined genomic segments were called micro-aberrations (<64 contiguous probes, ∼ 15 kb). Results Our data showed that primary tumor breast cancer genomes frequently contained many small-scale copy number gains and losses, termed micro-aberrations, most of which are undetectable using typical-density genome-wide aCGH arrays. The basal-like subtype exhibited the highest incidence of these events. These micro-aberrations sometimes altered expression of the involved gene. We confirmed the presence of the PTEN micro-amplification in SUM149 and by mRNA-seq showed that this resulted in loss of expression of all exons downstream of this event. Micro-aberrations disproportionately affected the 5′ regions of the affected genes, including the promoter region, and high frequency of micro-aberrations was associated with poor survival. Conclusion Using a high-probe-density, gene-centric aCGH microarray, we present evidence of small-scale genomic aberrations that can contribute to

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

    PubMed

    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

    2016-02-23

    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.

  14. GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes.

    PubMed

    van den Broek, Evert; van Lieshout, Stef; Rausch, Christian; Ylstra, Bauke; van de Wiel, Mark A; Meijer, Gerrit A; Fijneman, Remond J A; Abeln, Sanne

    2016-01-01

    Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs) of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large) series of tumor samples. 'GeneBreak' is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH) or by (low-pass) whole genome sequencing (WGS). First, 'GeneBreak' collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, 'GeneBreak', is implemented in R ( www.cran.r-project.org) and is available from Bioconductor ( www.bioconductor.org/packages/release/bioc/html/GeneBreak.html).

  15. Mitochondrial DNA Copy Number and Pancreatic Cancer in the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study

    PubMed Central

    Lynch, Shannon M.; Weinstein, Stephanie J.; Virtamo, Jarmo; Lan, Qing; Lui, Chin-San; Cheng, Wen-Ling; Rothman, Nathaniel; Albanes, Demetrius; Stolzenberg-Solomon, Rachael Z.

    2011-01-01

    Background Diabetes, obesity, and cigarette smoke, consistent risk factors for pancreatic cancer, are sources of oxidative stress in humans that could cause mitochondrial DNA (mtDNA) damage and increase mtDNA copy number. Methods To test whether higher mtDNA copy number is associated with increased incident pancreatic cancer, we conducted a nested case-control study in the Alpha-Tocopherol Beta Carotene Cancer Prevention (ATBC) Study cohort of male smokers, aged 50-69 years at baseline. Between 1992 and 2004, 203 incident cases of pancreatic adenocarcinoma occurred (follow-up: 12 years) among participants with whole blood samples used for mtDNA extraction. For these cases and 656 controls, we calculated odds ratios (OR) and 95% confidence intervals using unconditional logistic regression, adjusting for age, smoking, and diabetes history. All statistical tests were two-sided. Results Higher mtDNA copy number was significantly associated with increased pancreatic cancer risk (highest vs. lowest mtDNA copy number quintile, OR=1.64, 95%CI=1.01-2.67, continuous OR=1.14, 95% CI 1.06-1.23), particularly for cases diagnosed during the first 7 years of follow-up (OR=2.14,95% CI=1.16-3.96, p-trend=0.01, continuous OR=1.21, 95% CI 1.10-1.33), but not for cases occurring during follow-up of 7 years or greater (OR= 1.14, 95% CI=0.53-2.45, continuous OR=1.05, 95% CI 0.93-1.18). Conclusion Our results support the hypothesis that mtDNA copy number is associated with pancreatic cancer and could possibly serve as a biomarker for pancreatic cancer development. PMID:21859925

  16. Low copy number DNA profiling from isolated sperm using the aureka®-micromanipulation system.

    PubMed

    Schneider, C; Müller, U; Kilper, R; Siebertz, B

    2012-07-01

    A new cell isolation technique linked to the aureka® micromanipulation system (aureka®) was used to pick sperm from mixed samples containing sperm and epithelial cells. Both cell types were stained using the HY-LITER™ high-resolution, fluorescent staining kit. To isolate a single sperm of interest under a fluorescent microscope, a specific microsphere picking technique was used. This sensitive and reliable cell identification and isolation technique enables low-copy-number (LCN) DNA profiling, as few as 20 sperm are sufficient for obtaining a full short tandem repeat (STR) profile without any allelic drop out. The presented protocol covers the whole workflow, from sample staining and cell pick up to STR analysis.

  17. Copy number variation of ribosomal DNA and Pokey transposons in natural populations of Daphnia

    PubMed Central

    2012-01-01

    possibility that many rDNA units do not contain a copy of both 18S and 28S genes suggests that rDNA is much more complicated than once thought, and warrants further study. In addition, the lack of correlation between rPokey, gPokey and rDNA unit numbers suggests that Pokey transposition rate is generally very low, and that recombination, in combination with natural selection, eliminates rPokey much faster than gPokey. Our results suggest that further research to determine the mechanisms by which Pokey has escaped complete inactivation by its host (the usual fate of DNA transposons), would provide important insights into transposon biology. PMID:22390386

  18. Genetic variations related to maternal whole blood mitochondrial DNA copy number: a genome-wide and candidate gene study.

    PubMed

    Workalemahu, Tsegaselassie; Enquobahrie, Daniel A; Tadesse, Mahlet G; Hevner, Karin; Gelaye, Bizu; Sanchez, Sixto E; Williams, Michelle A

    2017-10-01

    We conducted genome-wide (GWAS) and candidate gene association studies of maternal mitochondrial DNA copy number. Maternal peripheral blood was collected during labor and delivery admission from 471 participants of a placental abruption case-control study conducted in Lima, Peru. Single nucleotide polymorphism (SNP) genotyping was performed using the Illumina Cardio-Metabo Chip. Whole blood mitochondrial DNA (mtDNA) copy number was measured using qRT-PCR techniques. We evaluated 119,629 SNPs in the GWAS and 161 SNPs (in 29 mitochondrial biogenesis and oxidative phosphorylation genes) in the candidate association study. Top hits from GWAS and the candidate gene study were selected to compute weighted genetic risk scores (wGRS). Linear regression models were used to calculate effect size estimates and related nominal p values. The top hit in our GWAS was chr19:51063065 in FOXA3 (empirical p values = 2.20e - 6). A total of 134 SNPs had p values < 0.001 including rs17111633 in CNNM1 (p values = 6.32e - 6) and chr19:51083059 in MYPOP (p values = 3.23e - 5). In the candidate association study, several SNPs in PPARG, PRKCA, SP1 and THRB were associated with mtDNA copy number (p values < 0.05). mtDNA copy number was significantly associated with wGRS based on top GWAS hits (β = 0.49, 95% CI:0.38-0.60, p < 0.001). Variations in nuclear DNA are potentially associated with maternal mtDNA copy number.

  19. Decreased Peripheral Mitochondrial DNA Copy Number is Associated with the Risk of Heart Failure and Long-term Outcomes.

    PubMed

    Huang, Jin; Tan, Lun; Shen, Rufei; Zhang, Lina; Zuo, Houjuan; Wang, Dao W

    2016-04-01

    Mitochondrial DNA (mtDNA) copy number variation (CNV), which reflects the oxidant-induced cell damage, has been observed in a wide range of human diseases. However, whether it correlates with heart failure, which is closely related to oxidative stress, has never been elucidated before. We aimed to systematically investigate the associations between leukocyte mtDNA CNV and heart failure risk and prognosis. A total of 1700 hospitalized patients with heart failure and 1700 age- and sex-matched community population were consecutively enrolled in this observational study, as well as 1638 (96.4%) patients were followed prospectively for a median of 17 months (12-24 months). The relative mtDNA copy number of leukocyte of peripheral blood or cardiac tissue was measured in triplicate by quantitative real-time PCR method. Patients with heart failure possessed much lower relative mtDNA copy number compared with control subjects (median 0.83, interquartile range [IQR] 0.60-1.16 vs median 1.00, IQR 0.47-2.20; P < 0.001), especially for the patients with ischemic etiology (median, 0.77 for ischemic and 0.91 for non-ischemic, P < 0.001). Patients with lower mtDNA copy number exhibited 1.7 times higher risk of heart failure (odds ratio 1.71, 95% confidence interval [CI] 1.48-1.97, P < 0.001). Long-term follow-up (median of 17 months) showed that decreased mtDNA copy number was significant associated with both increased cardiovascular deaths (hazard ratio [HR] 1.58, 95% CI 1.16-2.16, P = 0.004) and cardiovascular rehospitalization (HR 1.48, 95% CI 1.21-1.82, P < 0.001). After adjusting for the conventional risk factors and medications, lower mtDNA copy numbers were still significantly associated with 50% higher cardiovascular mortality (P = 0.035). In conclusion, mtDNA copy number depletion is an independent risk factor for heart failure and predicts higher cardiovascular mortality in patients with heart failure.

  20. DNA copy number variants of known glaucoma genes in relation to primary open-angle glaucoma.

    PubMed

    Liu, Yutao; Garrett, Melanie E; Yaspan, Brian L; Bailey, Jessica Cooke; Loomis, Stephanie J; Brilliant, Murray; Budenz, Donald L; Christen, William G; Fingert, John H; Gaasterland, Douglas; Gaasterland, Terry; Kang, Jae H; Lee, Richard K; Lichter, Paul; Moroi, Sayoko E; Realini, Anthony; Richards, Julia E; Schuman, Joel S; Scott, William K; Singh, Kuldev; Sit, Arthur J; Vollrath, Douglas; Weinreb, Robert; Wollstein, Gadi; Zack, Donald J; Zhang, Kang; Pericak-Vance, Margaret A; Haines, Jonathan L; Pasquale, Louis R; Wiggs, Janey L; Allingham, R Rand; Ashley-Koch, Allison E; Hauser, Michael A

    2014-11-20

    We examined the role of DNA copy number variants (CNVs) of known glaucoma genes in relation to primary open angle glaucoma (POAG). Our study included DNA samples from two studies (NEIGHBOR and GLAUGEN). All the samples were genotyped with the Illumina Human660W_Quad_v1 BeadChip. After removing non-blood-derived and amplified DNA samples, we applied quality control steps based on the mean Log R Ratio and the mean B allele frequency. Subsequently, data from 3057 DNA samples (1599 cases and 1458 controls) were analyzed with PennCNV software. We defined CNVs as those ≥5 kilobases (kb) in size and interrogated by ≥5 consecutive probes. We further limited our investigation to CNVs in known POAG-related genes, including CDKN2B-AS1, TMCO1, SIX1/SIX6, CAV1/CAV2, the LRP12-ZFPM2 region, GAS7, ATOH7, FNDC3B, CYP1B1, MYOC, OPTN, WDR36, SRBD1, TBK1, and GALC. Genomic duplications of CDKN2B-AS1 and TMCO1 were each found in a single case. Two cases carried duplications in the GAS7 region. Genomic deletions of SIX6 and ATOH7 were each identified in one case. One case carried a TBK1 deletion and another case carried a TBK1 duplication. No controls had duplications or deletions in these six genes. A single control had a duplication in the MYOC region. Deletions of GALC were observed in five cases and two controls. The CNV analysis of a large set of cases and controls revealed the presence of rare CNVs in known POAG susceptibility genes. Our data suggest that these rare CNVs may contribute to POAG pathogenesis and merit functional evaluation. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  1. DNA Copy Number Variants of Known Glaucoma Genes in Relation to Primary Open-Angle Glaucoma

    PubMed Central

    Liu, Yutao; Garrett, Melanie E.; Yaspan, Brian L.; Bailey, Jessica Cooke; Loomis, Stephanie J.; Brilliant, Murray; Budenz, Donald L.; Christen, William G.; Fingert, John H.; Gaasterland, Douglas; Gaasterland, Terry; Kang, Jae H.; Lee, Richard K.; Lichter, Paul; Moroi, Sayoko E.; Realini, Anthony; Richards, Julia E.; Schuman, Joel S.; Scott, William K.; Singh, Kuldev; Sit, Arthur J.; Vollrath, Douglas; Weinreb, Robert; Wollstein, Gadi; Zack, Donald J.; Zhang, Kang; Pericak-Vance, Margaret A.; Haines, Jonathan L.; Pasquale, Louis R.; Wiggs, Janey L.; Allingham, R. Rand; Ashley-Koch, Allison E.; Hauser, Michael A.

    2014-01-01

    Purpose. We examined the role of DNA copy number variants (CNVs) of known glaucoma genes in relation to primary open angle glaucoma (POAG). Methods. Our study included DNA samples from two studies (NEIGHBOR and GLAUGEN). All the samples were genotyped with the Illumina Human660W_Quad_v1 BeadChip. After removing non–blood-derived and amplified DNA samples, we applied quality control steps based on the mean Log R Ratio and the mean B allele frequency. Subsequently, data from 3057 DNA samples (1599 cases and 1458 controls) were analyzed with PennCNV software. We defined CNVs as those ≥5 kilobases (kb) in size and interrogated by ≥5 consecutive probes. We further limited our investigation to CNVs in known POAG-related genes, including CDKN2B-AS1, TMCO1, SIX1/SIX6, CAV1/CAV2, the LRP12-ZFPM2 region, GAS7, ATOH7, FNDC3B, CYP1B1, MYOC, OPTN, WDR36, SRBD1, TBK1, and GALC. Results. Genomic duplications of CDKN2B-AS1 and TMCO1 were each found in a single case. Two cases carried duplications in the GAS7 region. Genomic deletions of SIX6 and ATOH7 were each identified in one case. One case carried a TBK1 deletion and another case carried a TBK1 duplication. No controls had duplications or deletions in these six genes. A single control had a duplication in the MYOC region. Deletions of GALC were observed in five cases and two controls. Conclusions. The CNV analysis of a large set of cases and controls revealed the presence of rare CNVs in known POAG susceptibility genes. Our data suggest that these rare CNVs may contribute to POAG pathogenesis and merit functional evaluation. PMID:25414181

  2. Elevation of Pollen Mitochondrial DNA Copy Number by WHIRLY2: Altered Respiration and Pollen Tube Growth in Arabidopsis.

    PubMed

    Cai, Qiang; Guo, Liang; Shen, Zhao-Rui; Wang, Dan-Yang; Zhang, Quan; Sodmergen

    2015-09-01

    In plants, the copy number of the mitochondrial DNA (mtDNA) can be much lower than the number of mitochondria. The biological significance and regulatory mechanisms of this phenomenon remain poorly understood. Here, using the pollen vegetative cell, we examined the role of the Arabidopsis (Arabidopsis thaliana) mtDNA-binding protein WHIRLY2 (AtWHY2). AtWHY2 decreases during pollen development, in parallel with the rapid degradation of mtDNA; to examine the importance of this decrease, we used the pollen vegetative cell-specific promoter Lat52 to express AtWHY2. The transgenic plants (LWHY2) had very high mtDNA levels in pollen, more than 10 times more than in the wild type (ecotype Columbia-0). LWHY2 plants were fertile, morphologically normal, and set seeds; however, reciprocal crosses with heterozygous plants showed reduced transmission of LWHY2-1 through the male and slower growth of LWHY2-1 pollen tubes. We found that LWHY2-1 pollen had significantly more reactive oxygen species and less ATP compared with the wild type, indicating an effect on mitochondrial respiration. These findings reveal that AtWHY2 affects mtDNA copy number in pollen and suggest that low mtDNA copy numbers might be the normal means by which plant cells maintain mitochondrial genetic information. © 2015 American Society of Plant Biologists. All Rights Reserved.

  3. Elevation of Pollen Mitochondrial DNA Copy Number by WHIRLY2: Altered Respiration and Pollen Tube Growth in Arabidopsis1

    PubMed Central

    Cai, Qiang; Guo, Liang; Shen, Zhao-Rui; Wang, Dan-Yang; Zhang, Quan; Sodmergen

    2015-01-01

    In plants, the copy number of the mitochondrial DNA (mtDNA) can be much lower than the number of mitochondria. The biological significance and regulatory mechanisms of this phenomenon remain poorly understood. Here, using the pollen vegetative cell, we examined the role of the Arabidopsis (Arabidopsis thaliana) mtDNA-binding protein WHIRLY2 (AtWHY2). AtWHY2 decreases during pollen development, in parallel with the rapid degradation of mtDNA; to examine the importance of this decrease, we used the pollen vegetative cell-specific promoter Lat52 to express AtWHY2. The transgenic plants (LWHY2) had very high mtDNA levels in pollen, more than 10 times more than in the wild type (ecotype Columbia-0). LWHY2 plants were fertile, morphologically normal, and set seeds; however, reciprocal crosses with heterozygous plants showed reduced transmission of LWHY2-1 through the male and slower growth of LWHY2-1 pollen tubes. We found that LWHY2-1 pollen had significantly more reactive oxygen species and less ATP compared with the wild type, indicating an effect on mitochondrial respiration. These findings reveal that AtWHY2 affects mtDNA copy number in pollen and suggest that low mtDNA copy numbers might be the normal means by which plant cells maintain mitochondrial genetic information. PMID:26195569

  4. Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

    PubMed

    Pineda, Gina M; Montgomery, Anne H; Thompson, Robyn; Indest, Brooke; Carroll, Marion; Sinha, Sudhir K

    2014-11-01

    There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss

  5. A Broadly Applicable Assay for Rapidly and Accurately Quantifying DNA Surface Coverage on Diverse Particles.

    PubMed

    Yu, Haixiang; Xu, Xiaowen; Liang, Pingping; Loh, Kang Yong; Guntupalli, Bhargav; Roncancio, Daniel; Xiao, Yi

    2017-04-19

    DNA-modified particles are used extensively for applications in sensing, material science, and molecular biology. The performance of such DNA-modified particles is greatly dependent on the degree of surface coverage, but existing methods for quantitation can only be employed for certain particle compositions and/or conjugation chemistries. We have developed a simple and broadly applicable exonuclease III (Exo III) digestion assay based on the cleavage of phosphodiester bonds-a universal feature of DNA-modified particles-to accurately quantify DNA probe surface coverage on diverse, commonly used particles of different compositions, conjugation chemistries, and sizes. Our assay utilizes particle-conjugated, fluorophore-labeled probes that incorporate two abasic sites; these probes are hybridized to a complementary DNA (cDNA) strand, and quantitation is achieved via cleavage and digestion of surface-bound probe DNA via Exo III's apurinic endonucleolytic and exonucleolytic activities. The presence of the two abasic sites in the probe greatly speeds up the enzymatic reaction without altering the packing density of the probes on the particles. Probe digestion releases a signal-generating fluorophore and liberates the intact cDNA strand to start a new cycle of hybridization and digestion, until all fluorophore tags have been released. Since the molar ratio of fluorophore to immobilized DNA is 1:1, DNA surface coverage can be determined accurately based on the complete release of fluorophores. Our method delivers accurate, rapid, and reproducible quantitation of thiolated DNA on the surface of gold nanoparticles, and also performs equally well with other conjugation chemistries, substrates, and particle sizes, and thus offers a broadly useful assay for quantitation of DNA surface coverage.

  6. Excessive genomic DNA copy number variation in the Li–Fraumeni cancer predisposition syndrome

    PubMed Central

    Shlien, Adam; Tabori, Uri; Marshall, Christian R.; Pienkowska, Malgorzata; Feuk, Lars; Novokmet, Ana; Nanda, Sonia; Druker, Harriet; Scherer, Stephen W.; Malkin, David

    2008-01-01

    DNA copy number variations (CNVs) are a significant and ubiquitous source of inherited human genetic variation. However, the importance of CNVs to cancer susceptibility and tumor progression has not yet been explored. Li–Fraumeni syndrome (LFS) is an autosomal dominantly inherited disorder characterized by a strikingly increased risk of early-onset breast cancer, sarcomas, brain tumors and other neoplasms in individuals harboring germline TP53 mutations. Known genetic determinants of LFS do not fully explain the variable clinical phenotype in affected family members. As part of a wider study of CNVs and cancer, we conducted a genome-wide profile of germline CNVs in LFS families. Here, by examining DNA from a large healthy population and an LFS cohort using high-density oligonucleotide arrays, we show that the number of CNVs per genome is well conserved in the healthy population, but strikingly enriched in these cancer-prone individuals. We found a highly significant increase in CNVs among carriers of germline TP53 mutations with a familial cancer history. Furthermore, we identified a remarkable number of genomic regions in which known cancer-related genes coincide with CNVs, in both LFS families and healthy individuals. Germline CNVs may provide a foundation that enables the more dramatic chromosomal changes characteristic of TP53-related tumors to be established. Our results suggest that screening families predisposed to cancer for CNVs may identify individuals with an abnormally high number of these events. PMID:18685109

  7. High-resolution genomic copy number profiling of glioblastoma multiforme by single nucleotide polymorphism DNA microarray.

    PubMed

    Yin, Dong; Ogawa, Seishi; Kawamata, Norihiko; Tunici, Patrizia; Finocchiaro, Gaetano; Eoli, Marica; Ruckert, Christian; Huynh, Thien; Liu, Gentao; Kato, Motohiro; Sanada, Masashi; Jauch, Anna; Dugas, Martin; Black, Keith L; Koeffler, H Phillip

    2009-05-01

    Glioblastoma multiforme (GBM) is an extremely malignant brain tumor. To identify new genomic alterations in GBM, genomic DNA of tumor tissue/explants from 55 individuals and 6 GBM cell lines were examined using single nucleotide polymorphism DNA microarray (SNP-Chip). Further gene expression analysis relied on an additional 56 GBM samples. SNP-Chip results were validated using several techniques, including quantitative PCR (Q-PCR), nucleotide sequencing, and a combination of Q-PCR and detection of microsatellite markers for loss of heterozygosity with normal copy number [acquired uniparental disomy (AUPD)]. Whole genomic DNA copy number in each GBM sample was profiled by SNP-Chip. Several signaling pathways were frequently abnormal. Either the p16(INK4A)/p15(INK4B)-CDK4/6-pRb or p14(ARF)-MDM2/4-p53 pathways were abnormal in 89% (49 of 55) of cases. Simultaneous abnormalities of both pathways occurred in 84% (46 of 55) samples. The phosphoinositide 3-kinase pathway was altered in 71% (39 of 55) GBMs either by deletion of PTEN or amplification of epidermal growth factor receptor and/or vascular endothelial growth factor receptor/platelet-derived growth factor receptor alpha. Deletion of chromosome 6q26-27 often occurred (16 of 55 samples). The minimum common deleted region included PARK2, PACRG, QKI, and PDE10A genes. Further reverse transcription Q-PCR studies showed that PARK2 expression was decreased in another collection of GBMs at a frequency of 61% (34 of 56) of samples. The 1p36.23 region was deleted in 35% (19 of 55) of samples. Notably, three samples had homozygous deletion encompassing this site. Also, a novel internal deletion of a putative tumor suppressor gene, LRP1B, was discovered causing an aberrant protein. AUPDs occurred in 58% (32 of 55) of the GBM samples and five of six GBM cell lines. A common AUPD was found at chromosome 17p13.3-12 (included p53 gene) in 13 of 61 samples and cell lines. Single-strand conformational polymorphism and nucleotide

  8. Effect of location, organization, and repeat-copy number in satellite-DNA evolution.

    PubMed

    Navajas-Pérez, R; Quesada del Bosque, M E; Garrido-Ramos, M A

    2009-10-01

    Here, we analyze the evolutionary dynamics of a satellite-DNA family in an attempt to understand the effect of factors such as location, organization, and repeat-copy number in the molecular drive process leading to the concerted-evolution pattern found in this type of repetitive sequences. The presence of RAE180 satellite-DNA in the dioecious species of the plant genus Rumex is a noteworthy feature at this respect, as RAE180 satellite repeats have accumulated differentially, showing a distinct distribution pattern in different species. The evolution of dioecious Rumex gave rise to two phylogenetic clades: one clade composed of species with an ancestral XX/XY sex chromosome system and a second, derived clade of species with a multiple sex-chromosome system XX/XY(1)Y(2). While in the XX/XY dioecious species, the RAE180 satellite-DNA is located only in a small autosomal locus, the RAE180 repeats are present also in a small autosomal locus and additionally have been massively amplified in the Y chromosomes of XX/XY(1)Y(2) species. Here, we have found that the RAE180 repeats of the autosomal locus of XX/XY species are characterized by intra-specific sequence homogeneity and inter-specific divergence and that the comparison of individual nucleotide positions between related species shows a general pattern of concerted evolution. On the contrary, both in the autosomal and the Y-linked loci of XX/XY(1)Y(2) species, ancestral variability has remained with reduced rates of sequence homogenization and of evolution. Thus, this study demonstrates that molecular mechanisms of non-reciprocal exchange are key factors in the molecular drive process; the satellite DNAs in the non-recombining Y chromosomes show low rates of concerted evolution and intra-specific variability increase with no inter-specific divergence. By contrast, freely recombining loci undergo concerted evolution with genetic differentiation between species as occurred in the autosomal locus of XX/XY species

  9. Reduced rDNA copy number does not affect "competitive" chromosome pairing in XYY males of Drosophila melanogaster.

    PubMed

    Maggert, Keith A

    2014-03-20

    The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a "competitive" situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments.

  10. Reduced rDNA Copy Number Does Not Affect “Competitive” Chromosome Pairing in XYY Males of Drosophila melanogaster

    PubMed Central

    Maggert, Keith A.

    2014-01-01

    The ribosomal DNA (rDNA) arrays are causal agents in X-Y chromosome pairing in meiosis I of Drosophila males. Despite broad variation in X-linked and Y-linked rDNA copy number, polymorphisms in regulatory/spacer sequences between rRNA genes, and variance in copy number of interrupting R1 and R2 retrotransposable elements, there is little evidence that different rDNA arrays affect pairing efficacy. I investigated whether induced rDNA copy number polymorphisms affect chromosome pairing in a “competitive” situation in which complex pairing configurations were possible using males with XYY constitution. Using a common normal X chromosome, one of two different full-length Y chromosomes, and a third chromosome from a series of otherwise-isogenic rDNA deletions, I detected no differences in X-Y or Y-Y pairing or chromosome segregation frequencies that could not be attributed to random variation alone. This work was performed in the context of an undergraduate teaching program at Texas A&M University, and I discuss the pedagogical utility of this and other such experiments. PMID:24449686

  11. The effects of a wheat germ rich diet on oxidative mtDNA damage, mtDNA copy number and antioxidant enzyme activities in aging Drosophila.

    PubMed

    Mutlu, Ayse Gul

    2013-03-01

    The free radical theory of aging posits that the accumulation of macromolecular damage induced by toxic reactive oxygen species plays a central role in the aging process. Therefore consumption of dietary antioxidants appears to be of great importance. Wheat germ have strong antioxidant properties. Aim of this study is investigate the effects of a wheat germ rich diet on oxidative mtDNA damage, mtDNA copy number and antioxidant enzyme activities in Drosophila. Current results suggested that dietary wheat germ enhances the activities of antioxidant enzymes in Drosophila. There was no statistically difference in mtDNA damage and mtDNA copy number results of "Wheat Germ" and "Refined White Flour" feed groups. mtDNA damage slightly increased with aging in both groups but these changes were no statistically different.

  12. FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing

    PubMed Central

    Shen, Ronglai; Seshan, Venkatraman E.

    2016-01-01

    Allele-specific copy number analysis (ASCN) from next generation sequencing (NGS) data can greatly extend the utility of NGS beyond the identification of mutations to precisely annotate the genome for the detection of homozygous/heterozygous deletions, copy-neutral loss-of-heterozygosity (LOH), allele-specific gains/amplifications. In addition, as targeted gene panels are increasingly used in clinical sequencing studies for the detection of ‘actionable’ mutations and copy number alterations to guide treatment decisions, accurate, tumor purity-, ploidy- and clonal heterogeneity-adjusted integer copy number calls are greatly needed to more reliably interpret NGS-based cancer gene copy number data in the context of clinical sequencing. We developed FACETS, an ASCN tool and open-source software with a broad application to whole genome, whole-exome, as well as targeted panel sequencing platforms. It is a fully integrated stand-alone pipeline that includes sequencing BAM file post-processing, joint segmentation of total- and allele-specific read counts, and integer copy number calls corrected for tumor purity, ploidy and clonal heterogeneity, with comprehensive output and integrated visualization. We demonstrate the application of FACETS using The Cancer Genome Atlas (TCGA) whole-exome sequencing of lung adenocarcinoma samples. We also demonstrate its application to a clinical sequencing platform based on a targeted gene panel. PMID:27270079

  13. Specific patterns of DNA copy number gains and losses in eight new glioblastoma multiforme cell lines.

    PubMed

    Ramirez, Tzutzuy; Thoma, Karen; Taja-Chayeb, Lucia; Efferth, Thomas; Herrera, Luis A; Halatsch, Marc-Eric; Gebhart, Erich

    2003-08-01

    Eight cell lines newly established from glioblastoma multiforme were examined by comparative genomic hybridization for their patterns of genomic imbalance. The total number of DNA copy number alterations (CNAs) found in the eight cell lines varied between 15 and 24. This characterized the examined cell lines (or the tumors they were derived from) as distinctly progressed in karyotypic evolution. The most frequent CNAs were gains of the entire chromosome 6 or, at least, parts of it, and of 7p22, which were found in all eight cell lines. Other changes present in seven of the eight cell lines were gains of 3q26qter and the entire chromosome 7 and losses of segments on chromosome 4q (e.g., 4q34q35) and of the short arm of chromosome 10. Enh(3q21q25), dim(4q22q33) and dim(4qter), dim(13q22), enh(15q14), and enh(18q22q23) were found in six of the eight cell lines. Several other CNAs [e.g., dim(9p21)] were found in common in five or less of the eight lines. Using a hierarchical cluster analysis, the specific patterns of genomic imbalance allowed various groupings of the examined cell lines. Although a close relation could be confirmed among all examined lines on the basis of shared CNAs, two main groups could be roughly differentiated. Among those there were also more or less closely related subgroups. However, also alterations which were restricted to one single cell line each were found, e.g., dim(1q41qter), dim(2q22qter), enh(4p), dim(5p), dim(4p13pter), dim(8q21qter), enh(9p), dim(9q), dim(11p14pter), enh(12q15q23), enh(13q21), dim(14q21qter), dim(15q21qter), dim(19q), and enh(22q). The comparison of the obtained data on gains and losses of DNA copy numbers in specific chromosomal segments with the data on localization of genes possibly associated with the biology of glioblastoma multiforme additionally shows high conformity but also disparity of the examined cell lines among each other, as well as compared to primary glioblastoma multiforme. Eventually, each of the

  14. Transcription reporters that shuttle cloned DNA between high-copy Escherichia coli plasmids and low-copy broad-host-range plasmids.

    PubMed

    Ouimet, M C; Marczynski, G T

    2000-09-01

    We describe and apply lacZ transcription reporter plasmids designed for both biochemical analyses requiring high DNA yield and physiological studies requiring low gene dosage. Standard DNA ligations are performed at seven unique restriction sites 5' to the lacZ gene on high-copy ColE1 plasmids suitable for double- or single-strand DNA sequencing. A divergent gusA transcription reporter is included and serves as an internal control. Rec(+) Escherichia coli cells readily shuttle DNA placed between gusA and lacZ by allelic exchange with pRK290-based plasmids that subsequently conjugate and replicate in most gram-negative bacteria. We applied this system to study Caulobacter crescentus cell cycle promoters directed by the CtrA response-regulator protein. Synthetic oligonucleotides were ligated to create altered CtrA binding sites and corresponding promoters with varied transcription strength. We also document the phenomenon of long-range promoter interference. A strong promoter can repress up to twofold the transcription from a divergent promoter located 100 bp away. However, the cell cycle timing of both promoters is not changed. Additional applications of our system and theoretical aspects of promoter organization are discussed. Copyright 2000 Academic Press.

  15. Accurately Assessing the Risk of Schizophrenia Conferred by Rare Copy-Number Variation Affecting Genes with Brain Function

    PubMed Central

    Raychaudhuri, Soumya; Korn, Joshua M.; McCarroll, Steven A.; Altshuler, David; Sklar, Pamela; Purcell, Shaun; Daly, Mark J.

    2010-01-01

    Investigators have linked rare copy number variation (CNVs) to neuropsychiatric diseases, such as schizophrenia. One hypothesis is that CNV events cause disease by affecting genes with specific brain functions. Under these circumstances, we expect that CNV events in cases should impact brain-function genes more frequently than those events in controls. Previous publications have applied “pathway” analyses to genes within neuropsychiatric case CNVs to show enrichment for brain-functions. While such analyses have been suggestive, they often have not rigorously compared the rates of CNVs impacting genes with brain function in cases to controls, and therefore do not address important confounders such as the large size of brain genes and overall differences in rates and sizes of CNVs. To demonstrate the potential impact of confounders, we genotyped rare CNV events in 2,415 unaffected controls with Affymetrix 6.0; we then applied standard pathway analyses using four sets of brain-function genes and observed an apparently highly significant enrichment for each set. The enrichment is simply driven by the large size of brain-function genes. Instead, we propose a case-control statistical test, cnv-enrichment-test, to compare the rate of CNVs impacting specific gene sets in cases versus controls. With simulations, we demonstrate that cnv-enrichment-test is robust to case-control differences in CNV size, CNV rate, and systematic differences in gene size. Finally, we apply cnv-enrichment-test to rare CNV events published by the International Schizophrenia Consortium (ISC). This approach reveals nominal evidence of case-association in neuronal-activity and the learning gene sets, but not the other two examined gene sets. The neuronal-activity genes have been associated in a separate set of schizophrenia cases and controls; however, testing in independent samples is necessary to definitively confirm this association. Our method is implemented in the PLINK software package

  16. Accurately assessing the risk of schizophrenia conferred by rare copy-number variation affecting genes with brain function.

    PubMed

    Raychaudhuri, Soumya; Korn, Joshua M; McCarroll, Steven A; Altshuler, David; Sklar, Pamela; Purcell, Shaun; Daly, Mark J

    2010-09-09

    Investigators have linked rare copy number variation (CNVs) to neuropsychiatric diseases, such as schizophrenia. One hypothesis is that CNV events cause disease by affecting genes with specific brain functions. Under these circumstances, we expect that CNV events in cases should impact brain-function genes more frequently than those events in controls. Previous publications have applied "pathway" analyses to genes within neuropsychiatric case CNVs to show enrichment for brain-functions. While such analyses have been suggestive, they often have not rigorously compared the rates of CNVs impacting genes with brain function in cases to controls, and therefore do not address important confounders such as the large size of brain genes and overall differences in rates and sizes of CNVs. To demonstrate the potential impact of confounders, we genotyped rare CNV events in 2,415 unaffected controls with Affymetrix 6.0; we then applied standard pathway analyses using four sets of brain-function genes and observed an apparently highly significant enrichment for each set. The enrichment is simply driven by the large size of brain-function genes. Instead, we propose a case-control statistical test, cnv-enrichment-test, to compare the rate of CNVs impacting specific gene sets in cases versus controls. With simulations, we demonstrate that cnv-enrichment-test is robust to case-control differences in CNV size, CNV rate, and systematic differences in gene size. Finally, we apply cnv-enrichment-test to rare CNV events published by the International Schizophrenia Consortium (ISC). This approach reveals nominal evidence of case-association in neuronal-activity and the learning gene sets, but not the other two examined gene sets. The neuronal-activity genes have been associated in a separate set of schizophrenia cases and controls; however, testing in independent samples is necessary to definitively confirm this association. Our method is implemented in the PLINK software package.

  17. Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly

    PubMed Central

    Yaroslavsky, Anastasia I.; Smolina, Irina V.

    2013-01-01

    SUMMARY We present a novel approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single copy sensitivity and single base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this new technique by successfully detecting six unique target sites on human mitochondrial and autosomal DNA. We also demonstrate the high specificity of this method by detecting X- and Y- specific sequences on human sex chromosomes and by simultaneously detecting three unique target sites. Finally, we discriminate two target sites that differ by two nucleotides. The PNA-RCA-FISH approach is a unique in situ hybridization method capable of multi-target visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific. PMID:23521801

  18. Simultaneous quantification of mitochondrial DNA copy number and deletion ratio: A multiplex real-time PCR assay

    PubMed Central

    Phillips, Nicole R.; Sprouse, Marc L.; Roby, Rhonda K.

    2014-01-01

    Mitochondrial dysfunction is implicated in a vast array of diseases and conditions, such as Alzheimer's disease, cancer, and aging. Alterations in mitochondrial DNA (mtDNA) may provide insight into the processes that either initiate or propagate this dysfunction. Here, we describe a unique multiplex assay which simultaneously provides assessments of mtDNA copy number and the proportion of genomes with common large deletions by targeting two mitochondrial sites and one nuclear locus. This probe-based, single-tube multiplex provides high specificity while eliminating well-to-well variability that results from assaying nuclear and mitochondrial targets individually. PMID:24463429

  19. Intragenomic polymorphisms among high-copy loci: a genus-wide study of nuclear ribosomal DNA in Asclepias (Apocynaceae)

    PubMed Central

    Straub, Shannon C.K.; Fishbein, Mark; Liston, Aaron

    2015-01-01

    Despite knowledge that concerted evolution of high-copy loci is often imperfect, studies that investigate the extent of intragenomic polymorphisms and comparisons across a large number of species are rarely made. We present a bioinformatic pipeline for characterizing polymorphisms within an individual among copies of a high-copy locus. Results are presented for nuclear ribosomal DNA (nrDNA) across the milkweed genus, Asclepias. The 18S-26S portion of the nrDNA cistron of Asclepias syriaca served as a reference for assembly of the region from 124 samples representing 90 species of Asclepias. Reads were mapped back to each individual’s consensus and at each position reads differing from the consensus were tallied using a custom perl script. Low frequency polymorphisms existed in all individuals (mean = 5.8%). Most nrDNA positions (91%) were polymorphic in at least one individual, with polymorphic sites being less frequent in subunit regions and loops. Highly polymorphic sites existed in each individual, with highest abundance in the “noncoding” ITS regions. Phylogenetic signal was present in the distribution of intragenomic polymorphisms across the genus. Intragenomic polymorphisms in nrDNA are common in Asclepias, being found at higher frequency than any other study to date. The high and variable frequency of polymorphisms across species highlights concerns that phylogenetic applications of nrDNA may be error-prone. The new analytical approach provided here is applicable to other taxa and other high-copy regions characterized by low coverage genome sequencing (genome skimming). PMID:25653903

  20. Accurate measurement of psoralen-crosslinked DNA: direct biochemical measurements and indirect measurement by hybridization

    SciTech Connect

    Matsuo, N.; Ross, P.M.

    1988-11-01

    This paper evaluates methods to measure crosslinkage due to psoralen plus light in total DNA and in specific sequences. DNA exposed in cells or in vitro to a bifunctional psoralen and near ultraviolet light accumulates interstrand crosslinks. Crosslinkage is the DNA mass fraction that is attached in both strands to a crosslink. We show here biochemical methods to measure psoralen photocrosslinkage accurately in total DNA. We also describe methods to measure photocrosslinkage indirectly, in specific sequences, by nucleic acid hybridization. We show that a single 4,5',8-trimethylpsoralen (TMP) crosslink causes at least 50 kbp of alkali-denatured DNA contiguous in both strands with it to snap back into the duplex form when the denatured preparation is returned to neutral pH. This process was so efficient that the DNA was not nicked by the single-strand nuclease S1 at 100-fold excess after snapping back. Uncrosslinked DNA was digested to acid-soluble material by the enzyme. Crosslinkage therefore equals the fraction of S1-resistant nucleotide in this kind of experiment. We alkali-denatured DNA samples crosslinked to varying degrees by varying TMP concentration at constant light exposure. We then measured crosslinkage by ethidium bromide (EtBr) fluorometry at pH 11.8; by EtBr fluorometry at neutral pH of S1 digests of the DNA; and by the fraction of radioactivity remaining acid insoluble in S1-digests of DNA labeled uniformly with (3H)deoxythymidine. These assays measure distinct physical properties of crosslinked DNA. Numerical agreement is expected only when all three measurements are accurate. Under optimum conditions, the three methods yielded identical results over the range of measurement. Using alkaline EtBr fluorescence in crude cell lysates, we detected crosslinks at frequencies in the range of 1.6 X 10(-7) per base pair.

  1. An accurate DNA marker assay for stem rust resistance gene Sr2 in wheat

    USDA-ARS?s Scientific Manuscript database

    The stem rust resistance gene Sr2 has provided broad-spectrum protection against stem rust (Puccinia graminis) since its wide spread deployment in wheat from the 1940s. Because Sr2 confers partial resistance which is difficult to select under field conditions, a DNA marker is desirable that accurate...

  2. Increased mitochondrial DNA copy number in occupations associated with low-dose benzene exposure.

    PubMed

    Carugno, Michele; Pesatori, Angela Cecilia; Dioni, Laura; Hoxha, Mirjam; Bollati, Valentina; Albetti, Benedetta; Byun, Hyang-Min; Bonzini, Matteo; Fustinoni, Silvia; Cocco, Pierluigi; Satta, Giannina; Zucca, Mariagrazia; Merlo, Domenico Franco; Cipolla, Massimo; Bertazzi, Pier Alberto; Baccarelli, Andrea

    2012-02-01

    Benzene is an established leukemogen at high exposure levels. Although low-level benzene exposure is widespread and may induce oxidative damage, no mechanistic biomarkers are available to detect biological dysfunction at low doses. Our goals were to determine in a large multicenter cross-sectional study whether low-level benzene is associated with increased blood mitochondrial DNA copy number (mtDNAcn, a biological oxidative response to mitochondrial DNA damage and dysfunction) and to explore potential links between mtDNAcn and leukemia-related epigenetic markers. We measured blood relative mtDNAcn by real-time polymerase chain reaction in 341 individuals selected from various occupational groups with low-level benzene exposures (> 100 times lower than the Occupational Safety and Health Administration/European Union standards) and 178 referents from three Italian cities (Genoa, Milan, Cagliari). In each city, benzene-exposed participants showed higher mtDNAcn than referents: mtDNAcn was 0.90 relative units in Genoa bus drivers and 0.75 in referents (p = 0.019); 0.90 in Milan gas station attendants, 1.10 in police officers, and 0.75 in referents (p-trend = 0.008); 1.63 in Cagliari petrochemical plant workers, 1.25 in referents close to the plant, and 0.90 in referents farther from the plant (p-trend = 0.046). Using covariate-adjusted regression models, we estimated that an interquartile range increase in personal airborne benzene was associated with percent increases in mtDNAcn equal to 10.5% in Genoa (p = 0.014), 8.2% (p = 0.008) in Milan, 7.5% in Cagliari (p = 0.22), and 10.3% in all cities combined (p < 0.001). Using methylation data available for the Milan participants, we found that mtDNAcn was associated with LINE-1 hypomethylation (-2.41%; p = 0.007) and p15 hypermethylation (+15.95%, p = 0.008). Blood MtDNAcn was increased in persons exposed to low benzene levels, potentially reflecting mitochondrial DNA damage and dysfunction.

  3. Increased Mitochondrial DNA Copy Number in Occupations Associated with Low-Dose Benzene Exposure

    PubMed Central

    Pesatori, Angela Cecilia; Dioni, Laura; Hoxha, Mirjam; Bollati, Valentina; Albetti, Benedetta; Byun, Hyang-Min; Bonzini, Matteo; Fustinoni, Silvia; Cocco, Pierluigi; Satta, Giannina; Zucca, Mariagrazia; Merlo, Domenico Franco; Cipolla, Massimo; Bertazzi, Pier Alberto; Baccarelli, Andrea

    2011-01-01

    Background: Benzene is an established leukemogen at high exposure levels. Although low-level benzene exposure is widespread and may induce oxidative damage, no mechanistic biomarkers are available to detect biological dysfunction at low doses. Objectives: Our goals were to determine in a large multicenter cross-sectional study whether low-level benzene is associated with increased blood mitochondrial DNA copy number (mtDNAcn, a biological oxidative response to mitochondrial DNA damage and dysfunction) and to explore potential links between mtDNAcn and leukemia-related epigenetic markers. Methods: We measured blood relative mtDNAcn by real-time polymerase chain reaction in 341 individuals selected from various occupational groups with low-level benzene exposures (> 100 times lower than the Occupational Safety and Health Administration/European Union standards) and 178 referents from three Italian cities (Genoa, Milan, Cagliari). Results: In each city, benzene-exposed participants showed higher mtDNAcn than referents: mtDNAcn was 0.90 relative units in Genoa bus drivers and 0.75 in referents (p = 0.019); 0.90 in Milan gas station attendants, 1.10 in police officers, and 0.75 in referents (p-trend = 0.008); 1.63 in Cagliari petrochemical plant workers, 1.25 in referents close to the plant, and 0.90 in referents farther from the plant (p-trend = 0.046). Using covariate-adjusted regression models, we estimated that an interquartile range increase in personal airborne benzene was associated with percent increases in mtDNAcn equal to 10.5% in Genoa (p = 0.014), 8.2% (p = 0.008) in Milan, 7.5% in Cagliari (p = 0.22), and 10.3% in all cities combined (p < 0.001). Using methylation data available for the Milan participants, we found that mtDNAcn was associated with LINE-1 hypomethylation (–2.41%; p = 0.007) and p15 hypermethylation (+15.95%, p = 0.008). Conclusions: Blood MtDNAcn was increased in persons exposed to low benzene levels, potentially reflecting mitochondrial

  4. DNA copy number aberrations in small-cell lung cancer reveal activation of the focal adhesion pathway

    PubMed Central

    Ocak, S; Yamashita, H; Udyavar, AR; Miller, AN; Gonzalez, AL; Zou, Y; Jiang, A; Yi, Y; Shyr, Y; Estrada, L; Quaranta, V; Massion, PP

    2015-01-01

    Small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer in its clinical behavior, with a 5-year overall survival as low as 5%. Despite years of research in the field, molecular determinants of SCLC behavior are still poorly understood, and this deficiency has translated into an absence of specific diagnostics and targeted therapeutics. We hypothesized that tumor DNA copy number alterations would allow the identification of molecular pathways involved in SCLC progression. Array comparative genomic hybridization was performed on DNA extracted from 46 formalin-fixed paraffin-embedded SCLC tissue specimens. Genomic profiling of tumor and sex-matched control DNA allowed the identification of 70 regions of copy number gain and 55 regions of copy number loss. Using molecular pathway analysis, we found a strong enrichment in these regions of copy number alterations for 11 genes associated with the focal adhesion pathway. We verified these findings at the genomic, gene expression and protein level. Focal Adhesion Kinase (FAK), one of the central genes represented in this pathway, was commonly expressed in SCLC tumors and constitutively phosphorylated in SCLC cell lines. Those were poorly adherent to most substrates but not to laminin-322. Inhibition of FAK phosphorylation at Tyr397 by a small-molecule inhibitor, PF-573,228, induced a dose-dependent decrease of adhesion and an increase of spreading in SCLC cell lines on laminin-322. Cells that tended to spread also showed a decrease in focal adhesions, as demonstrated by a decreased vinculin expression. These results support the concept that pathway analysis of genes in regions of copy number alterations may uncover molecular mechanisms of disease progression and demonstrate a new role of FAK and associated adhesion pathways in SCLC. Further investigations of FAK at the functional level may lead to a better understanding of SCLC progression and may have therapeutic implications. PMID:20802517

  5. Genome-wide copy number profiling of single cells in S-phase reveals DNA-replication domains

    PubMed Central

    Van der Aa, Niels; Cheng, Jiqiu; Mateiu, Ligia; Esteki, Masoud Zamani; Kumar, Parveen; Dimitriadou, Eftychia; Vanneste, Evelyne; Moreau, Yves; Vermeesch, Joris Robert; Voet, Thierry

    2013-01-01

    Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell’s copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes. PMID:23295674

  6. Genome-wide copy number profiling of single cells in S-phase reveals DNA-replication domains.

    PubMed

    Van der Aa, Niels; Cheng, Jiqiu; Mateiu, Ligia; Zamani Esteki, Masoud; Kumar, Parveen; Dimitriadou, Eftychia; Vanneste, Evelyne; Moreau, Yves; Vermeesch, Joris Robert; Voet, Thierry

    2013-04-01

    Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell's copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes.

  7. Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias.

    PubMed

    Malik, Afshan N; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

    2011-08-19

    Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a "dilution bias" when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Ultra-deep Illumina sequencing accurately identifies MHC class IIb alleles and provides evidence for copy number variation in the guppy (Poecilia reticulata).

    PubMed

    Lighten, Jackie; van Oosterhout, Cock; Paterson, Ian G; McMullan, Mark; Bentzen, Paul

    2014-07-01

    We address the bioinformatic issue of accurately separating amplified genes of the major histocompatibility complex (MHC) from artefacts generated during high-throughput sequencing workflows. We fit observed ultra-deep sequencing depths (hundreds to thousands of sequences per amplicon) of allelic variants to expectations from genetic models of copy number variation (CNV). We provide a simple, accurate and repeatable method for genotyping multigene families, evaluating our method via analyses of 209 b of MHC class IIb exon 2 in guppies (Poecilia reticulata). Genotype repeatability for resequenced individuals (N = 49) was high (100%) within the same sequencing run. However, repeatability dropped to 83.7% between independent runs, either because of lower mean amplicon sequencing depth in the initial run or random PCR effects. This highlights the importance of fully independent replicates. Significant improvements in genotyping accuracy were made by greatly reducing type I genotyping error (i.e. accepting an artefact as a true allele), which may occur when using low-depth allele validation thresholds used by previous methods. Only a small amount (4.9%) of type II error (i.e. rejecting a genuine allele as an artefact) was detected through fully independent sequencing runs. We observed 1-6 alleles per individual, and evidence of sharing of alleles across loci. Variation in the total number of MHC class II loci among individuals, both among and within populations was also observed, and some genotypes appeared to be partially hemizygous; total allelic dosage added up to an odd number of allelic copies. Collectively, observations provide evidence of MHC CNV and its complex basis in natural populations.

  9. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

    SciTech Connect

    Malik, Afshan N.; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

    2011-08-19

    Highlights: {yields} Mitochondrial dysfunction is central to many diseases of oxidative stress. {yields} 95% of the mitochondrial genome is duplicated in the nuclear genome. {yields} Dilution of untreated genomic DNA leads to dilution bias. {yields} Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as {beta}-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  10. Relationship between mitochondrial DNA Copy Number and SIRT1 Expression in Porcine Oocytes

    PubMed Central

    Sato, Daichi; Itami, Nobuhiko; Tasaki, Hidetaka; Takeo, Shun; Kuwayama, Takehito; Iwata, Hisataka

    2014-01-01

    The present study assessed the effect of resveratrol on the expression of SIRT1 and mitochondrial quality and quantity in porcine oocytes. Supplementing the maturation medium with 20 µM resveratrol increased the expression of SIRT1, and enhanced mitochondrial functions, as observed from the increased ATP content and mitochondrial membrane potential. Addition of resveratrol also improved the ability of oocytes to develop into the blastocyst stage following activation. The effects of resveratrol on mitochondrial number were examined by comparing the mitochondrial DNA copy number (Mt number) between group of oocytes collected from the same donor gilt ovaries. Supplementing the maturation medium with only resveratrol did not affect the Mt number in the oocytes. However, supplementing the maturation medium with 10 µM MG132, a proteasome inhibitor, significantly increased the amount of ubiquitinated proteins and Mt number by 12 and 14%, respectively. In addition, when resveratrol was added to the medium containing MG132, the Mt number increased significantly by 39%, this effect was diminished by the addition of the SIRT1 inhibitor EX527. Furthermore, supplementing the medium with MG132 and EX527 did not affect Mt number. The mean SIRT1 expression in 20 oocytes was significantly and positively correlated with the Mt number in oocytes collected from the same donor. This study suggests that the expression of SIRT1 is associated with the Mt number in oocytes. In addition, activation of SIRT1 by resveratrol enhances the biosynthesis and degradation of mitochondria in oocytes, thereby replenishing and improving mitochondrial function and the developmental ability of oocytes. PMID:24747689

  11. Disentangling sources of variation in SSU rDNA sequences from single cell analyses of ciliates: impact of copy number variation and experimental error.

    PubMed

    Wang, Chundi; Zhang, Tengteng; Wang, Yurui; Katz, Laura A; Gao, Feng; Song, Weibo

    2017-07-26

    Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation. Therefore, using a polymerase with high fidelity is essential for surveys of sequence variation. Another source of variation results from errors during amplification of SSU rDNA within the polyploidy somatic macronuclei of ciliates. To investigate further the impact of SSU rDNA copy number variation, we use a high-fidelity polymerase to examine the intra-individual SSU rDNA polymorphism in ciliates with varying levels of macronuclear amplification: Halteria grandinella, Blepharisma americanum and Strombidium stylifer We estimate the rDNA copy numbers of these three species by single-cell quantitative PCR. The results indicate that: (i) sequence variation of SSU rDNA within a single cell is authentic in ciliates, but the level of intra-individual SSU rDNA polymorphism varies greatly among species; (ii) rDNA copy numbers vary greatly among species, even those within the same class; (iii) the average rDNA copy number of Halteria grandinella is about 567 893 (s.d. = 165 481), which is the highest record of rDNA copy number in ciliates to date; and (iv) based on our data and the records from previous studies, it is not always true in ciliates that rDNA copy numbers are positively correlated with cell or genome size. © 2017 The Author(s).

  12. Determination of the molecular mass of bacterial genomic DNA and plasmid copy number by high-pressure liquid chromatography.

    PubMed Central

    Genthner, F J; Hook, L A; Strohl, W R

    1985-01-01

    Relatively rapid methods for the determination of relative genome molecular mass (Mr) and the estimation of plasmid copy number have been developed. These methods are based on the ability of the Bio-Rad high-pressure liquid chromatography hydroxylapatite column to separate and quantify single-stranded DNA, double-stranded DNA, and plasmid DNA. Genome Mr values were calculated from reassociation kinetics of single-stranded DNA as measured with the hydroxylapatite column. Bacteriophage T4 DNA was used to establish a C0t (moles of nucleotides times seconds per liter), or standard reassociation value. From this C0t value, C0t values for Escherichia coli B, Beggiatoa alba B18LD, and Streptomyces coelicolor were determined by comparative calculations. From those calculated C0t values, the Mr values of 1.96 X 10(9) for E. coli, 2.02 X 10(9) for B. alba, and 3.28 X 10(9) for S. coelicolor were estimated. Plasmid concentration was determined from cleared lysates by comparing the integrated area under the phosphate buffer-eluted plasmid peak to values obtained with known amounts of plasmid. The plasmid copy number was estimated by multiplying the ratio between the amounts of plasmid and chromosomal DNA by the ratio between the Mr values of the chromosome and the plasmid. A copy number of 29 was obtained from a culture of E. coli HB101 harboring pBR322 grown to a culture density of 1.6 X 10(9) CFU . ml-1. PMID:4083871

  13. International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

    PubMed

    Yoo, Hee-Bong; Park, Sang-Ryoul; Dong, Lianhua; Wang, Jing; Sui, Zhiwei; Pavšič, Jernej; Milavec, Mojca; Akgoz, Muslum; Mozioğlu, Erkan; Corbisier, Philippe; Janka, Mátrai; Cosme, Bruno; de V Cavalcante, Janaina J; Flatshart, Roberto Becht; Burke, Daniel; Forbes-Smith, Michael; McLaughlin, Jacob; Emslie, Kerry; Whale, Alexandra S; Huggett, Jim F; Parkes, Helen; Kline, Margaret C; Harenza, Jo Lynne; Vallone, Peter M

    2016-12-20

    Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

  14. Mitochondrial DNA copy number in peripheral blood cell and hypertension risk among mining workers: a case-control study in Chinese coal miners.

    PubMed

    Lei, L; Guo, J; Shi, X; Zhang, G; Kang, H; Sun, C; Huang, J; Wang, T

    2017-09-01

    Alteration of mitochondrial DNA (mtDNA) copy number, which reflects oxidant-induced cell damage, has been observed in a wide range of human diseases. However, whether it correlates with hypertension has not been elucidated. We aimed to explore the association between mtDNA copy number and the risk of hypertension in Chinese coal miners. A case-control study was performed with 378 hypertension patients and 325 healthy controls in a large coal mining group located in North China. Face-to-face interviews were conducted by trained staffs with necessary medical knowledge. The mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral blood. No significant differences in mtDNA copy number were observed between hypertension patients and healthy controls. However, in both case and control groups, the mtDNA copy number was statistically significantly lower in the elder population (≥45 years old) compared with the younger subjects (<45 years old; 7.17 vs 6.64, P=0.005 and 7.21 vs 6.84, P=0.036). A significantly higher mtDNA copy number could be found in hypertension patients consuming alcohol regularly compared with no alcohol consumption patients (7.09 vs 6.69); mtDNA copy number was also positively correlated with age and alcohol consumption. Hypertension was found significantly correlated with factors such as age, work duration, monthly family income and drinking status. Our results suggest that the mtDNA copy number is not associated with hypertension in coal miners.

  15. High Mitochondrial DNA Copy Number Is a Protective Factor From Vision Loss in Heteroplasmic Leber's Hereditary Optic Neuropathy (LHON).

    PubMed

    Bianco, Angelica; Bisceglia, Luigi; Russo, Luciana; Palese, Luigi L; D'Agruma, Leonardo; Emperador, Sonia; Montoya, Julio; Guerriero, Silvana; Petruzzella, Vittoria

    2017-04-01

    Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease that typically causes bilateral blindness in young men. It is characterized by as yet undisclosed genetic and environmental factors affecting the incomplete penetrance. We identified 27 LHON subjects who possess heteroplasmic primary LHON mutations. Mitochondrial DNA (mtDNA) copy number was evaluated. The presence of centrocecal scotoma, an edematous, hyperemic optic nerve head, and vascular tortuosity, as well as telangiectasia was recognized in affected subjects. We found higher cellular mtDNA content in peripheral blood cells of unaffected heteroplasmic mutation carriers with respect to the affected. The increase of cellular mtDNA content prevents complete loss of vision despite the presence of a heteroplasmic state of LHON primary mutation, suggesting that it is a key factor responsible for penetrance of LHON.

  16. Ribosomal DNA copy number amplification and loss in human cancers is linked to tumor genetic context, nucleolus activity, and proliferation

    PubMed Central

    2017-01-01

    Ribosomal RNAs (rRNAs) are transcribed from two multicopy DNA arrays: the 5S ribosomal DNA (rDNA) array residing in a single human autosome and the 45S rDNA array residing in five human autosomes. The arrays are among the most variable segments of the genome, exhibit concerted copy number variation (cCNV), encode essential components of the ribosome, and modulate global gene expression. Here we combined whole genome data from >700 tumors and paired normal tissues to provide a portrait of rDNA variation in human tissues and cancers of diverse mutational signatures, including stomach and lung adenocarcinomas, ovarian cancers, and others of the TCGA panel. We show that cancers undergo coupled 5S rDNA array expansion and 45S rDNA loss that is accompanied by increased estimates of proliferation rate and nucleolar activity. These somatic changes in rDNA CN occur in a background of over 10-fold naturally occurring rDNA CN variation across individuals and cCNV of 5S-45S arrays in some but not all tissues. Analysis of genetic context revealed associations between cancer rDNA CN amplification or loss and the presence of specific somatic alterations, including somatic SNPs and copy number gain/losses in protein coding genes across the cancer genome. For instance, somatic inactivation of the tumor suppressor gene TP53 emerged with a strong association with coupled 5S expansion / 45S loss in several cancers. Our results uncover frequent and contrasting changes in the 5S and 45S rDNA along rapidly proliferating cell lineages with high nucleolar activity. We suggest that 5S rDNA amplification facilitates increased proliferation, nucleolar activity, and ribosomal synthesis in cancer, whereas 45S rDNA loss emerges as a byproduct of transcription-replication conflict in rapidly replicating tumor cells. The observations raise the prospects of using the rDNA arrays as re-emerging targets for the design of novel strategies in cancer therapy. PMID:28880866

  17. RUBIC identifies driver genes by detecting recurrent DNA copy number breaks

    PubMed Central

    van Dyk, Ewald; Hoogstraat, Marlous; ten Hoeve, Jelle; Reinders, Marcel J. T.; Wessels, Lodewyk F. A.

    2016-01-01

    The frequent recurrence of copy number aberrations across tumour samples is a reliable hallmark of certain cancer driver genes. However, state-of-the-art algorithms for detecting recurrent aberrations fail to detect several known drivers. In this study, we propose RUBIC, an approach that detects recurrent copy number breaks, rather than recurrently amplified or deleted regions. This change of perspective allows for a simplified approach as recursive peak splitting procedures and repeated re-estimation of the background model are avoided. Furthermore, we control the false discovery rate on the level of called regions, rather than at the probe level, as in competing algorithms. We benchmark RUBIC against GISTIC2 (a state-of-the-art approach) and RAIG (a recently proposed approach) on simulated copy number data and on three SNP6 and NGS copy number data sets from TCGA. We show that RUBIC calls more focal recurrent regions and identifies a much larger fraction of known cancer genes. PMID:27396759

  18. CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing

    PubMed Central

    Shain, A. Hunter; Botton, Thomas; Bastian, Boris C.

    2016-01-01

    Germline copy number variants (CNVs) and somatic copy number alterations (SCNAs) are of significant importance in syndromic conditions and cancer. Massively parallel sequencing is increasingly used to infer copy number information from variations in the read depth in sequencing data. However, this approach has limitations in the case of targeted re-sequencing, which leaves gaps in coverage between the regions chosen for enrichment and introduces biases related to the efficiency of target capture and library preparation. We present a method for copy number detection, implemented in the software package CNVkit, that uses both the targeted reads and the nonspecifically captured off-target reads to infer copy number evenly across the genome. This combination achieves both exon-level resolution in targeted regions and sufficient resolution in the larger intronic and intergenic regions to identify copy number changes. In particular, we successfully inferred copy number at equivalent to 100-kilobase resolution genome-wide from a platform targeting as few as 293 genes. After normalizing read counts to a pooled reference, we evaluated and corrected for three sources of bias that explain most of the extraneous variability in the sequencing read depth: GC content, target footprint size and spacing, and repetitive sequences. We compared the performance of CNVkit to copy number changes identified by array comparative genomic hybridization. We packaged the components of CNVkit so that it is straightforward to use and provides visualizations, detailed reporting of significant features, and export options for integration into existing analysis pipelines. CNVkit is freely available from https://github.com/etal/cnvkit. PMID:27100738

  19. CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing.

    PubMed

    Talevich, Eric; Shain, A Hunter; Botton, Thomas; Bastian, Boris C

    2016-04-01

    Germline copy number variants (CNVs) and somatic copy number alterations (SCNAs) are of significant importance in syndromic conditions and cancer. Massively parallel sequencing is increasingly used to infer copy number information from variations in the read depth in sequencing data. However, this approach has limitations in the case of targeted re-sequencing, which leaves gaps in coverage between the regions chosen for enrichment and introduces biases related to the efficiency of target capture and library preparation. We present a method for copy number detection, implemented in the software package CNVkit, that uses both the targeted reads and the nonspecifically captured off-target reads to infer copy number evenly across the genome. This combination achieves both exon-level resolution in targeted regions and sufficient resolution in the larger intronic and intergenic regions to identify copy number changes. In particular, we successfully inferred copy number at equivalent to 100-kilobase resolution genome-wide from a platform targeting as few as 293 genes. After normalizing read counts to a pooled reference, we evaluated and corrected for three sources of bias that explain most of the extraneous variability in the sequencing read depth: GC content, target footprint size and spacing, and repetitive sequences. We compared the performance of CNVkit to copy number changes identified by array comparative genomic hybridization. We packaged the components of CNVkit so that it is straightforward to use and provides visualizations, detailed reporting of significant features, and export options for integration into existing analysis pipelines. CNVkit is freely available from https://github.com/etal/cnvkit.

  20. Specific functions of the Rep and Rep' proteins of porcine circovirus during copy-release and rolling-circle DNA replication

    USDA-ARS?s Scientific Manuscript database

    The roles of two porcine circovirus replication initiator proteins, Rep and Rep', in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replica...

  1. Leukocyte mitochondrial DNA copy number, anthropometric indices, and weight change in US women

    PubMed Central

    Meng, Shasha; Wu, Shaowei; Liang, Liming; Liang, Geyu; Giovannucci, Edward; Vivo, Immaculata De; Nan, Hongmei

    2016-01-01

    Objectives To examine the association between leukocyte mitochondrial DNA copy number (mtCN) and different anthropometric indices as well as weight changes; and to compare mtCN and telomere length with respect to their associations with BMI and age. Design Population based cohort study. Setting Nurses' Health Study, an ongoing prospective cohort study of 121,700 nurses enrolled in 1976; in 1989-1990 a subset of 32,826 women provided blood samples. Participants 1,700 disease-free US women from case-control studies nested within the Nurses' Health Study with mtCN and telomere length measured who also have anthropometric measurements. Main outcome measure Relative mtCN and telomere lengths in peripheral blood leukocytes measured by quantitative real time polymerase chain reaction and various anthropometric measurements data from initial questionnaire. Results Leukocyte mtCN was inversely associated with current weight (LS means Q1-Q4: 0.07, 0.04, 0.03, −0.17; P trend =0.002), waist size (LS means Q1-Q4: 0.06, 0.05, −0.04, −0.06; P trend = 0.04), BMI (LS means normal light, normal heavy, overweight, pre-obese, obese: 0.11, −0.01, −0.04, 0.04, −0.25; P trend<0.0001), and waist-hip ratio (WHR) (LS means Q1-Q4: 0.06, 0.08, −0.04, −0.06; P trend = 0.03). A one-unit decrease in mtCN z score was equivalent to approximately 3.5 pounds of weight gain for an adult of 5′10″. In addition, weight gain was bi-directionally and inversely associated with mtCN. Moreover, mtCN was strongly positively correlated with telomere length (LS means Q1-Q4: −0.02, 0.09, 0.11, 0.33; P trend <0.0001). MtCN was inversely associated with BMI even after adjusting for telomere length (P trend =0.003), while telomere length was not associated with BMI. On the other hand, telomere length was inversely associated with age after adjusting for mtCN (P trend =0.04), while mtCN was not associated with age. Conclusions Our results provide compelling evidence for a potential bi

  2. Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif

    PubMed Central

    Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

    2013-01-01

    DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

  3. Coordinating DNA replication to produce one copy of the genome requires genes that act in ubiquitin metabolism.

    PubMed Central

    Singer, J D; Manning, B M; Formosa, T

    1996-01-01

    We have developed a genetic screen of the yeast Saccharomyces cerevisiae to identify genes that act to coordinate DNA replication so that each part of the genome is copied exactly once per cell cycle. A mutant was recovered in this screen that accumulates aberrantly high DNA contents but does not complete a second round of synthesis. The mutation principally responsible for this phenotype is in the DOA4 gene, which encodes a ubiquitin hydrolase, one of several yeast genes that encode enzymes that can remove the signalling polypeptide ubiquitin hydrolase, one of several yeast genes that encode enzymes that can remove the signaling polypeptide ubiquitin from its covalently linked conjugated forms. DOA4 is nonessential, and deleting this gene causes uncoordinated replication. Overreplication does not occur in cells with limiting amounts of Cdc7 protein kinase, suggesting that entry into S phase is required for this phenotype. The DNA formed in doa4 mutants is not highly unusual in the sense that mitotic recombination rates are normal, implying that a high level of repair is not induced. The temperature sensitivity of doa4 mutations is partially suppressed by extra copies of the polyubiquitin gene UB14, but overreplication still occurs in the presence of this suppressor. Mutations in DOA4 cause loss of the free ubiquitin pool in cells under heat stress conditions, and extra copies of UB14 restore this pool without restoring coordination of replication. We conclude that a ubiquitin-mediated signaling event directly involving the ubiquitin hydrolase encoded by DOA4 is needed in S. cerevisiae to prevent uncoordinated DNA replication. PMID:8657109

  4. High-resolution analysis of DNA copy number alterations in patients with isolated sporadic keratoconus

    PubMed Central

    Hellani, Ali M.; Al Mansouri, Sameer M.; Kalantan, Hatem; Al-Muammar, Abdulrahman M.

    2011-01-01

    Purpose To determine whether patients with sporadic, non-familial keratoconus and no pathogenic mutations in the visual system homeobox 1 (VSX1) gene have evidence of chromosomal copy number alterations. Methods Twenty Saudi Arabian patients with isolated keratoconus, no family history of the disease and no mutations in VSX1 were recruited. Additionally, 10 ethnically-matched healthy controls were also recruited for this study. We screened patients for chromosomal copy number aberrations using the Agilent Human Genome CGH 244A Oligo Microarray Chip. Results None of the keratoconus patients screened had evidence of chromosomal copy number alterations when compared to normal ethnically matched controls. Conclusions Chromosomal deletions and/or duplications were not detected in any of the patients tested here. Other chromosomal imbalances such as translocations, inversions, and some ploidies cannot be detected by current array CGH technology and other nuclear genetic or epigenetic factors cannot be excluded as a possible contributing factor to keratoconus pathogenesis. PMID:21528002

  5. High-resolution analysis of DNA copy number alterations in patients with isolated sporadic keratoconus.

    PubMed

    Abu-Amero, Khaled K; Hellani, Ali M; Al Mansouri, Sameer M; Kalantan, Hatem; Al-Muammar, Abdulrahman M

    2011-03-30

    To determine whether patients with sporadic, non-familial keratoconus and no pathogenic mutations in the visual system homeobox 1 (VSX1) gene have evidence of chromosomal copy number alterations. Twenty Saudi Arabian patients with isolated keratoconus, no family history of the disease and no mutations in VSX1 were recruited. Additionally, 10 ethnically-matched healthy controls were also recruited for this study. We screened patients for chromosomal copy number aberrations using the Agilent Human Genome CGH 244A Oligo Microarray Chip. None of the keratoconus patients screened had evidence of chromosomal copy number alterations when compared to normal ethnically matched controls. Chromosomal deletions and/or duplications were not detected in any of the patients tested here. Other chromosomal imbalances such as translocations, inversions, and some ploidies cannot be detected by current array CGH technology and other nuclear genetic or epigenetic factors cannot be excluded as a possible contributing factor to keratoconus pathogenesis.

  6. Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors

    PubMed Central

    2013-01-01

    Background The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer. Results We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein. Conclusions Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer. PMID:24257477

  7. High copy and stable expression of the xylanase XynHB in Saccharomyces cerevisiae by rDNA-mediated integration.

    PubMed

    Fang, Cheng; Wang, Qinhong; Selvaraj, Jonathan Nimal; Zhou, Yuling; Ma, Lixin; Zhang, Guimin; Ma, Yanhe

    2017-08-18

    Xylanase is a widely-used additive in baking industry for enhancing dough and bread quality. Several xylanases used in baking industry were expressed in different systems, but their expression in antibiotic free vector system is highly essential and safe. In the present study, an alternative rDNA-mediated technology was developed to increase the copy number of target gene by integrating it into Saccharomyces cerevisiae genome. A xylanase-encoding gene xynHB from Bacillus sp. was cloned into pHBM367H and integrated into S. cerevisiae genome through rDNA-mediated recombination. Exogenous XynHB expressed by recombinant S. cerevisiae strain A13 exhibited higher degradation activity towards xylan than other transformants. The real-time PCR analysis on A13 genome revealed the presence of 13.64 copies of xynHB gene. Though no antibiotics have been used, the genetic stability and the xylanase activity of xynHB remained stable up to 1,011 generations of cultivation. S. cerevisiae strain A13 expressing xylanase reduced the required kneading time and increased the height and diameter of the dough size, which would be safe and effective in baking industry as no antibiotics-resistance risk. The new effective rDNA-mediated technology without using antibiotics here provides a way to clone other food related industrial enzymes for applications.

  8. Somatic Mutation Allelic Ratio Test Using ddPCR (SMART-ddPCR): An Accurate Method for Assessment of Preferential Allelic Imbalance in Tumor DNA

    PubMed Central

    de Smith, Adam J.; Walsh, Kyle M.; Hansen, Helen M.; Endicott, Alyson A.; Wiencke, John K.; Metayer, Catherine; Wiemels, Joseph L.

    2015-01-01

    The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19–142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a

  9. Rare DNA copy number variants in cardiovascular malformations with extracardiac abnormalities

    USDA-ARS?s Scientific Manuscript database

    Clinically significant cardiovascular malformations (CVMs) occur in 5-8 per 1000 live births. Recurrent copy number variations (CNVs) are among the known causes of syndromic CVMs, accounting for an important fraction of cases. We hypothesized that many additional rare CNVs also cause CVMs and can be...

  10. Induction of direct repeat recombination by psoralen-DNA adducts in Saccharomyces cerevisiae: defects in DNA repair increase gene copy number variation.

    PubMed

    Saffran, Wilma A; Ahmed, Anam; Binyaminov, Olga; Gonzalez, Cynthia; Gupta, Amita; Fajardo, Manuel A; Kishun, Devindra; Nandram, Ashana; Reyes, Kenneth; Scalercio, Karina; Senior, Charles W

    2014-09-01

    Psoralen photoreaction produces covalent monoadducts and interstrand crosslinks in DNA. The interstrand DNA crosslinks are complex double strand lesions that require the involvement of multiple pathways for repair. Homologous recombination, which can carry out error-free repair, is a major pathway for crosslink repair; however, some recombination pathways can also produce DNA rearrangements. Psoralen photoreaction-induced recombination in yeast was measured using direct repeat substrates that can detect gene conversions, a form of conservative recombination, as well as deletions and triplications, which generate gene copy number changes. In repair-proficient cells the major products of recombination were gene conversions, along with substantial fractions of deletions. Deficiencies in DNA repair pathways increased non-conservative recombination products. Homologous recombination-deficient rad51, rad54, and rad57 strains had low levels of crosslink-induced recombination, and most products were deletions produced by single strand annealing. Nucleotide excision repair-deficient rad1 and rad2 yeast had increased levels of triplications, and rad1 cells had lower crosslink-induced recombination. Deficiencies in post-replication repair increased crosslink-induced recombination and gene copy number changes. Loss of REV3 function, in the error-prone branch, and of RAD5 and UBC13, in the error-free branch, produced moderate increases in deletions and triplications; rad18 cells, deficient in both post-replication repair sub-pathways, exhibited hyperrecombination, with primarily non-conservative products. Proper functioning of all the DNA repair pathways tested was required to maintain genomic stability and avoid gene copy number variation in response to interstrand crosslinks. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number, oxidative phosphorylation and ROS detoxification pathways

    PubMed Central

    Giordano, L; Deceglie, S; d'Adamo, P; Valentino, M L; La Morgia, C; Fracasso, F; Roberti, M; Cappellari, M; Petrosillo, G; Ciaravolo, S; Parente, D; Giordano, C; Maresca, A; Iommarini, L; Del Dotto, V; Ghelli, A M; Salomao, S R; Berezovsky, A; Belfort, R; Sadun, A A; Carelli, V; Loguercio Polosa, P; Cantatore, P

    2015-01-01

    Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that

  12. Effective use of the TSPY gene-specific copy number in determining fetal DNA in the maternal blood of cynomolgus monkeys.

    PubMed

    Yasmin, Lubna; Takano, Jun-Ichiro; Sankai, Tadashi

    2016-08-01

    Since the available concentration of single-copy fetal genes in maternal blood DNA is sometimes lower than detection limits by PCR methods, the development of specific and quantitative PCR detection methods for fetal DNA in maternal blood is anticipated, which may broaden the methods that can be used to monitor pregnancy. We used the TaqMan qPCR amplification for DYS14 multi-copy sequence and the SRY gene in maternal blood plasma (cell-free DNA) and fractional precipitated blood cells (cellular DNA) from individual cynomolgus monkeys at 22 weeks of pregnancy. The availability of cell-free fetal DNA was higher in maternal blood plasma than that of cellular DNA from fractional precipitated blood cells. There was a significantly higher (P < 0.001) mean copy number of fetal male DYS14 from maternal plasma (4.4 × 10(4) copies/mL) than that of detected fetal cellular DNA from fractional blood cell pellets. The sensitivity of the DYS14 PCR assay was found to be higher than that of the SRY assay for the detection of fetal DNA when its presence was at a minimum. The DYS14 assay is an improved method for quantifying male fetal DNA in circulating maternal blood in the primate model.

  13. SNP-microarrays can accurately identify the presence of an individual in complex forensic DNA mixtures.

    PubMed

    Voskoboinik, Lev; Ayers, Sheri B; LeFebvre, Aaron K; Darvasi, Ariel

    2015-05-01

    Common forensic and mass disaster scenarios present DNA evidence that comprises a mixture of several contributors. Identifying the presence of an individual in such mixtures has proven difficult. In the current study, we evaluate the practical usefulness of currently available "off-the-shelf" SNP microarrays for such purposes. We found that a set of 3000 SNPs specifically selected for this purpose can accurately identify the presence of an individual in complex DNA mixtures of various compositions. For example, individuals contributing as little as 5% to a complex DNA mixture can be robustly identified even if the starting DNA amount was as little as 5.0ng and had undergone whole-genome amplification (WGA) prior to SNP analysis. The work presented in this study represents proof-of-principle that our previously proposed approach, can work with real "forensic-type" samples. Furthermore, in the absence of a low-density focused forensic SNP microarray, the use of standard, currently available high-density SNP microarrays can be similarly used and even increase statistical power due to the larger amount of available information.

  14. Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner

    PubMed Central

    Lu, David V.; Brown, Randall H.; Arumugam, Manimozhiyan; Brent, Michael R.

    2009-01-01

    Motivation: The most accurate way to determine the intron–exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary determinant of alignment accuracy, while heuristics that prevent consideration of certain alignments are a primary determinant of runtime and memory usage. Both accuracy and speed are important considerations in choosing an alignment algorithm, but scoring systems have received much less attention than heuristics. Results: We present Pairagon, a pair hidden Markov model based cDNA-to-genome alignment program, as the most accurate aligner for sequences with high- and low-identity levels. We conducted a series of experiments testing alignment accuracy with varying sequence identity. We first created ‘perfect’ simulated cDNA sequences by splicing the sequences of exons in the reference genome sequences of fly and human. The complete reference genome sequences were then mutated to various degrees using a realistic mutation simulator and the perfect cDNAs were aligned to them using Pairagon and 12 other aligners. To validate these results with natural sequences, we performed cross-species alignment using orthologous transcripts from human, mouse and rat. We found that aligner accuracy is heavily dependent on sequence identity. For sequences with 100% identity, Pairagon achieved accuracy levels of >99.6%, with one quarter of the errors of any other aligner. Furthermore, for human/mouse alignments, which are only 85% identical, Pairagon achieved 87% accuracy, higher than any other aligner. Availability: Pairagon source and executables are freely available at http://mblab.wustl.edu/software/pairagon/ Contact: davidlu@wustl.edu; brent@cse.wustl.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19414532

  15. Integrated DNA methylation and copy-number profiling identify three clinically and biologically relevant groups of anaplastic glioma.

    PubMed

    Wiestler, Benedikt; Capper, David; Sill, Martin; Jones, David T W; Hovestadt, Volker; Sturm, Dominik; Koelsche, Christian; Bertoni, Anna; Schweizer, Leonille; Korshunov, Andrey; Weiß, Elisa K; Schliesser, Maximilian G; Radbruch, Alexander; Herold-Mende, Christel; Roth, Patrick; Unterberg, Andreas; Hartmann, Christian; Pietsch, Torsten; Reifenberger, Guido; Lichter, Peter; Radlwimmer, Bernhard; Platten, Michael; Pfister, Stefan M; von Deimling, Andreas; Weller, Michael; Wick, Wolfgang

    2014-10-01

    The outcome of patients with anaplastic gliomas varies considerably. Whether a molecular classification of anaplastic gliomas based on large-scale genomic or epigenomic analyses is superior to histopathology for reflecting distinct biological groups, predicting outcomes and guiding therapy decisions has yet to be determined. Epigenome-wide DNA methylation analysis, using a platform which also allows the detection of copy-number aberrations, was performed in a cohort of 228 patients with anaplastic gliomas (astrocytomas, oligoastrocytomas, and oligodendrogliomas), including 115 patients of the NOA-04 trial. We further compared these tumors with a group of 55 glioblastomas. Unsupervised clustering of DNA methylation patterns revealed two main groups correlated with IDH status: CpG island methylator phenotype (CIMP) positive (77.5 %) or negative (22.5 %). CIMP(pos) (IDH mutant) tumors showed a further separation based on copy-number status of chromosome arms 1p and 19q. CIMP(neg) (IDH wild type) tumors showed hallmark copy-number alterations of glioblastomas, and clustered together with CIMP(neg) glioblastomas without forming separate groups based on WHO grade. Notably, there was no molecular evidence for a distinct biological entity representing anaplastic oligoastrocytoma. Tumor classification based on CIMP and 1p/19q status was significantly associated with survival, allowing a better prediction of outcome than the current histopathological classification: patients with CIMP(pos) tumors with 1p/19q codeletion (CIMP-codel) had the best prognosis, followed by patients with CIMP(pos) tumors but intact 1p/19q status (CIMP-non-codel). Patients with CIMP(neg) anaplastic gliomas (GBM-like) had the worst prognosis. Collectively, our data suggest that anaplastic gliomas can be grouped by IDH and 1p/19q status into three molecular groups that show clear links to underlying biology and a significant association with clinical outcome in a prospective trial cohort.

  16. Relation of Mitochondrial DNA Copy Number in Peripheral Blood to Postoperative Atrial Fibrillation After Isolated Off-Pump Coronary Artery Bypass Grafting.

    PubMed

    Zhang, Jian; Xu, Shu; Xu, Yinli; Liu, Yu; Li, Zhi; Zhang, Yuji; Jin, Yan; Xue, Xiaodong; Wang, Huishan

    2017-02-01

    Oxidative stress has been considered to be an important factor contributing to postoperative atrial fibrillation (PoAF). Mitochondrial DNA (mtDNA) copy number in peripheral blood has been found to be associated with a patient's oxidative stress. Therefore, we sought to determine whether there was association between mtDNA copy number and the onset of atrial fibrillation. mtDNA copy numbers were measured using the quantitative real-time polymerase chain reaction in peripheral blood from 485 consecutive patients with sinus rhythm undergoing coronary artery bypass grafting. The blood was collected before surgery. In the cohort, the incidence of PoAF was 20.8% (101/485). The mean mtDNA copy number was significantly higher in patents with PoAF than in those with sinus rhythm (36.43 vs 16.63, p <0.001). The receiver operating characteristic analysis proved that the mtDNA copy number could predict PoAF with good sensitivity and specificity (area under the curve = 0.814, cutoff = 20.91, sensitivity = 70.3%, specificity = 80.2%, p <0.001). On multivariate logistic and Cox regression analysis, mtDNA copy number was shown to be a significant independent risk factor for PoAF (odds ratio = 10.01, p <0.001 and hazard ratio = 7.011, p = 0.004). There was a strong positive correlation between mtDNA copy number and malondialdehyde in patients with PoAF (r = 0.449, p = 0.01). In conclusion, we showed that elevated mtDNA copy number in peripheral blood is associated with PoAF. Further investigation is needed to validate mtDNA copy number as a predictive biomarker for PoAF and to explore its potential role in arrhythmogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. A scale-space method for detecting recurrent DNA copy number changes with analytical false discovery rate control

    PubMed Central

    van Dyk, Ewald; Reinders, Marcel J.T.; Wessels, Lodewyk F.A.

    2013-01-01

    Tumor formation is partially driven by DNA copy number changes, which are typically measured using array comparative genomic hybridization, SNP arrays and DNA sequencing platforms. Many techniques are available for detecting recurring aberrations across multiple tumor samples, including CMAR, STAC, GISTIC and KC-SMART. GISTIC is widely used and detects both broad and focal (potentially overlapping) recurring events. However, GISTIC performs false discovery rate control on probes instead of events. Here we propose Analytical Multi-scale Identification of Recurrent Events, a multi-scale Gaussian smoothing approach, for the detection of both broad and focal (potentially overlapping) recurring copy number alterations. Importantly, false discovery rate control is performed analytically (no need for permutations) on events rather than probes. The method does not require segmentation or calling on the input dataset and therefore reduces the potential loss of information due to discretization. An important characteristic of the approach is that the error rate is controlled across all scales and that the algorithm outputs a single profile of significant events selected from the appropriate scales. We perform extensive simulations and showcase its utility on a glioblastoma SNP array dataset. Importantly, ADMIRE detects focal events that are missed by GISTIC, including two events involving known glioma tumor-suppressor genes: CDKN2C and NF1. PMID:23476020

  18. A scale-space method for detecting recurrent DNA copy number changes with analytical false discovery rate control.

    PubMed

    van Dyk, Ewald; Reinders, Marcel J T; Wessels, Lodewyk F A

    2013-05-01

    Tumor formation is partially driven by DNA copy number changes, which are typically measured using array comparative genomic hybridization, SNP arrays and DNA sequencing platforms. Many techniques are available for detecting recurring aberrations across multiple tumor samples, including CMAR, STAC, GISTIC and KC-SMART. GISTIC is widely used and detects both broad and focal (potentially overlapping) recurring events. However, GISTIC performs false discovery rate control on probes instead of events. Here we propose Analytical Multi-scale Identification of Recurrent Events, a multi-scale Gaussian smoothing approach, for the detection of both broad and focal (potentially overlapping) recurring copy number alterations. Importantly, false discovery rate control is performed analytically (no need for permutations) on events rather than probes. The method does not require segmentation or calling on the input dataset and therefore reduces the potential loss of information due to discretization. An important characteristic of the approach is that the error rate is controlled across all scales and that the algorithm outputs a single profile of significant events selected from the appropriate scales. We perform extensive simulations and showcase its utility on a glioblastoma SNP array dataset. Importantly, ADMIRE detects focal events that are missed by GISTIC, including two events involving known glioma tumor-suppressor genes: CDKN2C and NF1.

  19. Apparent Polyploidization after Gamma Irradiation: Pitfalls in the Use of Quantitative Polymerase Chain Reaction (qPCR) for the Estimation of Mitochondrial and Nuclear DNA Gene Copy Numbers

    PubMed Central

    Kam, Winnie W. Y.; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

    2013-01-01

    Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization. PMID:23722662

  20. Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology.

    PubMed

    Kirkizlar, Eser; Zimmermann, Bernhard; Constantin, Tudor; Swenerton, Ryan; Hoang, Bin; Wayham, Nicholas; Babiarz, Joshua E; Demko, Zachary; Pelham, Robert J; Kareht, Stephanie; Simon, Alexander L; Jinnett, Kristine N; Rabinowitz, Matthew; Sigurjonsson, Styrmir; Hill, Matthew

    2015-10-01

    We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Genome-wide DNA Copy-number Analysis in ACTS-CC Trial of Adjuvant Chemotherapy for Stage III Colonic Cancer.

    PubMed

    Ishikawa, Toshiaki; Uetake, Hiroyuki; Murotani, Kenta; Kobunai, Takashi; Ishiguro, Megumi; Matsui, Shigeyuki; Sugihara, Kenichi

    2016-03-01

    The adjuvant chemotherapy trial of TS-1 for colon cancer phase III trial was designed to validate the non-inferiority of the oral fluoropyrimidine S-1 to uracil and tegafur/leucovorin as adjuvant chemotherapy for stage III colonic cancer. As a prospective biomarker study of this trial, DNA copy number was studied using formalin-fixed, paraffin-embedded specimens. FFPE blocks were obtained from 795 patients of the 1,535 patients enrolled in the study. The quality of extracted DNA was assessed using arbitrarily primed polymerase chain reaction and microfluidic analysis. Genomic copy-number alterations in cancer were analyzed by high-density single-nucleotide polymorphism arrays. Copy-number changes in Japanese patients with colonic cancer were compared with those in Western countries using data from a previously reported meta-analysis. We then compared genome-wide segment copy number and clinicopathological features of colorectal cancer. Genome-wide copy number was analyzed in 161 samples and DNA copy-number alteration profiles showed frequent DNA copy-number gains at chromosome 7, 8q and 13, and losses at 4, 5q, 8p, 17p and 18q. The weighted kappa statistic from comparing copy-number alteration status with data from Western countries was 0.828 (95% confidence interval=0.786 -0.871). DNA copy-number alterations of 8,684 segments were compared with clinicopathological features in 161 patients. Location of the tumor correlated with genomic segments of chromosome 4, 5, 7, 8, 13, 14, 18 and 20. Differentiation of the tumor correlated with segments in chromosome 4, 6, 8, 11, 13, 14,15, 16, 17 and 20. Somatic copy-number alteration profiles of stage III colonic cancer in the Japanese ACTS-CC trial closely agreed with the results of previous Western studies. Location and differentiation of the tumor correlated with DNA copy-number alterations. Our findings will facilitate understanding the characteristics of colonic cancer. Further investigation may contribute to the

  2. Detection of the free living amoeba Naegleria fowleri by using conventional and real-time PCR based on a single copy DNA sequence.

    PubMed

    Régoudis, Estelle; Pélandakis, Michel

    2016-02-01

    The amoeba-flagellate Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis (PAM). This thermophilic species occurs worldwide and tends to proliferate in warm aquatic environment. The PAM cases remain rare but this infection is mostly fatal. Here, we describe a single copy region which has been cloned and sequenced, and was used for both conventional and real-time PCR. Targeting a single-copy DNA sequence allows to directly quantify the N. fowleri cells. The real-time PCR results give a detection limit of 1 copy per reaction with high reproducibility without the need of a Taqman probe. This procedure is of interest as compared to other procedures which are mostly based on the detection of multi-copy DNA associated with a Taqman probe.

  3. Optical Imaging of Paramagnetic Bead-DNA Aggregation Inhibition Allows for Low Copy Number Detection of Infectious Pathogens

    PubMed Central

    DuVall, Jacquelyn A.; Borba, Juliane C.; Shafagati, Nazly; Luzader, Deborah; Shukla, Nishant; Li, Jingyi; Kehn-Hall, Kylene; Kendall, Melissa M.; Feldman, Sanford H.; Landers, James P.

    2015-01-01

    DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer ‘trigger’ DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use. PMID:26068926

  4. Optical Imaging of Paramagnetic Bead-DNA Aggregation Inhibition Allows for Low Copy Number Detection of Infectious Pathogens.

    PubMed

    DuVall, Jacquelyn A; Borba, Juliane C; Shafagati, Nazly; Luzader, Deborah; Shukla, Nishant; Li, Jingyi; Kehn-Hall, Kylene; Kendall, Melissa M; Feldman, Sanford H; Landers, James P

    2015-01-01

    DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer 'trigger' DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use.

  5. Proliferation of mitochondria in chronically stimulated rabbit skeletal muscle--transcription of mitochondrial genes and copy number of mitochondrial DNA.

    PubMed

    Schultz, J; Wiesner, R J

    2000-12-01

    Mitochondrial proliferation was studied in chronically stimulated rabbit skeletal muscle over a period of 50 days. After this time, subunits of COX had increased about fourfold. Corresponding mRNAs, encoded on mitochondrial DNA as well as on nuclear genes, were unchanged when related to total tissue RNA, however, they were elevated two- to fivefold when the massive increase of ribosomes per unit mass of muscle was taken into account. The same was true for the mRNA encoding mitochondrial transcription factor A. Surprisingly, tissue levels of mtTFA protein were reduced about twofold, together with mitochondrial DNA. In conclusion, mitochondria are able to maintain high rates of mitochondrial transcription even in the presence of reduced mtTFA protein and mtDNA levels. Therefore, stimulated mtTFA gene expression accompanies stimulated mitochondrial transcription, as in other models, but it is not sufficient for an increase of mtDNA copy number and other, yet unknown, factors have to be postulated.

  6. Measurement and relevance of neuroblastoma DNA copy number changes in the post-genome era.

    PubMed

    Mosse, Yael P; Greshock, Joel; Weber, Barbara L; Maris, John M

    2005-10-18

    The completion of the human genome sequence and the development of high throughput technology present exciting opportunities for the study of cancer cells. High-resolution analysis of chromosomal aberrations provides a global framework for understanding complex patterns in cancer cells, allowing us to ask hypothesis-driven questions. Genome-wide analysis of amplification and deletion of genomic regions is a critical step to resolving the mechanisms of neuroblastoma tumorigenesis. We used a high-resolution aCGH system that has over 4000 human BAC clones, resulting in an average coverage of 1Mb across the genome, to define whole genome copy number aberrations (CNAs) in a panel of human neuroblastoma-derived cell lines. By combining the aCGH data with meticulous regional validation studies, we showed that array CGH could reliably detect known aberrations including single copy gain or loss, that data correlate well with standard techniques used for the detection of these genetic changes, and that this technique can be used to identify novel regions of genomic imbalance.

  7. Decreased mitochondrial DNA copy number in the hippocampus and peripheral blood during opiate addiction is mediated by autophagy and can be salvaged by melatonin.

    PubMed

    Feng, Yue-Mei; Jia, Yun-Fang; Su, Ling-Yan; Wang, Dong; Lv, Li; Xu, Lin; Yao, Yong-Gang

    2013-09-01

    Drug addiction is a chronic brain disease that is a serious social problem and causes enormous financial burden. Because mitochondrial abnormalities have been associated with opiate addiction, we examined the effect of morphine on mtDNA levels in rat and mouse models of addiction and in cultured cells. We found that mtDNA copy number was significantly reduced in the hippocampus and peripheral blood of morphine-addicted rats and mice compared with control animals. Concordantly, decreased mtDNA copy number and elevated mtDNA damage were observed in the peripheral blood from opiate-addicted patients, indicating detrimental effects of drug abuse and stress. In cultured rat pheochromocytoma (PC12) cells and mouse neurons, morphine treatment caused many mitochondrial defects, including a reduction in mtDNA copy number that was mediated by autophagy. Knockdown of the Atg7 gene was able to counteract the loss of mtDNA copy number induced by morphine. The mitochondria-targeted antioxidant melatonin restored mtDNA content and neuronal outgrowth and prevented the increase in autophagy upon morphine treatment. In mice, coadministration of melatonin with morphine ameliorated morphine-induced behavioral sensitization, analgesic tolerance and mtDNA content reduction. During drug withdrawal in opiate-addicted patients and improvement of protracted abstinence syndrome, we observed an increase of serum melatonin level. Taken together, our study indicates that opioid addiction is associated with mtDNA copy number reduction and neurostructural remodeling. These effects appear to be mediated by autophagy and can be salvaged by melatonin.

  8. Tetramethylpyrazine blocks TFAM degradation and up-regulates mitochondrial DNA copy number by interacting with TFAM

    PubMed Central

    Lan, Linhua; Guo, Miaomiao; Ai, Yong; Chen, Fuhong; Zhang, Ya; Xia, Lei; Huang, Dawei; Niu, Lili; Zheng, Ying; Suzuki, Carolyn K.

    2017-01-01

    The natural small molecule compound: 2,3,5,6-tetramethylpyrazine (TMP), is a major component of the Chinese medicine Chuanxiong, which has wide clinical applications in dilating blood vessels, inhibiting platelet aggregation and treating thrombosis. Recent work suggests that TMP is also an antitumour agent. Despite its chemotherapeutic potential, the mechanism(s) underlying TMP action are unknown. Herein, we demonstrate that TMP binds to mitochondrial transcription factor A (TFAM) and blocks its degradation by the mitochondrial Lon protease. TFAM is a key regulator of mtDNA replication, transcription and transmission. Our previous work showed that when TFAM is not bound to DNA, it is rapidly degraded by the ATP-dependent Lon protease, which is essential for mitochondrial proteostasis. In cultured cells, TMP specifically blocks Lon-mediated degradation of TFAM, leading to TFAM accumulation and subsequent up-regulation of mtDNA content in cells with substantially low levels of mtDNA. In vitro protease assays show that TMP does not directly inhibit mitochondrial Lon, rather interacts with TFAM and blocks degradation. Pull-down assays show that biotinylated TMP interacts with TFAM. These findings suggest a novel mechanism whereby TMP stabilizes TFAM and confers resistance to Lon-mediated degradation, thereby promoting mtDNA up-regulation in cells with low mtDNA content. PMID:28465355

  9. Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus).

    PubMed

    Morin, P A; Chambers, K E; Boesch, C; Vigilant, L

    2001-07-01

    Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate microsatellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.

  10. The effect of leaving groups on binding and reactivity in enzyme-free copying of DNA and RNA

    PubMed Central

    Kervio, Eric; Sosson, Marilyne; Richert, Clemens

    2016-01-01

    The template-directed incorporation of nucleotides at the terminus of a growing primer is the basis of the transmission of genetic information. Nature uses polymerases-catalyzed reactions, but enzyme-free versions exist that employ nucleotides with organic leaving groups. The leaving group affects yields, but it was not clear whether inefficient extensions are due to poor binding, low reactivity toward the primer, or rapid hydrolysis. We have measured the binding of a total of 15 different activated nucleotides to DNA or RNA sequences. Further, we determined rate constants for the chemical step of primer extension involving methylimidazolides or oxyazabenzotriazolides of deoxynucleotides or ribonucleotides. Binding constants range from 10 to >500 mM and rate constants from 0.1 to 370 M−1 h−1. For aminoterminal primers, a fast covalent step and slow hydrolysis are the main factors leading to high yields. For monomers with weakly pairing bases, the leaving group can improve binding significantly. A detailed mechanistic picture emerges that explains why some enzyme-free primer extensions occur in high yield, while others remain recalcitrant to copying without enzymatic catalysis. A combination of tight binding and rapid extension, coupled with slow hydrolysis induces efficient enzyme-free copying. PMID:27235418

  11. DNA copy number profiling reveals extensive genomic loss in hereditary BRCA1 and BRCA2 ovarian carcinomas

    PubMed Central

    Kamieniak, M M; Muñoz-Repeto, I; Rico, D; Osorio, A; Urioste, M; García-Donas, J; Hernando, S; Robles-Díaz, L; Ramón y Cajal, T; Cazorla, A; Sáez, R; García-Bueno, J M; Domingo, S; Borrego, S; Palacios, J; van de Wiel, M A; Ylstra, B; Benítez, J; García, M J

    2013-01-01

    Background: Few studies have attempted to characterise genomic changes occurring in hereditary epithelial ovarian carcinomas (EOCs) and inconsistent results have been obtained. Given the relevance of DNA copy number alterations in ovarian oncogenesis and growing clinical implications of the BRCA-gene status, we aimed to characterise the genomic profiles of hereditary and sporadic ovarian tumours. Methods: High-resolution array Comparative Genomic Hybridisation profiling of 53 familial (21 BRCA1, 6 BRCA2 and 26 non-BRCA1/2) and 15 sporadic tumours in combination with supervised and unsupervised analysis was used to define common and/or specific copy number features. Results: Unsupervised hierarchical clustering did not stratify tumours according to their familial or sporadic condition or to their BRCA1/2 mutation status. Common recurrent changes, spanning genes potentially fundamental for ovarian carcinogenesis, regardless of BRCA mutations, and several candidate subtype-specific events were defined. Despite similarities, greater contribution of losses was revealed to be a hallmark of BRCA1 and BRCA2 tumours. Conclusion: Somatic alterations occurring in the development of familial EOCs do not differ substantially from the ones occurring in sporadic carcinomas. However, some specific features like extensive genomic loss observed in BRCA1/2 tumours may be of clinical relevance helping to identify BRCA-related patients likely to respond to PARP inhibitors. PMID:23558894

  12. An accurate DNA marker assay for stem rust resistance gene Sr2 in wheat.

    PubMed

    Mago, R; Simkova, H; Brown-Guedira, G; Dreisigacker, S; Breen, J; Jin, Y; Singh, R; Appels, R; Lagudah, E S; Ellis, J; Dolezel, J; Spielmeyer, W

    2011-03-01

    The stem rust resistance gene Sr2 has provided broad-spectrum protection against stem rust (Puccinia graminis Pers. f. sp. tritici) since its wide spread deployment in wheat from the 1940s. Because Sr2 confers partial resistance which is difficult to select under field conditions, a DNA marker is desirable that accurately predicts Sr2 in diverse wheat germplasm. Using DNA sequence derived from the vicinity of the Sr2 locus, we developed a cleaved amplified polymorphic sequence (CAPS) marker that is associated with the presence or absence of the gene in 115 of 122 (95%) diverse wheat lines. The marker genotype predicted the absence of the gene in 100% of lines which were considered to lack Sr2. Discrepancies were observed in lines that were predicted to carry Sr2 but failed to show the CAPS marker. Given the high level of accuracy observed, the marker provides breeders with a selection tool for one of the most important disease resistance genes of wheat.

  13. Polycyclic aromatic hydrocarbons exposure decreased sperm mitochondrial DNA copy number: A cross-sectional study (MARHCS) in Chongqing, China.

    PubMed

    Ling, Xi; Zhang, Guowei; Sun, Lei; Wang, Zhi; Zou, Peng; Gao, Jianfang; Peng, Kaige; Chen, Qing; Yang, Huan; Zhou, Niya; Cui, Zhihong; Zhou, Ziyuan; Liu, Jinyi; Cao, Jia; Ao, Lin

    2017-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants that have adverse effects on the male reproductive function. Many studies have confirmed that PAHs preferentially accumulate in mitochondria DNA relative to nuclear DNA and disrupt mitochondrial functions. However, it is rare whether exposure to PAHs is associated with mitochondrial damage and dysfunction in sperm. To evaluate the effects of PAHs on sperm mitochondria, we measured mitochondrial membrane potential (MMP), mitochondrial DNA copy number (mtDNAcn) and mtDNA integrity in 666 individuals from the Male Reproductive Health in Chongqing College Students (MARHCS) study. PAHs exposure was estimated by measuring eight urinary PAH metabolites (1-OHNap, 2-OHNap, 1-OHPhe, 2-OHPhe, 3-OHPhe, 4-OHPhe, 2-OHFlu and 1-OHPyr). The subjects were divided into low, median and high exposure groups using the tertile levels of urinary PAH metabolites. In univariate analyses, the results showed that increased levels of 2-OHPhe, 3-OHPhe, ∑Phe metabolites and 2-OHFlu were found to be associated with decreased sperm mtDNAcn. After adjusting for potential confounders, significantly negative associations of these metabolites remained (p = 0.039, 0.012, 0.01, 0.035, respectively). Each 1 μg/g creatinine increase in 2-OHPhe, 3-OHPhe, ∑Phe metabolites and 2-OHFlu was associated with a decrease in sperm mtDNAcn of 9.427%, 11.488%, 9.635% and 11.692%, respectively. There were no significant associations between urinary PAH metabolites and sperm MMP or mtDNA integrity. The results indicated that the low exposure levels of PAHs can cause abnormities in sperm mitochondria.

  14. The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA.

    PubMed

    Tamariz, Jeannie; Voynarovska, Kristina; Prinz, Mechthild; Caragine, Theresa

    2006-07-01

    Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the

  15. Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome

    PubMed Central

    Reeves, Analise Z; Lesser, Cammie F

    2017-01-01

    Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives. PMID:28203614

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

    PubMed Central

    Forero-Castro, Maribel; Robledo, Cristina; Benito, Rocío; Abáigar, María; África Martín, Ana; Arefi, Maryam; Fuster, José Luis; de las Heras, Natalia; Rodríguez, Juan N.; Quintero, Jonathan; Riesco, Susana; Hermosín, Lourdes; de la Fuente, Ignacio; Recio, Isabel; Ribera, Jordi; Labrador, Jorge; Alonso, José M.; Olivier, Carmen; Sierra, Magdalena; Megido, Marta; Corchete-Sánchez, Luis A.; Ciudad Pizarro, Juana; García, Juan Luis; Ribera, José M.; Hernández-Rivas, Jesús M.

    2016-01-01

    Identifying additional genetic alterations associated with poor prognosis in acute lymphoblastic leukemia (ALL) is still a challenge. Aims: To characterize the presence of additional DNA copy number alterations (CNAs) in children and adults with ALL by whole-genome oligonucleotide array (aCGH) analysis, and to identify their associations with clinical features and outcome. Array-CGH was carried out in 265 newly diagnosed ALLs (142 children and 123 adults). The NimbleGen CGH 12x135K array (Roche) was used to analyze genetic gains and losses. CNAs were analyzed with GISTIC and aCGHweb software. Clinical and biological variables were analyzed. Three of the patients showed chromothripsis (cth6, cth14q and cth15q). CNAs were associated with age, phenotype, genetic subtype and overall survival (OS). In the whole cohort of children, the losses on 14q32.33 (p = 0.019) and 15q13.2 (p = 0.04) were related to shorter OS. In the group of children without good- or poor-risk cytogenetics, the gain on 1p36.11 was a prognostic marker independently associated with shorter OS. In adults, the gains on 19q13.2 (p = 0.001) and Xp21.1 (p = 0.029), and the loss of 17p (p = 0.014) were independent markers of poor prognosis with respect to OS. In summary, CNAs are frequent in ALL and are associated with clinical parameters and survival. Genome-wide DNA copy number analysis allows the identification of genetic markers that predict clinical outcome, suggesting that detection of these genetic lesions will be useful in the management of patients newly diagnosed with ALL. PMID:26872047

  17. Recurrent DNA copy number alterations in intestinal-type sinonasal adenocarcinoma.

    PubMed

    Perez-Escuredo, J; Lopez-Hernandez, A; Costales, M; Lopez, F; Ares, S P; Vivanco, B; Llorente, J L; Hermsen, M A

    2016-09-01

    Intestinal-type sinonasal adenocarcinoma (ITAC) is a rare tumour related to occupational wood dust exposure. Few studies have described recurrent genetic changes on a genome-wide scale. The aim of this study was to obtain a high resolution map of recurrent genetic alterations in ITAC. Copy number alterations were evaluated by microarray CGH and MLPA in 37 primary tumours. The results were correlated with pathological characteristics and clinical outcome. Microarray CGH identified the following recurrent aberrations, in descending order: gains at 5p15 (22 cases, 60%), 8q24 (21 cases, 57%), 20q13 (20 cases, 54%), 20q11, and 8q21 (19 cases, 51%), 20p13, and 7p11 (16 cases, 43%), and losses at 5q11-qter, 8p12-pter, and 18q12-23 (15 cases, 40%), and 17p13, and 19p13 (13 cases, 35%). MLPA analysis confirmed this global pattern of gains and losses. Chromosomal loss at 4q32-ter and gains at 1q22, 6p22 and 3q29, as well as deletion of TIMP2 and CRK correlated with unfavourable clinical outcome. ITACs have a unique pattern of chromosomal abnormalities. The four different histological subtypes of ITAC appeared genetically similar. Four chromosomal gains and losses and two specific genes showed prognostic value and may be involved in tumour progression.

  18. Deficiency of the multi-copy mouse Y gene Sly causes sperm DNA damage and abnormal chromatin packaging.

    PubMed

    Riel, Jonathan M; Yamauchi, Yasuhiro; Sugawara, Atsushi; Li, Ho Yan J; Ruthig, Victor; Stoytcheva, Zoia; Ellis, Peter J I; Cocquet, Julie; Ward, Monika A

    2013-02-01

    In mouse and man Y chromosome deletions are frequently associated with spermatogenic defects. Mice with extensive deletions of non-pairing Y chromosome long arm (NPYq) are infertile and produce sperm with grossly misshapen heads, abnormal chromatin packaging and DNA damage. The NPYq-encoded multi-copy gene Sly controls the expression of sex chromosome genes after meiosis and Sly deficiency results in a remarkable upregulation of sex chromosome genes. Sly deficiency has been shown to be the underlying cause of the sperm head anomalies and infertility associated with NPYq gene loss, but it was not known whether it recapitulates sperm DNA damage phenotype. We produced and examined mice with transgenically (RNAi) silenced Sly and demonstrated that these mice have increased incidence of sperm with DNA damage and poorly condensed and insufficiently protaminated chromatin. We also investigated the contribution of each of the two Sly-encoded transcript variants and noted that the phenotype was only observed when both variants were knocked down, and that the phenotype was intermediate in severity compared with mice with severe NPYq deficiency. Our data demonstrate that Sly deficiency is responsible for the sperm DNA damage/chromatin packaging defects observed in mice with NPYq deletions and point to SLY proteins involvement in chromatin reprogramming during spermiogenesis, probably through their effect on the post-meiotic expression of spermiogenic genes. Considering the importance of the sperm epigenome for embryonic and fetal development and the possibility of its inter-generational transmission, our results are important for future investigations of the molecular mechanisms of this biologically and clinically important process.

  19. Phylogeny of the genus Chrysanthemum L.: evidence from single-copy nuclear gene and chloroplast DNA sequences.

    PubMed

    Liu, Ping-Li; Wan, Qian; Guo, Yan-Ping; Yang, Ji; Rao, Guang-Yuan

    2012-01-01

    Chrysanthemum L. (Asteraceae-Anthemideae) is a genus with rapid speciation. It comprises about 40 species, most of which are distributed in East Asia. Many of these are narrowly distributed and habitat-specific. Considerable variations in morphology and ploidy are found in this genus. Some species have been the subjects of many studies, but the relationships between Chrysanthemum and its allies and the phylogeny of this genus remain poorly understood. In the present study, 32 species/varieties from Chrysanthemum and 11 from the allied genera were analyzed using DNA sequences of the single-copy nuclear CDS gene and seven cpDNA loci (psbA-trnH, trnC-ycf6, ycf6-psbM, trnY-rpoB, rpS4-trnT, trnL-F, and rpL16). The cpDNA and nuclear CDS gene trees both suggest that 1) Chrysanthemum is not a monophyletic taxon, and the affinity between Chrysanthemum and Ajania is so close that these two genera should be incorporated taxonomically; 2) Phaeostigma is more closely related to the Chrysanthemum+Ajania than other generic allies. According to pollen morphology and to the present cpDNA and CDS data, Ajania purpurea is a member of Phaeostigma. Species differentiation in Chrysanthemum appears to be correlated with geographic and environmental conditions. The Chinese Chrysanthemum species can be divided into two groups, the C. zawadskii group and the C. indicum group. The former is distributed in northern China and the latter in southern China. Many polyploid species, such as C. argyrophyllum, may have originated from allopolyploidization involving divergent progenitors. Considering all the evidence from present and previous studies, we conclude that geographic and ecological factors as well as hybridization and polyploidy play important roles in the divergence and speciation of the genus Chrysanthemum.

  20. Gene copy silencing and DNA methylation in natural and artificially produced allopolyploid fish.

    PubMed

    Matos, Isa M N; Coelho, Maria M; Schartl, Manfred

    2016-10-01

    Allelic silencing is an important mechanism for coping with gene dosage changes in polyploid organisms that is well known in allopolyploid plants. Only recently, it was shown in the allotriploid fish Squalius alburnoides that this process also occurs in vertebrates. However, it is still unknown whether this silencing mechanism is common to other allopolyploid fish, and which mechanisms might be responsible for allelic silencing. We addressed these questions in a comparative study between Squalius alburnoides and another allopolyploid complex, the Amazon molly (Poecilia formosa). We examined the allelic expression patterns for three target genes in four somatic tissues of natural allo-anorthoploids and laboratory-produced tri-genomic hybrids of S. alburnoides and P. formosa. Also, for both complexes, we evaluated the correlation between total DNA methylation level and the ploidy status and genomic composition of the individuals. We found that allelic silencing also occurs in other allopolyploid organisms besides the single one that was previously known. We found and discuss disparities within and between the two considered complexes concerning the pattern of allele-specific expression and DNA methylation levels. Disparities might be due to intrinsic characteristics of each genome involved in the hybridization process. Our findings also support the idea that long-term evolutionary processes have an effect on the allele expression patterns and possibly also on DNA methylation levels. © 2016. Published by The Company of Biologists Ltd.

  1. Laser capture microdissection of cervical human papillomavirus infections: copy number of the virus in cancerous and normal tissue and heterogeneous DNA methylation.

    PubMed

    Kalantari, Mina; Garcia-Carranca, Alejandro; Morales-Vazquez, Claudia Dalia; Zuna, Rosemary; Montiel, Delia Perez; Calleja-Macias, Itzel E; Johansson, Bo; Andersson, Sonia; Bernard, Hans-Ulrich

    2009-08-01

    Research on the pathogenicity of human papillomaviruses (HPVs) during cervical carcinogenesis often relies on the study of homogenized tissue or cultured cells. This approach does not detect molecular heterogeneities within the infected tissue. It is desirable to understand molecular properties in specific histological contexts. We asked whether laser capture microdissection (LCM) of archival cervical tumors in combination with real-time polymerase chain reaction and bisulfite sequencing permits (i) sensitive DNA diagnosis of small clusters of formalin-fixed cells, (ii) quantification of HPV DNA in neoplastic and normal cells, and (iii) analysis of HPV DNA methylation, a marker of tumor progression. We analyzed 26 tumors containing HPV-16 or 18. We prepared DNA from LCM dissected thin sections of 100 to 2000 cells, and analyzed aliquots corresponding to between nine and 70 cells. We detected nine to 630 HPV-16 genome copies and one to 111 HPV-18 genome copies per tumor cell, respectively. In 17 of the 26 samples, HPV DNA existed in histologically normal cells distant from the margins of the tumors, but at much lower concentrations than in the tumor, suggesting that HPVs can infect at low levels without pathogenic changes. Methylation of HPV DNA, a biomarker of integration of the virus into cellular DNA, could be measured only in few samples due to limited sensitivity, and indicated heterogeneous methylation patterns in small clusters of cancerous and normal cells. LCM is powerful to study molecular parameters of cervical HPV infections like copy number, latency and epigenetics.

  2. Rare DNA copy number variants in cardiovascular malformations with extracardiac abnormalities

    PubMed Central

    Lalani, Seema R; Shaw, Chad; Wang, Xueqing; Patel, Ankita; Patterson, Lance W; Kolodziejska, Katarzyna; Szafranski, Przemyslaw; Ou, Zhishuo; Tian, Qi; Kang, Sung-Hae L; Jinnah, Amina; Ali, Sophia; Malik, Aamir; Hixson, Patricia; Potocki, Lorraine; Lupski, James R; Stankiewicz, Pawel; Bacino, Carlos A; Dawson, Brian; Beaudet, Arthur L; Boricha, Fatima M; Whittaker, Runako; Li, Chumei; Ware, Stephanie M; Cheung, Sau Wai; Penny, Daniel J; Jefferies, John Lynn; Belmont, John W

    2013-01-01

    Clinically significant cardiovascular malformations (CVMs) occur in 5–8 per 1000 live births. Recurrent copy number variations (CNVs) are among the known causes of syndromic CVMs, accounting for an important fraction of cases. We hypothesized that many additional rare CNVs also cause CVMs and can be detected in patients with CVMs plus extracardiac anomalies (ECAs). Through a genome-wide survey of 203 subjects with CVMs and ECAs, we identified 55 CNVs >50 kb in length that were not present in children without known cardiovascular defects (n=872). Sixteen unique CNVs overlapping these variants were found in an independent CVM plus ECA cohort (n=511), which were not observed in 2011 controls. The study identified 12/16 (75%) novel loci including non-recurrent de novo 16q24.3 loss (4/714) and de novo 2q31.3q32.1 loss encompassing PPP1R1C and PDE1A (2/714). The study also narrowed critical intervals in three well-recognized genomic disorders of CVM, such as the cat-eye syndrome region on 22q11.1, 8p23.1 loss encompassing GATA4 and SOX7 and 17p13.3-p13.2 loss. An analysis of protein-interaction databases shows that the rare inherited and de novo CNVs detected in the combined cohort are enriched for genes encoding proteins that are direct or indirect partners of proteins known to be required for normal cardiac development. Our findings implicate rare variants such as 16q24.3 loss and 2q31.3-q32.1 loss, and delineate regions within previously reported structural variants known to cause CVMs. PMID:22929023

  3. Specific functions of the Rep and Rep׳ proteins of porcine circovirus during copy-release and rolling-circle DNA replication.

    PubMed

    Cheung, Andrew K

    2015-07-01

    The roles of two porcine circovirus replication initiator proteins, Rep and Rep׳, in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replication) and generates the single-stranded circular (ssc) genome from the displaced DNA strand. In the process, a minus-genome primer (MGP) necessary for complementary-strand synthesis, from ssc to ccc, is synthesized. Rep׳ cleaves the growing nascent-strand to regenerate the parent ccc molecule. In the process, a Rep׳-DNA hybrid containing the right palindromic sequence (at the origin of DNA replication) is generated. Analysis of the virus particle showed that it is composed of four components: ssc, MGP, capsid protein and a novel Rep-related protein (designated Protein-3).

  4. Mitochondrial DNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the doubly uniparental inheritance of mitochondrial DNA system in the blue mussel Mytilus galloprovincialis.

    PubMed

    Sano, Natsumi; Obata, Mayu; Ooie, Yosiyasu; Komaru, Akira

    2011-08-01

    Doubly uniparental inheritance (DUI) of mitochondrial (mt) DNA has been reported in the blue mussel Mytilus galloprovincialis. In DUI, males inherit both paternal (M type) and maternal (F type) mtDNA. Here we investigated changes in M type mtDNA copy numbers and mitochondrial mass in testicular cells by real-time polymerase chain reaction and flow cytometry. The ratios of M type mtDNA copy numbers to nuclear DNA content were not different between haploid (1n), diploid (2n) and tetraploid (4n) spermatogenic cells. The mitochondrial mass decreased gradually during spermatogenesis. These results suggest that mtDNA and mitochondrial mass are maintained during spermatogenesis. We then traced M type mtDNA in larvae after fertilization. M type mtDNA was maintained up to 24 h after fertilization in the male-biased crosses, but decreased significantly in female-biased crosses (predicted by Mito Tracker staining pattern). These results are strikingly different from those reported for mammals and fish, where it is well known that the mitochondria and mtDNA are reduced during spermatogenesis and that sperm mitochondria and mtDNA are eliminated soon after fertilization. Thus, the M type mtDNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the DUI system in the blue mussel.

  5. DNA Copy Number Aberrations, and Human Papillomavirus Status in Penile Carcinoma. Clinico-Pathological Correlations and Potential Driver Genes

    PubMed Central

    Lambros, Maryou; Stankiewicz, Elzbieta; Ng, Charlotte K. Y.; Weigelt, Britta; Rajab, Ramzi; Tinwell, Brendan; Corbishley, Cathy; Watkin, Nick; Berney, Dan; Reis-Filho, Jorge S.

    2016-01-01

    Penile squamous cell carcinoma is a rare disease, in which somatic genetic aberrations have yet to be characterized. We hypothesized that gene copy aberrations might correlate with human papillomavirus status and clinico-pathological features. We sought to determine the spectrum of gene copy number aberrations in a large series of PSCCs and to define their correlations with human papillomavirus, histopathological subtype, and tumor grade, stage and lymph node status. Seventy formalin-fixed, paraffin embedded penile squamous cell carcinomas were centrally reviewed by expert uropathologists. DNA was extracted from micro-dissected samples, subjected to PCR-based human papillomavirus assessment and genotyping (INNO-LiPA human papillomavirus Genotyping Extra Assay) and microarray-based comparative genomic hybridization using a 32K Bacterial Artificial Chromosome array platform. Sixty-four samples yielded interpretable results. Recurrent gains were observed in chromosomes 1p13.3-q44 (88%), 3p12.3-q29 (86%), 5p15.33-p11 (67%) and 8p12-q24.3 (84%). Amplifications of 5p15.33-p11 and 11p14.1-p12 were found in seven (11%) and four (6%) cases, respectively. Losses were observed in chromosomes 2q33-q37.3 (86%), 3p26.3-q11.1 (83%) and 11q12.2-q25 (81%). Although many losses and gains were similar throughout the cohort, there were small significant differences observed at specific loci, between human papillomavirus positive and negative tumors, between tumor types, and tumor grade and nodal status. These results demonstrate that despite the diversity of genetic aberrations in penile squamous cell carcinomas, there are significant correlations between the clinico-pathological data and the genetic changes that may play a role in disease natural history and progression and highlight potential driver genes, which may feature in molecular pathways for existing therapeutic agents. PMID:26901676

  6. Efficacy and limits of genotyping low copy number (LCN) DNA samples by multiplex PCR of STR loci.

    PubMed

    Kloosterman, Ate D; Kersbergen, Paula

    2003-01-01

    In this study, we have evaluated the efficacy and the validity of the AmpFISTR SGM plus multiplex PCR typing system when Low Copy Number (LCN) amounts of DNA are processed. The characteristics of SGM plus profiles produced under LCN conditions were studied on the basis of heterozygote balance, between loci balance and stutter proportion based on profiles that were obtained from a variety of mock casework samples. These experiments clearly showed that LCN DNA profiles carry their own characteristic features, which must be taken into account during interpretation. Herewith, we confirmed the data of recent other studies that a comprehensive interpretation strategy is dependent upon multiple replication of the PCR using the same extract together with the proper use of extraction and amplification controls. The limitations of LCN DNA analysis were further studied in a series of single cell PCR experiments using an amplification regime of 34 PCR cycles. The allele dropout phenomenon was demonstrated to its full extent when single cells were analysed. However, the "consensus profile" which was obtained from separate single cell PCR experiments matched the actual profile of the cell donor. Single cell PCR experiments also showed that a further increase of the number of PCR cycles did not result in enhanced sensitivity and had a highly negative effect on the balance of this multiplex PCR system which hampered correct interpretation of the profile. Also, the potential of LCN typing in analysing mixtures of DNA was investigated. It was clearly shown that LCN typing had no advantages over 28 cycles amplification in the detection of the minor component of DNA-mixtures. In addition to the 34 cycles PCR amplification regime, the utility of a new approach that involved reamplification of the 28 cycle SGM plus PCR products with an extra 6 PCR cycles after the addition of fresh AmpliTaq Gold DNA Polymerase was investigated. This approach provides the scientist with an extra typing

  7. Effects of acetyl-L-carnitine on lamb oocyte blastocyst rate, ultrastructure, and mitochondrial DNA copy number.

    PubMed

    Reader, Karen L; Cox, Neil R; Stanton, Jo-Ann L; Juengel, Jennifer L

    2015-06-01

    Viable lambs can be produced after transfer of in vitro-derived embryos from oocytes harvested from prepubertal lambs. However, this occurs at a much lower efficiency than from adult ewe oocyte donors. The reduced competence of prepubertal oocytes is believed to be due, at least in part, to deficiencies in cytoplasmic maturation. Differences in the cytoplasmic ultrastructure between prepubertal and adult oocytes have been described in the sheep, pig, and cow. Prepubertal lamb oocytes have been shown to have a different distribution of mitochondria and lipid droplets, and less mitochondria and storage vesicles than their adult counterparts. L-carnitine plays a role in supplying energy to the cell by transporting long-chain fatty acids into mitochondria for β-oxidation to produce ATP. Both L-carnitine and its derivative acetyl-L-carnitine have been reported to increase the blastocyst rate of oocytes from mice, cows, and pigs, treated during IVM. L-carnitine has also been shown to increase mitochondrial biogenesis in adipose cells. Therefore, the aims of this study were to determine if treatment of oocytes from prepubertal lambs with acetyl-L-carnitine during IVM could increase the blastocyst rate and alter mitochondria, vesicle, or lipid droplet number, volume, or distribution. The blastocyst rate was doubled in prepubertal lamb oocytes treated with acetyl-L-carnitine when compared to untreated oocytes (10.0% and 4.6%, respectively; P = 0.028). Light microscopy, scanning electron microscopy, and stereology techniques were used to quantify organelles in untreated and acetyl-L-carnitine-treated lamb oocytes, and quantitative polymerase chain reaction methods were used to measure the mitochondrial DNA copy number. There were no differences in mitochondrial volume, number, or mitochondrial DNA copy number. Acetyl-L-carnitine treatment increased the cytoplasmic volume (P = 0.015) of the oocytes, and there were trends toward an increase in the vesicle volume (P = 0

  8. Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection.

    PubMed

    Cohen, Richard N; van der Aa, Marieke A E M; Macaraeg, Nichole; Lee, Ai Ping; Szoka, Francis C

    2009-04-17

    Nuclear uptake of plasmid DNA is one of the many cellular barriers that limit the efficiency of non-viral gene delivery systems. We have determined the number of plasmids that reach the nucleus of a transfected cell using an internally standardized quantitative PCR (qPCR) assay. We isolated nuclei using two different protocols: a density gradient technique and a detergent-based method. The density gradient procedure yielded nuclei with substantially less adhering plasmids on the outside of the nuclei. Using the density gradient protocol we determined that cells transfected with Lipofectamine lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus, depending on the applied plasmid dose. Any increase above 3000 plasmids/nucleus resulted in only marginal increases in transgene expression. Furthermore, lipoplex-delivered plasmids were more efficiently expressed, on the basis of protein expression per plasmid number in the nucleus, than polyplex-delivered plasmids. This indicates that polymer may remain bound to some plasmids in the nucleus. Lastly, by sorting transfected cells into high- and low-expressing sub-populations, we observe that a sub-population of cells contain 3x greater plasmids/nucleus but express nearly 100x more transgene than other cells within a single transfection reaction. Taken together these results suggest the importance of considering the processes downstream from nuclear entry for strategies to improve the efficiency of gene transfer reagents.

  9. Preferential access to genetic information from endogenous hominin ancient DNA and accurate quantitative SNP-typing via SPEX

    PubMed Central

    Brotherton, Paul; Sanchez, Juan J.; Cooper, Alan; Endicott, Phillip

    2010-01-01

    The analysis of targeted genetic loci from ancient, forensic and clinical samples is usually built upon polymerase chain reaction (PCR)-generated sequence data. However, many studies have shown that PCR amplification from poor-quality DNA templates can create sequence artefacts at significant levels. With hominin (human and other hominid) samples, the pervasive presence of highly PCR-amplifiable human DNA contaminants in the vast majority of samples can lead to the creation of recombinant hybrids and other non-authentic artefacts. The resulting PCR-generated sequences can then be difficult, if not impossible, to authenticate. In contrast, single primer extension (SPEX)-based approaches can genotype single nucleotide polymorphisms from ancient fragments of DNA as accurately as modern DNA. A single SPEX-type assay can amplify just one of the duplex DNA strands at target loci and generate a multi-fold depth-of-coverage, with non-authentic recombinant hybrids reduced to undetectable levels. Crucially, SPEX-type approaches can preferentially access genetic information from damaged and degraded endogenous ancient DNA templates over modern human DNA contaminants. The development of SPEX-type assays offers the potential for highly accurate, quantitative genotyping from ancient hominin samples. PMID:19864251

  10. A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy number changes by array comparative genomic hybridization.

    PubMed

    Chung, Yeun-Jun; Jonkers, Jos; Kitson, Hannah; Fiegler, Heike; Humphray, Sean; Scott, Carol; Hunt, Sarah; Yu, Yuejin; Nishijima, Ichiko; Velds, Arno; Holstege, Henne; Carter, Nigel; Bradley, Allan

    2004-01-01

    Microarray-based comparative genomic hybridization (CGH) has become a powerful method for the genome-wide detection of chromosomal imbalances. Although BAC microarrays have been used for mouse CGH studies, the resolving power of these analyses was limited because high-density whole-genome mouse BAC microarrays were not available. We therefore developed a mouse BAC microarray containing 2803 unique BAC clones from mouse genomic libraries at 1-Mb intervals. For the general amplification of BAC clone DNA prior to spotting, we designed a set of three novel degenerate oligonucleotide-primed (DOP) PCR primers that preferentially amplify mouse genomic sequences while minimizing unwanted amplification of contaminating Escherichia coli DNA. The resulting 3K mouse BAC microarrays reproducibly identified DNA copy number alterations in cell lines and primary tumors, such as single-copy deletions, regional amplifications, and aneuploidy.

  11. Are benzo[a]pyrene-DNA adducts an accurate biomarker of long-term in utero exposure to smoking?

    PubMed

    Stéphan-Blanchard, Erwan; Chardon, Karen; Telliez, Frédéric; Arnould, Jean-Pierre; Léké, André; Ammari, Mohamed; Horne, Rosemary S C; Libert, Jean-Pierre; Bach, Véronique

    2011-06-01

    Maternal smoking during pregnancy is associated with adverse perinatal outcomes. In view of concerns about underreporting, benzo[a]pyrene (B[a]P)-DNA adducts could be used to provide information about long-term in utero exposure to smoking but have not previously been used with samples from neonates. This study aimed to verify whether B[a]P-DNA adducts could accurately assess tobacco smoke exposure during fetal life. The objectives were to correlate B[a]P-DNA adduct levels with active maternal and passive smoking and to determine the sensitivity and specificity of smoking and nonsmoking status by comparing neonatal B[a]P-DNA adduct levels with those of maternal self-reports. B[a]P-DNA adducts in neonatal buccal cell samples were determined by a competitive immunoassay. Three groups of neonates were constituted according to maternal self-reported smoking status during pregnancy: nonsmokers (n=25; control group), <10 cigarettes per day (n=18; S- group), or >10 cigarettes per day (n=21; S+ group). The mean B[a]P-DNA adduct level rose significantly when comparing the controls with the S- and S+ groups. Maternal active smoking had the strongest effect on B[a]P-DNA adduct levels in neonates. A cross analysis between B[a]P-DNA adduct levels and maternal self-reported levels revealed high sensitivity and specificity. This preliminary study suggests that B[a]P-DNA adducts are reliable biomarkers for the screening of long-term in utero exposure to smoking and are accurate when compared with maternal self-reported levels of active smoking. Detection of B[a]P-DNA adducts in neonates could provide a useful, noninvasive tool in clinical risk assessment studies but would benefit from further confirmation with another validated biomarker.

  12. DRNApred, fast sequence-based method that accurately predicts and discriminates DNA- and RNA-binding residues.

    PubMed

    Yan, Jing; Kurgan, Lukasz

    2017-06-02

    Protein-DNA and protein-RNA interactions are part of many diverse and essential cellular functions and yet most of them remain to be discovered and characterized. Recent research shows that sequence-based predictors of DNA-binding residues accurately find these residues but also cross-predict many RNA-binding residues as DNA-binding, and vice versa. Most of these methods are also relatively slow, prohibiting applications on the whole-genome scale. We describe a novel sequence-based method, DRNApred, which accurately and in high-throughput predicts and discriminates between DNA- and RNA-binding residues. DRNApred was designed using a new dataset with both DNA- and RNA-binding proteins, regression that penalizes cross-predictions, and a novel two-layered architecture. DRNApred outperforms state-of-the-art predictors of DNA- or RNA-binding residues on a benchmark test dataset by substantially reducing the cross predictions and predicting arguably higher quality false positives that are located nearby the native binding residues. Moreover, it also more accurately predicts the DNA- and RNA-binding proteins. Application on the human proteome confirms that DRNApred reduces the cross predictions among the native nucleic acid binders. Also, novel putative DNA/RNA-binding proteins that it predicts share similar subcellular locations and residue charge profiles with the known native binding proteins. Webserver of DRNApred is freely available at http://biomine.cs.vcu.edu/servers/DRNApred/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Stability of single copy transgene expression in CHOK1 cells is affected by histone modifications but not by DNA methylation.

    PubMed

    Spencer, Shawal; Gugliotta, Agustina; Koenitzer, Jennifer; Hauser, Hansjörg; Wirth, Dagmar

    2015-02-10

    Intraclonal heterogeneity of genetically modified mammalian cells has been observed as a phenomenon that has a strong impact on overall transgene expression levels and that limits the predictability of transgene expression in genetically modified cells, thereby hampering single cell based screening approaches. The underlying mechanism(s) leading to this variance are poorly understood. To study the dynamics and mechanisms of heterogeneity of early stage silencing we analyzed the expression in more than 100 independent clones of CHOK1 cells that harbour genetically stable integrates of single copy reporter cassettes driven by EF1α and CMV promoters. Single cell analysis showed intraclonal variability with heterogeneity in expression in genetically uniform populations. DNA methylation is a well known mechanism responsible for silencing of gene expression. Interestingly, loss of expression was not associated with DNA methylation of the CMV promoter. However, in most of the clonal populations expression could be increased by inhibitors of the histone deacetylases (HDACi) suggesting that heterogeneity of transgene expression is crucially governed by histone modifications. Further, to determine if the epigenetic status of transgene expression is governed by the chromosomal integration locus we targeted heterologous expression cassettes into two chromosomal sites using recombinase mediated cassette exchange (RMCE). The expression status of a particular clone was faithfully re-established when the same promoter used. In this way the problem of early stage cell clone instability can be bypassed. However, upon introduction of an unrelated promoter methylation-independent silencing was observed. Together, these results suggest that histone modifications are the relevant mechanisms by which epigenetic modulation of transgene expression cassettes is governed in the early phase of clone generation.

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

    PubMed

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

    2011-07-28

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

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

    PubMed Central

    2011-01-01

    Background The rapidly growing amount of array CGH data requires improved visualization software supporting the process of identifying candidate cancer genes. Optimally, such software should work across multiple microarray platforms, should be able to cope with data from different sources and should be easy to operate. Results We have developed a web-based software FISH Oracle to visualize data from multiple array CGH experiments in a genomic context. Its fast visualization engine and advanced web and database technology supports highly interactive use. FISH Oracle comes with a convenient data import mechanism, powerful search options for genomic elements (e.g. gene names or karyobands), quick navigation and zooming into interesting regions, and mechanisms to export the visualization into different high quality formats. These features make the software especially suitable for the needs of life scientists. Conclusions FISH Oracle offers a fast and easy to use visualization tool for array CGH and SNP array data. It allows for the identification of genomic regions representing minimal common changes based on data from one or more experiments. FISH Oracle will be instrumental to identify candidate onco and tumor suppressor genes based on the frequency and genomic position of DNA copy number changes. The FISH Oracle application and an installed demo web server are available at http://www.zbh.uni-hamburg.de/fishoracle. PMID:21884636

  16. Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

    PubMed

    Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Natkanska, Urszula; Kwiatkowska, Aleksandra; Rawska, Ewa; Potocki, Leszek; Kuna, Ewelina; Panek, Anita; Lewinska, Anna; Wnuk, Maciej

    2016-09-01

    The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement.

  17. Identification of DNA copy-number aberrations by array-comparative genomic hybridization in patients with schizophrenia.

    PubMed

    Moon, Ho Jin; Yim, Sung-Vin; Lee, Woon Kyu; Jeon, Yang-Whan; Kim, Young Hoon; Ko, Young Jin; Lee, Kwang-Soo; Lee, Kweon-Haeng; Han, Sang-Ick; Rha, Hyoung Kyun

    2006-06-02

    Chromosomal abnormalities are implicated as important markers for the pathogenesis in patients with schizophrenia. In this study, with using bacterial artificial chromosome (BAC) array-based comparative genomic hybridization (CGH), we analyzed DNA copy-number changes among 30 patients with schizophrenia. The most frequent changes were partial gain of Xq23 (52%) and loss of 3q13.12 (32%). Other frequent gains were found in: 1p, 6q, 10p, 11p, 11q, 14p, and 15q regions, and frequent losses were found in: 2p, 9q, 10q, 14q, 20q, and 22q regions. The set of abnormal regions was confirmed by real-time PCR (9q12, 9q34.2, 11p15.4, 14q32.33, 15q15.1, 22q11.21, and Xq23). All real-time PCR results were consistent with the array-CGH results. Therefore, it is suggested that array-CGH and real-time PCR analysis could be used as powerful tools in screening for schizophrenia-related genes. Our results might be useful for further exploration of candidate genomic regions in the pathogenesis of schizophrenia.

  18. Lake sedimentary DNA accurately records 20(th) Century introductions of exotic conifers in Scotland.

    PubMed

    Sjögren, Per; Edwards, Mary E; Gielly, Ludovic; Langdon, Catherine T; Croudace, Ian W; Merkel, Marie Kristine Føreid; Fonville, Thierry; Alsos, Inger Greve

    2017-01-01

    Sedimentary DNA (sedDNA) has recently emerged as a new proxy for reconstructing past vegetation, but its taphonomy, source area and representation biases need better assessment. We investigated how sedDNA in recent sediments of two small Scottish lakes reflects a major vegetation change, using well-documented 20(th) Century plantations of exotic conifers as an experimental system. We used next-generation sequencing to barcode sedDNA retrieved from subrecent lake sediments. For comparison, pollen was analysed from the same samples. The sedDNA record contains 73 taxa (mainly genus or species), all but one of which are present in the study area. Pollen and sedDNA shared 35% of taxa, which partly reflects a difference in source area. More aquatic taxa were recorded in sedDNA, whereas taxa assumed to be of regional rather than local origin were recorded only as pollen. The chronology of the sediments and planting records are well aligned, and sedDNA of exotic conifers appears in high quantities with the establishment of plantations around the lakes. SedDNA recorded other changes in local vegetation that accompanied afforestation. There were no signs of DNA leaching in the sediments or DNA originating from pollen.

  19. The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro.

    PubMed

    Cotterill, Matthew; Harris, Sarah E; Collado Fernandez, Esther; Lu, Jianping; Huntriss, John D; Campbell, Bruce K; Picton, Helen M

    2013-07-01

    Mitochondria are responsible for the production of ATP, which drives cellular metabolic and biosynthetic processes. This is the first study to quantify the mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in germinal vesicle (GV) and metaphase II (MII) oocytes through JC1 staining. Primordial to early antral follicles (n = 249) were isolated from the sheep ovarian cortex following digestion at 37°C for 1 h and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n = 133) were aspirated from 3- to 5-mm diameter antral follicles, and mature MII oocytes (n = 71) were generated following in vitro maturation (IVM). The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605 ± 205, n = 8) to mature MII oocytes (744 633 ± 115 799, n = 13; P < 0.05). Mitochondrial activity (P > 0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in the mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganization during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the pre-ovulatory gonadotrophin surge.

  20. UNDR ROVER - a fast and accurate variant caller for targeted DNA sequencing.

    PubMed

    Park, Daniel J; Li, Roger; Lau, Edmund; Georgeson, Peter; Nguyen-Dumont, Tú; Pope, Bernard J

    2016-04-16

    Previously, we described ROVER, a DNA variant caller which identifies genetic variants from PCR-targeted massively parallel sequencing (MPS) datasets generated by the Hi-Plex protocol. ROVER permits stringent filtering of sequencing chemistry-induced errors by requiring reported variants to appear in both reads of overlapping pairs above certain thresholds of occurrence. ROVER was developed in tandem with Hi-Plex and has been used successfully to screen for genetic mutations in the breast cancer predisposition gene PALB2. ROVER is applied to MPS data in BAM format and, therefore, relies on sequence reads being mapped to a reference genome. In this paper, we describe an improvement to ROVER, called UNDR ROVER (Unmapped primer-Directed ROVER), which accepts MPS data in FASTQ format, avoiding the need for a computationally expensive mapping stage. It does so by taking advantage of the location-specific nature of PCR-targeted MPS data. The UNDR ROVER algorithm achieves the same stringent variant calling as its predecessor with a significant runtime performance improvement. In one indicative sequencing experiment, UNDR ROVER (in its fastest mode) required 8-fold less sequential computation time than the ROVER pipeline and 13-fold less sequential computation time than a variant calling pipeline based on the popular GATK tool. UNDR ROVER is implemented in Python and runs on all popular POSIX-like operating systems (Linux, OS X). It requires as input a tab-delimited format file containing primer sequence information, a FASTA format file containing the reference genome sequence, and paired FASTQ files containing sequence reads. Primer sequences at the 5' end of reads associate read-pairs with their targeted amplicon and, thus, their expected corresponding coordinates in the reference genome. The primer-intervening sequence of each read is compared against the reference sequence from the same location and variants are identified using the same algorithm as ROVER

  1. Development of a PCR free, fieldable, rapid, accurate, and sensitive bio-electronic DNA biosensor

    NASA Astrophysics Data System (ADS)

    Hill, Doyle; Chafin, David; Greco, Roberta; Jafri, Samina; Murante, Richard; Noonan, John; Pham, An; Seabridge, Scott; Tannous, Vera; VanDerMeid, Karl; Wang, Daguang; Wescott, Nate; McFarlane, Kristin; Shah, Sanjiv

    2005-05-01

    The objective of this study was to demonstrate proof of concept for the Integrated Nano-Technologies BioDetect Bacillus anthracis electronic DNA sensor. B. anthracis Ames strain DNA was successfully detected by the formation of DNA bridges on the sensor. The bridges were coated with metal, resulting in a significant drop in electrical resistance. In this small test, at a relatively high DNA concentration, the overall accuracy of the sensor was 90.7%. The technology shows significant promise for future development as a bio-agent detection system.

  2. A binding free energy decomposition approach for accurate calculations of the fidelity of DNA polymerases

    PubMed Central

    Rucker, Robert; Oelschlaeger, Peter; Warshel, Arieh

    2010-01-01

    DNA polymerase β (pol β) is a small eukaryotic enzyme with the ability to repair short single-stranded DNA gaps that has found use as a model system for larger replicative DNA polymerases. For all DNA polymerases, the factors determining their catalytic power and fidelity are the interactions between the bases of the base pair, amino acids near the active site, and the two magnesium ions. In this report, we study effects of all three aspects on human pol β transition state (TS) binding free energies by reproducing a consistent set of experimentally determined data for different structures. Our calculations comprise the combination of four different base pairs (incoming pyrimidine nucleotides incorporated opposite both matched and mismatched purines) with four different pol β structures (wild type and three separate mutations of ionized residues to alanine). We decompose the incoming deoxynucleoside 5′-triphosphate-TS, and run separate calculations for the neutral base part and the highly charged triphosphate part, using different dielectric constants in order to account for the specific electrostatic environments. This new approach improves our ability to predict the effect of matched and mismatched base pairing and of mutations in DNA polymerases on fidelity and may be a useful tool in studying the potential of DNA polymerase mutations in the development of cancer. It also supports our point of view with regards to the origin of the structural control of fidelity, allowing for a quantified description of the fidelity of DNA polymerases. PMID:19842163

  3. Performance of Molecular Inversion Probes (MIP) in Allele CopyNumber Determination

    SciTech Connect

    Wang, Yuker; Moorhead, Martin; Karlin-Neumann, George; Wang,Nicolas J.; Ireland, James; Lin, Steven; Chen, Chunnuan; Heiser, LauraM.; Chin, Koei; Esserman, Laura; Gray, Joe W.; Spellman, Paul T.; Faham,Malek

    2007-05-14

    We have developed a new protocol for using MolecularInversion Probes (MIP) to accurately and specifically measure allele copynumber (ACN). The new protocol provides for significant improvementsincluding the reduction of input DNA (from 2?g) by more than 25 fold (to75ng total genomic DNA), higher overall precision resulting in one orderof magnitude lower false positive rate, and greater dynamic range withaccurate absolute copy number up to 60 copies.

  4. A Double-Layered Mixture Model for the Joint Analysis of DNA Copy Number and Gene Expression Data

    PubMed Central

    Choi, Hyungwon; Qin, Zhaohui S.

    2010-01-01

    Abstract Copy number aberration is a common form of genomic instability in cancer. Gene expression is closely tied to cytogenetic events by the central dogma of molecular biology, and serves as a mediator of copy number changes in disease phenotypes. Accordingly, it is of interest to develop proper statistical methods for jointly analyzing copy number and gene expression data. This work describes a novel Bayesian inferential approach for a double-layered mixture model (DLMM) which directly models the stochastic nature of copy number data and identifies abnormally expressed genes due to aberrant copy number. Simulation studies were conducted to illustrate the robustness of DLMM under various settings of copy number aberration frequency, confounding effects, and signal-to-noise ratio in gene expression data. Analysis of a real breast cancer data shows that DLMM is able to identify expression changes specifically attributable to copy number aberration in tumors and that a sample-specific index built based on the selected genes is correlated with relevant clinical information. PMID:20170400

  5. Accurate Identification of Candida parapsilosis (Sensu Lato) by Use of Mitochondrial DNA and Real-Time PCR

    PubMed Central

    Souza, Ana Carolina R.; Ferreira, Renata C.; Gonçalves, Sarah S.; Quindós, Guillermo; Eraso, Elena; Bizerra, Fernando C.; Briones, Marcelo R. S.

    2012-01-01

    Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the “gold standard” for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species. PMID:22535986

  6. Copy number analysis by low coverage whole genome sequencing using ultra low-input DNA from formalin-fixed paraffin embedded tumor tissue.

    PubMed

    Kader, Tanjina; Goode, David L; Wong, Stephen Q; Connaughton, Jacquie; Rowley, Simone M; Devereux, Lisa; Byrne, David; Fox, Stephen B; Mir Arnau, Gisela; Tothill, Richard W; Campbell, Ian G; Gorringe, Kylie L

    2016-11-15

    Unlocking clinically translatable genomic information, including copy number alterations (CNA), from formalin-fixed paraffin-embedded (FFPE) tissue is challenging due to low yields and degraded DNA. We describe a robust, cost-effective low-coverage whole genome sequencing (LC WGS) method for CNA detection using 5 ng of FFPE-derived DNA. CN profiles using 100 ng or 5 ng input DNA were highly concordant and comparable with molecular inversion probe (MIP) array profiles. LC WGS improved CN profiles of samples that performed poorly using MIP arrays. Our technique enables identification of driver and prognostic CNAs in archival patient samples previously deemed unsuitable for genomic analysis due to DNA limitations.

  7. Accurate characterization of carcinogenic DNA adducts using MALDI tandem time-of-flight mass spectrometry

    NASA Astrophysics Data System (ADS)

    Barnes, Charles A.; Chiu, Norman H. L.

    2009-01-01

    Many chemical carcinogens and their in vivo activated metabolites react readily with genomic DNA, and form covalently bound carcinogen-DNA adducts. Clinically, carcinogen-DNA adducts have been linked to various cancer diseases. Among the current methods for DNA adduct analysis, mass spectroscopic method allows the direct measurement of unlabeled DNA adducts. The goal of this study is to explore the use of matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to determine the identity of carcinogen-DNA adducts. Two of the known carcinogenic DNA adducts, namely N-(2'-deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenyl-imidazo [4,5-b] pyridine (dG-C8-PhIP) and N-(2'-deoxyguanosin-8yl)-4-aminobiphenyl (dG-C8-ABP), were selected as our models. In MALDI-TOF MS measurements, the small matrix ion and its cluster ions did not interfere with the measurements of both selected dG adducts. To achieve a higher accuracy for the characterization of selected dG adducts, 1 keV collision energy in MALDI-TOF/TOF MS/MS was used to measure the adducts. In comparison to other MS/MS techniques with lower collision energies, more extensive precursor ion dissociations were observed. The detection of the corresponding fragment ions allowed the identities of guanine, PhIP or ABP, and the position of adduction to be confirmed. Some of the fragment ions of dG-C8-PhIP have not been reported by other MS/MS techniques.

  8. A repeat protein-based DNA polymerase inhibitor for an efficient and accurate gene amplification by PCR.

    PubMed

    Hwang, Da-Eun; Shin, Yong-Keol; Munashingha, Palinda Ruvan; Park, So-Yeon; Seo, Yeon-Soo; Kim, Hak-Sung

    2016-12-01

    A polymerase chain reaction (PCR) using a thermostable DNA polymerase is the most widely applied method in many areas of research, including life sciences, biotechnology, and medical sciences. However, a conventional PCR incurs an amplification of undesired genes mainly owing to non-specifically annealed primers and the formation of a primer-dimer complex. Herein, we present the development of a Taq DNA polymerase-specific repebody, which is a small-sized protein binder composed of leucine rich repeat (LRR) modules, as a thermolabile inhibitor for a precise and accurate gene amplification by PCR. We selected a repebody that specifically binds to the DNA polymerase through a phage display, and increased its affinity to up to 10 nM through a modular evolution approach. The repebody was shown to effectively inhibit DNA polymerase activity at low temperature and undergo thermal denaturation at high temperature, leading to a rapid and full recovery of the polymerase activity, during the initial denaturation step of the PCR. The performance and utility of the repebody was demonstrated through an accurate and efficient amplification of a target gene without nonspecific gene products in both conventional and real-time PCRs. The repebody is expected to be effectively utilized as a thermolabile inhibitor in a PCR. Biotechnol. Bioeng. 2016;113: 2544-2552. © 2016 Wiley Periodicals, Inc.

  9. Alterations in mitochondrial DNA copy number and the activities of electron transport chain complexes and pyruvate dehydrogenase in the frontal cortex from subjects with autism.

    PubMed

    Gu, F; Chauhan, V; Kaur, K; Brown, W T; LaFauci, G; Wegiel, J; Chauhan, A

    2013-09-03

    Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence suggests that mitochondrial dysfunction and oxidative stress may contribute to the etiology of autism. This is the first study to compare the activities of mitochondrial electron transport chain (ETC) complexes (I-V) and pyruvate dehydrogenase (PDH), as well as mitochondrial DNA (mtDNA) copy number in the frontal cortex tissues from autistic and age-matched control subjects. The activities of complexes I, V and PDH were most affected in autism (n=14) being significantly reduced by 31%, 36% and 35%, respectively. When 99% confidence interval (CI) of control group was taken as a reference range, impaired activities of complexes I, III and V were observed in 43%, 29% and 43% of autistic subjects, respectively. Reduced activities of all five ETC complexes were observed in 14% of autistic cases, and the activities of multiple complexes were decreased in 29% of autistic subjects. These results suggest that defects in complexes I and III (sites of mitochondrial free radical generation) and complex V (adenosine triphosphate synthase) are more prevalent in autism. PDH activity was also reduced in 57% of autistic subjects. The ratios of mtDNA of three mitochondrial genes ND1, ND4 and Cyt B (that encode for subunits of complexes I and III) to nuclear DNA were significantly increased in autism, suggesting a higher mtDNA copy number in autism. Compared with the 95% CI of the control group, 44% of autistic children showed higher copy numbers of all three mitochondrial genes examined. Furthermore, ND4 and Cyt B deletions were observed in 44% and 33% of autistic children, respectively. This study indicates that autism is associated with mitochondrial dysfunction in the brain.

  10. Safe, accurate, prenatal diagnosis of thanatophoric dysplasia using ultrasound and free fetal DNA.

    PubMed

    Chitty, Lyn S; Khalil, Asma; Barrett, Angela N; Pajkrt, Eva; Griffin, David R; Cole, Tim J

    2013-05-01

    To improve the prenatal diagnosis of thanatophoric dysplasia by defining the change in fetal size across gestation and the frequency of sonographic features, and developing non-invasive molecular genetic diagnosis based on cell-free fetal DNA (cffDNA) in maternal plasma. Fetuses with a confirmed diagnosis of thanatophoric dysplasia were ascertained, records reviewed, sonographic features and measurements determined. Charts of fetal size were then constructed using the LMS (lambda-mu-sigma) method and compared with charts used in normal pregnancies and those complicated by achondroplasia. Cases in this cohort referred to our Regional Genetics Laboratory for molecular diagnosis using cffDNA were identified and results reviewed. Forty-two cases were scanned in our units. Commonly reported sonographic features were very short and sometimes bowed femora, frontal bossing, cloverleaf skull, short fingers, a small chest and polyhydramnios. Limb shortening was obvious from as early as 13 weeks' gestation, with minimal growth after 20 weeks. Analysis of cffDNA in three of these pregnancies confirmed the presence of the c.742C>CT (p.Arg248Cys) or the c.1948A>AG (p.Lys650Glu) mutation in the fibroblast growth factor receptor 3 gene. These data should improve the accuracy of the sonographic diagnosis of thanatophoric dysplasia and have implications for reliable and safe targeted molecular confirmation using cffDNA. © 2013 John Wiley & Sons, Ltd.

  11. Safe, accurate, prenatal diagnosis of thanatophoric dysplasia using ultrasound and free fetal DNA

    PubMed Central

    Chitty, Lyn S; Khalil, Asma; Barrett, Angela N; Pajkrt, Eva; Griffin, David R; Cole, Tim J

    2013-01-01

    Objective To improve the prenatal diagnosis of thanatophoric dysplasia by defining the change in fetal size across gestation and the frequency of sonographic features, and developing non-invasive molecular genetic diagnosis based on cell-free fetal DNA (cffDNA) in maternal plasma. Methods Fetuses with a confirmed diagnosis of thanatophoric dysplasia were ascertained, records reviewed, sonographic features and measurements determined. Charts of fetal size were then constructed using the LMS (lambda-mu-sigma) method and compared with charts used in normal pregnancies and those complicated by achondroplasia. Cases in this cohort referred to our Regional Genetics Laboratory for molecular diagnosis using cffDNA were identified and results reviewed. Results Forty-two cases were scanned in our units. Commonly reported sonographic features were very short and sometimes bowed femora, frontal bossing, cloverleaf skull, short fingers, a small chest and polyhydramnios. Limb shortening was obvious from as early as 13 weeks' gestation, with minimal growth after 20 weeks. Analysis of cffDNA in three of these pregnancies confirmed the presence of the c.742C>CT (p.Arg248Cys) or the c.1948A>AG (p.Lys650Glu) mutation in the fibroblast growth factor receptor 3 gene. Conclusion These data should improve the accuracy of the sonographic diagnosis of thanatophoric dysplasia and have implications for reliable and safe targeted molecular confirmation using cffDNA. © 2013 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd. PMID:23408600

  12. Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.

    PubMed

    Kukat, Christian; Wurm, Christian A; Spåhr, Henrik; Falkenberg, Maria; Larsson, Nils-Göran; Jakobs, Stefan

    2011-08-16

    Mammalian mtDNA is packaged in DNA-protein complexes denoted mitochondrial nucleoids. The organization of the nucleoid is a very fundamental question in mitochondrial biology and will determine tissue segregation and transmission of mtDNA. We have used a combination of stimulated emission depletion microscopy, enabling a resolution well below the diffraction barrier, and molecular biology to study nucleoids in a panel of mammalian tissue culture cells. We report that the nucleoids labeled with antibodies against DNA, mitochondrial transcription factor A (TFAM), or incorporated BrdU, have a defined, uniform mean size of ∼100 nm in mammals. Interestingly, the nucleoid frequently contains only a single copy of mtDNA (average ∼1.4 mtDNA molecules per nucleoid). Furthermore, we show by molecular modeling and volume calculations that TFAM is a main constituent of the nucleoid, besides mtDNA. These fundamental insights into the organization of mtDNA have broad implications for understanding mitochondrial dysfunction in disease and aging.

  13. Toward a more accurate time scale for the human mitochondrial DNA tree.

    PubMed

    Hasegawa, M; Di Rienzo, A; Kocher, T D; Wilson, A C

    1993-10-01

    Several estimates of the time of occurrence of the most recent common mitochondrial DNA (mtDNA) ancestor of modern humans have been made. Estimates derived from noncoding regions based on a model that classifies sites into two categories (variable and invariable) have been consistently older than those derived from the third positions of codons. This discrepancy can be attributed to a violation of the assumption of rate homogeneity among variable sites when analyzing the noncoding regions. Additional data from the partial control region sequences allow us to take into account some of this further heterogeneity. By assigning the sites to three classes (highly variable, moderately variable, and invariable) and by assuming that the last common mtDNA ancestor of humans and chimpanzees lived 4 million years ago, the most recent common mtDNA ancestor of humans is estimated to have occurred 211,000 +/- 111,000 years ago (+/- 1 SE), consistent with the estimate, 101,000 +/- 52,000 years, made from third positions of codons and also with those proposed previously. We used the same technique to estimate when a putative expansion of modern humans out of Africa took place and estimated a time of 89,000 +/- 69,000 years ago. Even though the standard errors of these estimates are large, they allow us to reject the multiregional hypothesis of modern human origin.

  14. Using minimum DNA marker loci for accurate population classification in rice (Oryza sativa L.)

    USDA-ARS?s Scientific Manuscript database

    Using few DNA markers to classify genetic background of a germplasm pool will help breeders make a quick decision while saving time and resources. WHICHLOCI is a computer program that selects the best combination of loci for population assignment through empiric analysis of molecular marker data. Th...

  15. Accurate quantification of DNA methylation of DRD4 applying capillary gel electrophoresis with LIF detection.

    PubMed

    Goedecke, Simon; Schlosser, Sabrina; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

    2009-04-01

    Aberrant DNA methylation of gene promoters may be investigated by an array of different technologies. Besides DNA sequencing techniques following bisulfite treatment and determination of overall methylation by quantification of 5-methylcytosine/cytosine ratio following DNA hydrolysis, most approaches rely on PCR amplification of a defined template and subsequent analysis by conventional gel electrophoresis. As an additional analytical tool, a capillary gel electrophoresis method has been developed to quantify the methylation in combined bisulfite restriction analysis products of the gene dopamine receptor D4 (DRD4). Analyses were carried out in a bare fused-silica capillary dynamically coated with a 1% w/w solution of PVA (M(r)=72,000). A buffer (pH 7.3) containing 3% w/w 2-hydroxyethylcellulose (M(nu) approximately 90,000 g/mol) was used as sieving matrix. With 1/x weighted regression the accuracy (bias) of the method is within +/-10% and the precision (expressed as RSD) also meets the common acceptance criteria of 15% (20% near lower LOQ). It overcomes the limitations of standard gel electrophoresis, which allows only one single run per analysis and requires large amounts of DNA. Therefore, the method represents a valuable tool for routine quantitative analysis of the methylation status of DRD4 and other target genes.

  16. Exposure to Inorganic Arsenic Is Associated with Increased Mitochondrial DNA Copy Number and Longer Telomere Length in Peripheral Blood

    PubMed Central

    Ameer, Syeda S.; Xu, YiYi; Engström, Karin; Li, Huiqi; Tallving, Pia; Nermell, Barbro; Boemo, Analia; Parada, Luis A.; Peñaloza, Lidia G.; Concha, Gabriela; Harari, Florencia; Vahter, Marie; Broberg, Karin

    2016-01-01

    Background: Exposure to inorganic arsenic (iAs) through drinking water causes cancer. Alterations in mitochondrial DNA copy number (mtDNAcn) and telomere length in blood have been associated with cancer risk. We elucidated if arsenic exposure alters mtDNAcn and telomere length in individuals with different arsenic metabolizing capacity. Methods: We studied two groups in the Salta province, Argentina, one in the Puna area of the Andes (N = 264, 89% females) and one in Chaco (N = 169, 75% females). We assessed arsenic exposure as the sum of arsenic metabolites [iAs, methylarsonic acid (MMA), dimethylarsinic acid (DMA)] in urine (U-As) using high-performance liquid chromatography coupled with hydride generation and inductively coupled plasma mass spectrometry. Efficiency of arsenic metabolism was expressed as percentage of urinary metabolites. MtDNAcn and telomere length were determined in blood by real-time PCR. Results: Median U-As was 196 (5–95 percentile: 21–537) μg/L in Andes and 80 (5–95 percentile: 15–1637) μg/L in Chaco. The latter study group had less-efficient metabolism, with higher %iAs and %MMA in urine compared with the Andean group. U-As was significantly associated with increased mtDNAcn (log2 transformed to improve linearity) in Chaco (β = 0.027 per 100 μg/L, p = 0.0085; adjusted for age and sex), but not in Andes (β = 0.025, p = 0.24). U-As was also associated with longer telomere length in Chaco (β = 0.016, p = 0.0066) and Andes (β = 0.0075, p = 0.029). In both populations, individuals with above median %iAs showed significantly higher mtDNAcn and telomere length compared with individuals with below median %iAs. Conclusions: Arsenic was associated with increased mtDNAcn and telomere length, particularly in individuals with less-efficient arsenic metabolism, a group who may have increased risk for arsenic-related cancer. PMID:27597942

  17. Prevalence and pathogen load estimates for the fungus Batrachochytrium dendrobatidis are impacted by ITS DNA copy number variation.

    PubMed

    Rebollar, Eria A; Woodhams, Douglas C; LaBumbard, Brandon; Kielgast, Jos; Harris, Reid N

    2017-03-21

    The ribosomal gene complex is a multi-copy region that is widely used for phylogenetic analyses of organisms from all 3 domains of life. In fungi, the copy number of the internal transcribed spacer (ITS) is used to detect abundance of pathogens causing diseases such as chytridiomycosis in amphibians and white nose syndrome in bats. Chytridiomycosis is caused by the fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), and is responsible for declines and extinctions of amphibians worldwide. Over a decade ago, a qPCR assay was developed to determine Bd prevalence and pathogen load. Here, we demonstrate the effect that ITS copy number variation in Bd strains can have on the estimation of prevalence and pathogen load. We used data sets from different amphibian species to simulate how ITS copy number affects prevalence and pathogen load. In addition, we tested 2 methods (gBlocks® synthetic standards and digital PCR) to determine ITS copy number in Bd strains. Our results show that assumptions about the ITS copy number can lead to under- or overestimation of Bd prevalence and pathogen load. The use of synthetic standards replicated previously published estimates of ITS copy number, whereas dPCR resulted in estimates that were consistently lower than previously published estimates. Standardizing methods will assist with comparison across studies and produce reliable estimates of prevalence and pathogen load in the wild, while using the same Bd strain for exposure experiments and zoospore standards in qPCR remains the best method for estimating parameters used in epidemiological studies.

  18. Accurate zygote-specific discrimination of single-nucleotide polymorphisms using microfluidic electrochemical DNA melting curves.

    PubMed

    Yang, Allen H J; Hsieh, Kuangwen; Patterson, Adriana S; Ferguson, B Scott; Eisenstein, Michael; Plaxco, Kevin W; Soh, H Tom

    2014-03-17

    We report the first electrochemical system for the detection of single-nucleotide polymorphisms (SNPs) that can accurately discriminate homozygous and heterozygous genotypes using microfluidics technology. To achieve this, our system performs real-time melting-curve analysis of surface-immobilized hybridization probes. As an example, we used our sensor to analyze two SNPs in the apolipoprotein E (ApoE) gene, where homozygous and heterozygous mutations greatly affect the risk of late-onset Alzheimer's disease. Using probes specific for each SNP, we simultaneously acquired melting curves for probe-target duplexes at two different loci and thereby accurately distinguish all six possible ApoE allele combinations. Since the design of our device and probes can be readily adapted for targeting other loci, we believe that our method offers a modular platform for the diagnosis of SNP-based diseases and personalized medicine. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. A chromatin structure-based model accurately predicts DNA replication timing in human cells.

    PubMed

    Gindin, Yevgeniy; Valenzuela, Manuel S; Aladjem, Mirit I; Meltzer, Paul S; Bilke, Sven

    2014-03-28

    The metazoan genome is replicated in precise cell lineage-specific temporal order. However, the mechanism controlling this orchestrated process is poorly understood as no molecular mechanisms have been identified that actively regulate the firing sequence of genome replication. Here, we develop a mechanistic model of genome replication capable of predicting, with accuracy rivaling experimental repeats, observed empirical replication timing program in humans. In our model, replication is initiated in an uncoordinated (time-stochastic) manner at well-defined sites. The model contains, in addition to the choice of the genomic landmark that localizes initiation, only a single adjustable parameter of direct biological relevance: the number of replication forks. We find that DNase-hypersensitive sites are optimal and independent determinants of DNA replication initiation. We demonstrate that the DNA replication timing program in human cells is a robust emergent phenomenon that, by its very nature, does not require a regulatory mechanism determining a proper replication initiation firing sequence.

  20. Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR.

    PubMed

    Blanes, Milena S; Tsoi, Stephen C M; Dyck, Michael K

    2016-02-14

    Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo's sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.

  1. Basal-like Breast cancer DNA copy number losses identify genes involved in genomic instability, response to therapy, and patient survival.

    PubMed

    Weigman, Victor J; Chao, Hann-Hsiang; Shabalin, Andrey A; He, Xiaping; Parker, Joel S; Nordgard, Silje H; Grushko, Tatyana; Huo, Dezheng; Nwachukwu, Chika; Nobel, Andrew; Kristensen, Vessela N; Børresen-Dale, Anne-Lise; Olopade, Olufunmilayo I; Perou, Charles M

    2012-06-01

    Breast cancer is a heterogeneous disease with known expression-defined tumor subtypes. DNA copy number studies have suggested that tumors within gene expression subtypes share similar DNA Copy number aberrations (CNA) and that CNA can be used to further sub-divide expression classes. To gain further insights into the etiologies of the intrinsic subtypes, we classified tumors according to gene expression subtype and next identified subtype-associated CNA using a novel method called SWITCHdna, using a training set of 180 tumors and a validation set of 359 tumors. Fisher's exact tests, Chi-square approximations, and Wilcoxon rank-sum tests were performed to evaluate differences in CNA by subtype. To assess the functional significance of loss of a specific chromosomal region, individual genes were knocked down by shRNA and drug sensitivity, and DNA repair foci assays performed. Most tumor subtypes exhibited specific CNA. The Basal-like subtype was the most distinct with common losses of the regions containing RB1, BRCA1, INPP4B, and the greatest overall genomic instability. One Basal-like subtype-associated CNA was loss of 5q11-35, which contains at least three genes important for BRCA1-dependent DNA repair (RAD17, RAD50, and RAP80); these genes were predominantly lost as a pair, or all three simultaneously. Loss of two or three of these genes was associated with significantly increased genomic instability and poor patient survival. RNAi knockdown of RAD17, or RAD17/RAD50, in immortalized human mammary epithelial cell lines caused increased sensitivity to a PARP inhibitor and carboplatin, and inhibited BRCA1 foci formation in response to DNA damage. These data suggest a possible genetic cause for genomic instability in Basal-like breast cancers and a biological rationale for the use of DNA repair inhibitor related therapeutics in this breast cancer subtype.

  2. More comprehensive forensic genetic marker analyses for accurate human remains identification using massively parallel DNA sequencing.

    PubMed

    Ambers, Angie D; Churchill, Jennifer D; King, Jonathan L; Stoljarova, Monika; Gill-King, Harrell; Assidi, Mourad; Abu-Elmagd, Muhammad; Buhmeida, Abdelbaset; Al-Qahtani, Mohammed; Budowle, Bruce

    2016-10-17

    Although the primary objective of forensic DNA analyses of unidentified human remains is positive identification, cases involving historical or archaeological skeletal remains often lack reference samples for comparison. Massively parallel sequencing (MPS) offers an opportunity to provide biometric data in such cases, and these cases provide valuable data on the feasibility of applying MPS for characterization of modern forensic casework samples. In this study, MPS was used to characterize 140-year-old human skeletal remains discovered at a historical site in Deadwood, South Dakota, United States. The remains were in an unmarked grave and there were no records or other metadata available regarding the identity of the individual. Due to the high throughput of MPS, a variety of biometric markers could be typed using a single sample. Using MPS and suitable forensic genetic markers, more relevant information could be obtained from a limited quantity and quality sample. Results were obtained for 25/26 Y-STRs, 34/34 Y SNPs, 166/166 ancestry-informative SNPs, 24/24 phenotype-informative SNPs, 102/102 human identity SNPs, 27/29 autosomal STRs (plus amelogenin), and 4/8 X-STRs (as well as ten regions of mtDNA). The Y-chromosome (Y-STR, Y-SNP) and mtDNA profiles of the unidentified skeletal remains are consistent with the R1b and H1 haplogroups, respectively. Both of these haplogroups are the most common haplogroups in Western Europe. Ancestry-informative SNP analysis also supported European ancestry. The genetic results are consistent with anthropological findings that the remains belong to a male of European ancestry (Caucasian). Phenotype-informative SNP data provided strong support that the individual had light red hair and brown eyes. This study is among the first to genetically characterize historical human remains with forensic genetic marker kits specifically designed for MPS. The outcome demonstrates that substantially more genetic information can be obtained from

  3. Design of accurate predictors for DNA-binding sites in proteins using hybrid SVM-PSSM method.

    PubMed

    Ho, Shinn-Ying; Yu, Fu-Chieh; Chang, Chia-Yun; Huang, Hui-Ling

    2007-01-01

    In this paper, we investigate the design of accurate predictors for DNA-binding sites in proteins from amino acid sequences. As a result, we propose a hybrid method using support vector machine (SVM) in conjunction with evolutionary information of amino acid sequences in terms of their position-specific scoring matrices (PSSMs) for prediction of DNA-binding sites. Considering the numbers of binding and non-binding residues in proteins are significantly unequal, two additional weights as well as SVM parameters are analyzed and adopted to maximize net prediction (NP, an average of sensitivity and specificity) accuracy. To evaluate the generalization ability of the proposed method SVM-PSSM, a DNA-binding dataset PDC-59 consisting of 59 protein chains with low sequence identity on each other is additionally established. The SVM-based method using the same six-fold cross-validation procedure and PSSM features has NP=80.15% for the training dataset PDNA-62 and NP=69.54% for the test dataset PDC-59, which are much better than the existing neural network-based method by increasing the NP values for training and test accuracies up to 13.45% and 16.53%, respectively. Simulation results reveal that SVM-PSSM performs well in predicting DNA-binding sites of novel proteins from amino acid sequences.

  4. Binding of nuclear factors to a satellite DNA of retroviral origin with marked differences in copy number among species of the rodent Ctenomys.

    PubMed Central

    Pesce, C G; Rossi, M S; Muro, A F; Reig, O A; Zorzópulos, J; Kornblihtt, A R

    1994-01-01

    The major satellite DNA of the subterranean rodent Ctenomys, named RPCS, contains several consensus sequences characteristic of the U3 region of retroviral long terminal repeats (LTRs), such as a polypurine tract, CCAAT boxes, binding sites for the CCAAT/enhancer-binding protein (C/EBP), a TATA box and putative polyadenylation signals. RPCS presents an enormous variation in abundance between species of the same genus: while C. australis or C. talarum have approximately 3 x 10(6) copies per genome, C. opimus has none. A sequence (RPCS-I) with identity to the SV40-enhancer core element, present in all the repeating units of the satellite is specifically protected in DNase I footprintings. Competitions of band-shift assays with different transcription factor binding sites indicate that binding to RPCS-I is specific and involves CCAAT proteins related to NF-1, but not to C/EBP. By the use of quantitative protein/DNA binding assays we determined that, despite of their conspicuous difference in RPCS copy number, C. talarum and C. opimus have equivalent amounts and identical quality of RPCS-binding proteins. These results are consistent with the observation, by in situ hybridization, that RPCS is clustered in heterochromatic regions, where it might have restricted accessibility to transcription factors in vivo. This is the first report of the binding of transcription factors to a satellite DNA of retroviral origin. Images PMID:8127714

  5. Poisonous or non-poisonous plants? DNA-based tools and applications for accurate identification.

    PubMed

    Mezzasalma, Valerio; Ganopoulos, Ioannis; Galimberti, Andrea; Cornara, Laura; Ferri, Emanuele; Labra, Massimo

    2017-01-01

    Plant exposures are among the most frequently reported cases to poison control centres worldwide. This is a growing condition due to recent societal trends oriented towards the consumption of wild plants as food, cosmetics, or medicine. At least three general causes of plant poisoning can be identified: plant misidentification, introduction of new plant-based supplements and medicines with no controls about their safety, and the lack of regulation for the trading of herbal and phytochemical products. Moreover, an efficient screening for the occurrence of plants poisonous to humans is also desirable at the different stages of the food supply chain: from the raw material to the final transformed product. A rapid diagnosis of intoxication cases is necessary in order to provide the most reliable treatment. However, a precise taxonomic characterization of the ingested species is often challenging. In this review, we provide an overview of the emerging DNA-based tools and technologies to address the issue of poisonous plant identification. Specifically, classic DNA barcoding and its applications using High Resolution Melting (Bar-HRM) ensure high universality and rapid response respectively, whereas High Throughput Sequencing techniques (HTS) provide a complete characterization of plant residues in complex matrices. The pros and cons of each approach have been evaluated with the final aim of proposing a general user's guide to molecular identification directed to different stakeholder categories interested in the diagnostics of poisonous plants.

  6. Nucleobase-functionalized graphene nanoribbons for accurate high-speed DNA sequencing

    NASA Astrophysics Data System (ADS)

    Paulechka, Eugene; Wassenaar, Tsjerk A.; Kroenlein, Kenneth; Kazakov, Andrei; Smolyanitsky, Alex

    2016-01-01

    We propose a water-immersed nucleobase-functionalized suspended graphene nanoribbon as an intrinsically selective device for nucleotide detection. The proposed sensing method combines Watson-Crick selective base pairing with graphene's capacity for converting anisotropic lattice strain to changes in an electrical current at the nanoscale. Using detailed atomistic molecular dynamics (MD) simulations, we study sensor operation at ambient conditions. We combine simulated data with theoretical arguments to estimate the levels of measurable electrical signal variation in response to strains and determine that the proposed sensing mechanism shows significant promise for realistic DNA sensing devices without the need for advanced data processing, or highly restrictive operational conditions.We propose a water-immersed nucleobase-functionalized suspended graphene nanoribbon as an intrinsically selective device for nucleotide detection. The proposed sensing method combines Watson-Crick selective base pairing with graphene's capacity for converting anisotropic lattice strain to changes in an electrical current at the nanoscale. Using detailed atomistic molecular dynamics (MD) simulations, we study sensor operation at ambient conditions. We combine simulated data with theoretical arguments to estimate the levels of measurable electrical signal variation in response to strains and determine that the proposed sensing mechanism shows significant promise for realistic DNA sensing devices without the need for advanced data processing, or highly restrictive operational conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07061a

  7. Localization of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplification.

    PubMed

    Khrustaleva, L I; Kik, C

    2001-03-01

    The sensitivity of fluorescence in situ hybridization (FISH) for mapping plant chromosomes of single-copy DNA sequences is limited. We have adapted for plant cytogenetics a new signal-amplification method termed tyramide-FISH (Tyr-FISH). Until present this technique has only been applied to human chromosomes. The method is based on enzymatic deposition of fluorochrome-conjugated tyramide. With Tyr-FISH it was possible to detect target T-DNA sequences on plant metaphase chromosomes as small as 710 bp without using a cooled CCD camera. Short detection time and high sensitivity, in combination with a low background, make the Tyr-FISH method very suitable for routine application in plant cytogenetic research. With Tyr-FISH we analysed the position of T-DNA inserts in transgenic shallots. We found that the inserts were preferentially located in the distal region of metaphase chromosomes. Sequential fluorescence in situ hybridization with a 375 bp satellite sequence suggested that a specific T-DNA insert was located within the satellite sequence hybridization region on a metaphase chromosome. Analysis of less-condensed prophase and interphase chromosomes revealed that the T-DNA was integrated outside the satellite DNA-hybridization region in a more proximal euchromatin region.

  8. Integrative analysis of DNA copy number and gene expression in metastatic oral squamous cell carcinoma identifies genes associated with poor survival

    PubMed Central

    2010-01-01

    Background Lymphotropism in oral squamous cell carcinoma (OSCC) is one of the most important prognostic factors of 5-year survival. In an effort to identify genes that may be responsible for the initiation of OSCC lymphotropism, we examined DNA copy number gains and losses and corresponding gene expression changes from tumor cells in metastatic lymph nodes of patients with OSCC. Results We performed integrative analysis of DNA copy number alterations (CNA) and corresponding mRNA expression from OSCC cells isolated from metastatic lymph nodes of 20 patients using Affymetrix 250 K Nsp I SNP and U133 Plus 2.0 arrays, respectively. Overall, genome CNA accounted for expression changes in 31% of the transcripts studied. Genome region 11q13.2-11q13.3 shows the highest correlation between DNA CNA and expression. With a false discovery rate < 1%, 530 transcripts (461 genes) demonstrated a correlation between CNA and expression. Among these, we found two subsets that were significantly associated with OSCC (n = 122) when compared to controls, and with survival (n = 27), as tested using an independent dataset with genome-wide expression profiles for 148 primary OSCC and 45 normal oral mucosa. We fit Cox models to calculate a principal component analysis-derived risk-score for these two gene sets ('122-' or '27-transcript PC'). The models combining the 122- or 27-transcript PC with stage outperformed the model using stage alone in terms of the Area Under the Curve (AUC = 0.82 or 0.86 vs. 0.72, with p = 0.044 or 0.011, respectively). Conclusions Genes exhibiting CNA-correlated expression may have biological impact on carcinogenesis and cancer progression in OSCC. Determination of copy number-associated transcripts associated with clinical outcomes in tumor cells with an aggressive phenotype (i.e., cells metastasized to the lymph nodes) can help prioritize candidate transcripts from high-throughput data for further studies. PMID:20537188

  9. Cross-species DNA copy number analyses identifies multiple 1q21-q23 subtype-specific driver genes for breast cancer.

    PubMed

    Silva, Grace O; He, Xiaping; Parker, Joel S; Gatza, Michael L; Carey, Lisa A; Hou, Jack P; Moulder, Stacy L; Marcom, Paul K; Ma, Jian; Rosen, Jeffrey M; Perou, Charles M

    2015-07-01

    A large number of DNA copy number alterations (CNAs) exist in human breast cancers, and thus characterizing the most frequent CNAs is key to advancing therapeutics because it is likely that these regions contain breast tumor 'drivers' (i.e., cancer causal genes). This study aims to characterize the genomic landscape of breast cancer CNAs and identify potential subtype-specific drivers using a large set of human breast tumors and genetically engineered mouse (GEM) mammary tumors. Using a novel method called SWITCHplus, we identified subtype-specific DNA CNAs occurring at a 15% or greater frequency, which excluded many well-known breast cancer-related drivers such as amplification of ERBB2, and deletions of TP53 and RB1. A comparison of CNAs between mouse and human breast tumors identified regions with shared subtype-specific CNAs. Additional criteria that included gene expression-to-copy number correlation, a DawnRank network analysis, and RNA interference functional studies highlighted candidate driver genes that fulfilled these multiple criteria. Numerous regions of shared CNAs were observed between human breast tumors and GEM mammary tumor models that shared similar gene expression features. Specifically, we identified chromosome 1q21-23 as a Basal-like subtype-enriched region with multiple potential driver genes including PI4KB, SHC1, and NCSTN. This step-wise computational approach based on a cross-species comparison is applicable to any tumor type for which sufficient human and model system DNA copy number data exist, and in this instance, highlights that a single region of amplification may in fact harbor multiple driver genes.

  10. ArrayExplorer, a program in Visual Basic for robust and accurate filter cDNA array analysis.

    PubMed

    Patriotis, P C; Querec, T D; Gruver, B N; Brown, T R; Patriotis, C

    2001-10-01

    Determining the dynamics in the global regulation of gene expression holds the promise of bringing a better understanding of the processes that govern physiological cell growth regulation and its disruption during the development of disease. The advent for cDNA arrays has created the possibility for the parallel analysis of expression of thousands of genes in a given cell population, simultaneously. The level of expression of a given set of genes within the studied tissue corresponds to the intensity of a labeled cDNA probe synthesized from the studied tissue RNA and bound specifically to the cDNAs of the genes spotted on the array. The accurate extraction of gene expression intensity values is essential for further data analysis and the interpretation of the obtained results. Here, we describe a new array image-processing software developed in Microsoft Visual Basic, the ArrayExplorer, which provides a user-friendly, multiple-window interface and a number of automatic and manual features that facilitate a reliable, robust, and accurate extraction of gene intensity values from filter-array images.

  11. TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants

    PubMed Central

    Sulak, Michael; Fong, Lindsey; Mika, Katelyn; Chigurupati, Sravanthi; Yon, Lisa; Mongan, Nigel P; Emes, Richard D; Lynch, Vincent J

    2016-01-01

    A major constraint on the evolution of large body sizes in animals is an increased risk of developing cancer. There is no correlation, however, between body size and cancer risk. This lack of correlation is often referred to as 'Peto's Paradox'. Here, we show that the elephant genome encodes 20 copies of the tumor suppressor gene TP53 and that the increase in TP53 copy number occurred coincident with the evolution of large body sizes, the evolution of extreme sensitivity to genotoxic stress, and a hyperactive TP53 signaling pathway in the elephant (Proboscidean) lineage. Furthermore, we show that several of the TP53 retrogenes (TP53RTGs) are transcribed and likely translated. While TP53RTGs do not appear to directly function as transcription factors, they do contribute to the enhanced sensitivity of elephant cells to DNA damage and the induction of apoptosis by regulating activity of the TP53 signaling pathway. These results suggest that an increase in the copy number of TP53 may have played a direct role in the evolution of very large body sizes and the resolution of Peto's paradox in Proboscideans. DOI: http://dx.doi.org/10.7554/eLife.11994.001 PMID:27642012

  12. TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants.

    PubMed

    Sulak, Michael; Fong, Lindsey; Mika, Katelyn; Chigurupati, Sravanthi; Yon, Lisa; Mongan, Nigel P; Emes, Richard D; Lynch, Vincent J

    2016-09-19

    A major constraint on the evolution of large body sizes in animals is an increased risk of developing cancer. There is no correlation, however, between body size and cancer risk. This lack of correlation is often referred to as 'Peto's Paradox'. Here, we show that the elephant genome encodes 20 copies of the tumor suppressor gene TP53 and that the increase in TP53 copy number occurred coincident with the evolution of large body sizes, the evolution of extreme sensitivity to genotoxic stress, and a hyperactive TP53 signaling pathway in the elephant (Proboscidean) lineage. Furthermore, we show that several of the TP53 retrogenes (TP53RTGs) are transcribed and likely translated. While TP53RTGs do not appear to directly function as transcription factors, they do contribute to the enhanced sensitivity of elephant cells to DNA damage and the induction of apoptosis by regulating activity of the TP53 signaling pathway. These results suggest that an increase in the copy number of TP53 may have played a direct role in the evolution of very large body sizes and the resolution of Peto's paradox in Proboscideans.

  13. Accurate and Efficient Bypass of 8,5'-Cyclopurine-2'-Deoxynucleosides by Human and Yeast DNA Polymerase η

    PubMed Central

    Swanson, Ashley L.; Wang, Jianshuang; Wang, Yinsheng

    2012-01-01

    Reactive oxygen species (ROS), which can be produced during normal aerobic metabolism, can induce the formation of tandem DNA lesions, including 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA) and 8,5'-cyclo-2'-deoxyguanosine (cyclo-dG). Previous studies have shown that cyclo-dA and cyclo-dG accumulate in cells and can block mammalian RNA polymerase II and replicative DNA polymerases. Here, we used primer extension and steady-state kinetic assays to examine the efficiency and fidelity for polymerase η to insert nucleotides opposite, and extend primer past, these cyclopurine lesions. We found that Saccharomyces cerevisiae and human polymerase η inserted 2'-deoxynucleotides opposite cyclo-dA, cyclo-dG, and their adjacent 5' nucleosides at fidelities and efficiencies that were similar as their respective undamaged nucleosides. Moreover, the yeast enzyme exhibited similar processivity in DNA synthesis on templates housing a cyclo-dA or cyclo-dG as those carrying an unmodified dA or dG; the human polymerase, however, dissociated from the primer-template complex after inserting one or two additional nucleotides after the lesion. Pol η's accurate and efficient bypass of cyclo-dA and cyclo-dG indicate that this polymerase is likely responsible for error-free bypass of these lesions, whereas mutagenic bypass of these lesions may involve other translesion synthesis DNA polymerases. Together, our results suggested that pol η may have an additional function in cells, i.e., to alleviate the cellular burden of endogenously induced DNA lesions, including cyclo-dA and cyclo-dG. PMID:22768970

  14. Accurate and efficient bypass of 8,5'-cyclopurine-2'-deoxynucleosides by human and yeast DNA polymerase η.

    PubMed

    Swanson, Ashley L; Wang, Jianshuang; Wang, Yinsheng

    2012-08-20

    Reactive oxygen species (ROS), which can be produced during normal aerobic metabolism, can induce the formation of tandem DNA lesions, including 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA) and 8,5'-cyclo-2'-deoxyguanosine (cyclo-dG). Previous studies have shown that cyclo-dA and cyclo-dG accumulate in cells and can block mammalian RNA polymerase II and replicative DNA polymerases. Here, we used primer extension and steady-state kinetic assays to examine the efficiency and fidelity for polymerase η to insert nucleotides opposite, and extend primer past, these cyclopurine lesions. We found that Saccharomyces cerevisiae and human polymerase η inserted 2'-deoxynucleotides opposite cyclo-dA, cyclo-dG and their adjacent 5' nucleosides at fidelities and efficiencies that were similar to those of their respective undamaged nucleosides. Moreover, the yeast enzyme exhibited similar processivity in DNA synthesis on templates housing a cyclo-dA or cyclo-dG to those carrying an unmodified dA or dG; the human polymerase, however, dissociated from the primer-template complex after inserting one or two additional nucleotides after the lesion. Pol η's accurate and efficient bypass of cyclo-dA and cyclo-dG indicates that this polymerase is likely responsible for error-free bypass of these lesions, whereas mutagenic bypass of these lesions may involve other translesion synthesis DNA polymerases. Together, our results suggested that pol η may have an additional function in cells, i.e., to alleviate the cellular burden of endogenously induced DNA lesions, including cyclo-dA and cyclo-dG.

  15. Integrated Genome-wide DNA Copy Number and Expression Analysis Identifies Distinct Mechanisms of Primary Chemo-resistance in Ovarian Carcinomas

    PubMed Central

    Etemadmoghadam, Dariush; deFazio, Anna; Beroukhim, Rameen; Mermel, Craig; George, Joshy; Getz, Gaddy; Tothill, Richard; Okamoto, Aikou; Raeder, Maria B; Harnett, Paul; Lade, Stephen; Akslen, Lars A; Tinker, Anna; Locandro, Bianca; Alsop, Kathryn; Chiew, Yoke-Eng; Traficante, Nadia; Fereday, Sian; Johnson, Daryl; Fox, Stephen; Sellers, William; Urashima, Mitsuyoshi; Salvesen, Helga B; Meyerson, Matthew; Bowtell, David

    2009-01-01

    Purpose A significant number of women with serous ovarian cancer are intrinsically refractory to platinum-taxol based treatment. We analyzed somatic DNA copy number variation (CNV) and gene expression data to identify key mechanisms associated with primary resistance in advanced-stage serous cancers. Experimental Design Genome-wide CNV was measured in 118 ovarian tumors using high-resolution oligonucleotide microarrays. A well-defined subset of 85 advanced-stage serous tumors was then used to relate CNV to primary resistance to treatment. The discovery-based approach was complemented by quantitative-PCR copy number analysis of twelve candidate genes as independent validation of previously reported associations with clinical outcome. Likely CNV targets and tumor molecular subtypes were further characterized by gene expression profiling. Results Amplification of 19q12, containing Cyclin E (CCNE1) and 20q11.22-q13.12, mapping immediately adjacent to the steroid receptor co-activator NCOA3, were significantly associated with poor response to primary treatment. Other genes previously associated with CNV and clinical outcome in ovarian cancer were not associated with primary treatment resistance. Chemoresistant tumors with high CCNE1 copy number and protein expression were associated with increased cellular proliferation but so too were a subset of treatment responsive patients, suggesting a cell-cycle independent role for CCNE1 in modulating chemoresponse. Patients with a poor clinical outcome without CCNE1 amplification over expressed genes involved in extracellular matrix deposition. Conclusions We have identified two distinct mechanisms of primary treatment failure in serous ovarian cancer, involving CCNE1 amplification and enhanced extracellular matrix deposition. CCNE1 copy number is validated as a dominant marker of patient outcome in ovarian cancer. PMID:19193619

  16. Reticulate evolution in diploid and tetraploid species of Polystachya (Orchidaceae) as shown by plastid DNA sequences and low-copy nuclear genes

    PubMed Central

    Russell, Anton; Samuel, Rosabelle; Klejna, Verena; Barfuss, Michael H. J.; Rupp, Barbara; Chase, Mark W.

    2010-01-01

    Background and Aims Here evidence for reticulation in the pantropical orchid genus Polystachya is presented, using gene trees from five nuclear and plastid DNA data sets, first among only diploid samples (homoploid hybridization) and then with the inclusion of cloned tetraploid sequences (allopolyploids). Two groups of tetraploids are compared with respect to their origins and phylogenetic relationships. Methods Sequences from plastid regions, three low-copy nuclear genes and ITS nuclear ribosomal DNA were analysed for 56 diploid and 17 tetraploid accessions using maximum parsimony and Bayesian inference. Reticulation was inferred from incongruence between gene trees using supernetwork and consensus network analyses and from cloning and sequencing duplicated loci in tetraploids. Key Results Diploid trees from individual loci showed considerable incongruity but little reticulation signal when support from more than one gene tree was required to infer reticulation. This was coupled with generally low support in the individual gene trees. Sequencing the duplicated gene copies in tetraploids showed clearer evidence of hybrid evolution, including multiple origins of one group of tetraploids included in the study. Conclusions A combination of cloning duplicate gene copies in allotetraploids and consensus network comparison of gene trees allowed a phylogenetic framework for reticulation in Polystachya to be built. There was little evidence for homoploid hybridization, but our knowledge of the origins and relationships of three groups of allotetraploids are greatly improved by this study. One group showed evidence of multiple long-distance dispersals to achieve a pantropical distribution; another showed no evidence of multiple origins or long-distance dispersal but had greater morphological variation, consistent with hybridization between more distantly related parents. PMID:20525745

  17. Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR

    PubMed Central

    Humphrey, Bruce; McLeod, Neil; Turner, Carrie; Sutton, J. Mark; Dark, Paul M.; Warhurst, Geoffrey

    2015-01-01

    Background More than two decades after its discovery, contaminant microbial DNA in PCR reagents continues to impact the sensitivity and integrity of broad-range PCR diagnostic techniques. This is particularly relevant to their use in the setting of human sepsis, where a successful diagnostic on blood samples needs to combine universal bacterial detection with sensitivity to 1-2 genome copies, because low levels of a broad range of bacteria are implicated. Results We investigated the efficacy of ethidium monoazide (EMA) and propidium monoazide (PMA) treatment as emerging methods for the decontamination of PCR reagents. Both treatments were able to inactivate contaminating microbial DNA but only at concentrations that considerably affected assay sensitivity. Increasing amplicon length improved EMA/PMA decontamination efficiency but at the cost of assay sensitivity. The same was true for UV exposure as an alternative decontamination strategy, likely due to damage sustained by oligonucleotide primers which were a significant source of contamination. However, a simple combination strategy with UV-treated PCR reagents paired with EMA-treated primers produced an assay capable of two genome copy detection and a <5% contamination rate. This decontamination strategy could have important utility in developing improved pan-bacterial assays for rapid diagnosis of low pathogen burden conditions such as in the blood of patients with suspected blood stream infection. PMID:26172943

  18. Removal of Contaminant DNA by Combined UV-EMA Treatment Allows Low Copy Number Detection of Clinically Relevant Bacteria Using Pan-Bacterial Real-Time PCR.

    PubMed

    Humphrey, Bruce; McLeod, Neil; Turner, Carrie; Sutton, J Mark; Dark, Paul M; Warhurst, Geoffrey

    2015-01-01

    More than two decades after its discovery, contaminant microbial DNA in PCR reagents continues to impact the sensitivity and integrity of broad-range PCR diagnostic techniques. This is particularly relevant to their use in the setting of human sepsis, where a successful diagnostic on blood samples needs to combine universal bacterial detection with sensitivity to 1-2 genome copies, because low levels of a broad range of bacteria are implicated. We investigated the efficacy of ethidium monoazide (EMA) and propidium monoazide (PMA) treatment as emerging methods for the decontamination of PCR reagents. Both treatments were able to inactivate contaminating microbial DNA but only at concentrations that considerably affected assay sensitivity. Increasing amplicon length improved EMA/PMA decontamination efficiency but at the cost of assay sensitivity. The same was true for UV exposure as an alternative decontamination strategy, likely due to damage sustained by oligonucleotide primers which were a significant source of contamination. However, a simple combination strategy with UV-treated PCR reagents paired with EMA-treated primers produced an assay capable of two genome copy detection and a <5% contamination rate. This decontamination strategy could have important utility in developing improved pan-bacterial assays for rapid diagnosis of low pathogen burden conditions such as in the blood of patients with suspected blood stream infection.

  19. Chopping Copy.

    ERIC Educational Resources Information Center

    Bush, Don

    1994-01-01

    Discusses ways an editor can cut out words to help the reader understand quickly. Discusses dead wood, redundancy, redundancy in thought, smothered verbs, false precision, editing and academia, and making copy smoother. (SR)

  20. Mitochondrial DNA copy number augments performance of A1C and oral glucose tolerance testing in the prediction of type 2 diabetes

    PubMed Central

    Cho, Seong Beom; Koh, InSong; Nam, Hye-Young; Jeon, Jae-Pil; Lee, Hong Kyu; Han, Bok-Ghee

    2017-01-01

    Here, we tested the performance of the mitochondrial DNA copy number (mtDNA-CN) in predicting future type 2 diabetes (n = 1108). We used the baseline clinical data (age, sex, body mass index, waist-to-hip ratio, systolic and diastolic blood pressure) and the mtDNA-CN, hemoglobin A1c (A1C) levels and results of oral glucose tolerance test (OGTT) including fasting plasma glucose, 1-hour glucose, and 2-hour glucose levels, to predict future diabetes. We built a prediction model using the baseline data and the diabetes status at biannual follow-up of 8 years. The mean area under curve (AUC) for all follow-ups of the full model including all variables was 0.92 ± 0.04 (mean ± standard deviation), while that of the model excluding the mtDNA-CN was 0.90 ± 0.03. The sensitivity of the f4ull model was much greater than that of the model not including mtDNA-CN: the mean sensitivities of the model with and without mtDNA-CN were 0.60 ± 0.06 and 0.53 ± 0.04, respectively. We found that the mtDNA-CN of peripheral leukocytes is a biomarker that augments the predictive power for future diabetes of A1C and OGTT. We believe that these results could provide invaluable information for developing strategies for the management of diabetes. PMID:28251996

  1. Maternal age and ovarian stimulation independently affect oocyte mtDNA copy number and cumulus cell gene expression in bovine clones.

    PubMed

    Cree, Lynsey M; Hammond, Elizabeth R; Shelling, Andrew N; Berg, Martin C; Peek, John C; Green, Mark P

    2015-06-01

    Does maternal ageing and ovarian stimulation alter mitochondrial DNA (mtDNA) copy number and gene expression of oocytes and cumulus cells from a novel bovine model for human IVF? Oocytes collected from females with identical nuclear genetics show decreased mtDNA copy number and increased expression of an endoplasmic reticulum (ER) stress gene with repect to ovarian stimulation, whilst differences in the expression of genes involved in mitochondrial function, antioxidant protection and apoptosis were evident in relation to maternal ageing and the degree of ovarian stimulation in cumulus cells. Oocyte quality declines with advancing maternal age; however, the underlying mechanism, as well as the effects of ovarian stimulation are poorly understood. Human studies investigating these effects are often limited by differences in age and ovarian stimulation regimens within a patient cohort, as well as genetic and environmental variability. A novel bovine cross-sectional maternal age model for human IVF was undertaken. Follicles were aspirated from young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian clones following multiple unstimulated, mild and standard ovarian stimulation cycles. These bovine cloned females were generated by the process of somatic cell nuclear transfer (SCNT) from the same founder and represent a homogeneous population with reduced genetic and environmental variability. Maternal age and ovarian stimulation effects were investigated in relation to mtDNA copy number, and the expression of 19 genes involved in mitochondrial function, antioxidant protection, oocyte-cumulus cell signalling and follicle development in both oocytes and cumulus cells. Young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian bovine clones were maintained as one herd. Stimulation cycles were based on the long GnRH agonist down-regulation regimen used in human fertility clinics. Follicle growth

  2. Genome wide copy number analysis of single cells

    PubMed Central

    Baslan, Timour; Kendall, Jude; Rodgers, Linda; Cox, Hilary; Riggs, Mike; Stepansky, Asya; Troge, Jennifer; Ravi, Kandasamy; Esposito, Diane; Lakshmi, B.; Wigler, Michael; Navin, Nicholas; Hicks, James

    2016-01-01

    Summary Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells, where information regarding genetic heterogeneity is lost. Here, we present a protocol that allows for the genome wide copy number analysis of single nuclei isolated from mixed populations of cells. Single nucleus sequencing (SNS), combines flow sorting of single nuclei based on DNA content, whole genome amplification (WGA), followed by next generation sequencing to quantize genomic intervals in a genome wide manner. Multiplexing of single cells is discussed. Additionally, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ~3 days from flow cytometry to sequence-ready DNA libraries. PMID:22555242

  3. Evaluation of Faecalibacterium 16S rDNA genetic markers for accurate identification of swine faecal waste by quantitative PCR.

    PubMed

    Duan, Chuanren; Cui, Yamin; Zhao, Yi; Zhai, Jun; Zhang, Baoyun; Zhang, Kun; Sun, Da; Chen, Hang

    2016-10-01

    A genetic marker within the 16S rRNA gene of Faecalibacterium was identified for use in a quantitative PCR (qPCR) assay to detect swine faecal contamination in water. A total of 146,038 bacterial sequences were obtained using 454 pyrosequencing. By comparative bioinformatics analysis of Faecalibacterium sequences with those of numerous swine and other animal species, swine-specific Faecalibacterium 16S rRNA gene sequences were identified and Polymerase Chain Okabe (PCR) primer sets designed and tested against faecal DNA samples from swine and non-swine sources. Two PCR primer sets, PFB-1 and PFB-2, showed the highest specificity to swine faecal waste and had no cross-reaction with other animal samples. PFB-1 and PFB-2 amplified 16S rRNA gene sequences from 50 samples of swine with positive ratios of 86 and 90%, respectively. We compared swine-specific Faecalibacterium qPCR assays for the purpose of quantifying the newly identified markers. The quantification limits (LOQs) of PFB-1 and PFB-2 markers in environmental water were 6.5 and 2.9 copies per 100 ml, respectively. Of the swine-associated assays tested, PFB-2 was more sensitive in detecting the swine faecal waste and quantifying the microbial load. Furthermore, the microbial abundance and diversity of the microbiomes of swine and other animal faeces were estimated using operational taxonomic units (OTUs). The species specificity was demonstrated for the microbial populations present in various animal faeces. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Application of droplet digital PCR to determine copy number of endogenous genes and transgenes in sugarcane.

    PubMed

    Sun, Yue; Joyce, Priya Aiyar

    2017-08-28

    Droplet digital PCR combined with the low copy ACT allele as endogenous reference gene, makes accurate and rapid estimation of gene copy number in Q208 (A) and Q240 (A) attainable. Sugarcane is an important cultivated crop with both high polyploidy and aneuploidy in its 10 Gb genome. Without a known copy number reference gene, it is difficult to accurately estimate the copy number of any gene of interest by PCR-based methods in sugarcane. Recently, a new technology, known as droplet digital PCR (ddPCR) has been developed which can measure the absolute amount of the target DNA in a given sample. In this study, we deduced the true copy number of three endogenous genes, actin depolymerizing factor (ADF), adenine phosphoribosyltransferase (APRT) and actin (ACT) in three Australian sugarcane varieties, using ddPCR by comparing the absolute amounts of the above genes with a transgene of known copy number. A single copy of the ACT allele was detected in Q208 (A) , two copies in Q240 (A) , but was absent in Q117. Copy number variation was also observed for both APRT and ADF, and ranged from 9 to 11 in the three tested varieties. Using this newly developed ddPCR method, transgene copy number was successfully determined in 19 transgenic Q208 (A) and Q240 (A) events using ACT as the reference endogenous gene. Our study demonstrates that ddPCR can be used for high-throughput genetic analysis and is a quick, accurate and reliable alternative method for gene copy number determination in sugarcane. This discovered ACT allele would be a suitable endogenous reference gene for future gene copy number variation and dosage studies of functional genes in Q208 (A) and Q240 (A) .

  5. DNA copy number alterations, gene expression changes and disease-free survival in patients with colorectal cancer: a 10 year follow-up.

    PubMed

    Bigagli, Elisabetta; De Filippo, Carlotta; Castagnini, Cinzia; Toti, Simona; Acquadro, Francesco; Giudici, Francesco; Fazi, Marilena; Dolara, Piero; Messerini, Luca; Tonelli, Francesco; Luceri, Cristina

    2016-12-01

    DNA copy number alterations (CNAs) and gene expression changes have amply been encountered in colorectal cancers (CRCs), but the extent at which CNAs affect gene expression, as well as their relevance for tumor development, are still poorly defined. Here we aimed at assessing the clinical relevance of these parameters in a 10 year follow-up study. Tumors and normal adjacent colon mucosa, obtained at primary surgery from 21 CRC patients, were subjected to (i) high-resolution array CGH (a-CGH) for the detection of CNAs and (ii) microarray-based transcriptome profiling for the detection of gene expression (GE) changes. Correlations between these genomic and transcriptomic changes and their associations with clinical and histopathological parameters were assessed with the aim to identify molecular signatures associated with disease-free survival of the CRC patients during a 10 year follow-up. DNA copy number gains were frequently detected in chromosomes 7, 8q, 13, 19, 20q and X, whereas DNA copy number losses were frequently detected in chromosomes 1p, 4, 8p, 15, 17p, 18, 19 and 22q. None of these alterations were observed in all samples. In addition, we found that 2,498 genes were up- and that 1,094 genes were down-regulated in the tumor samples compared to their corresponding normal mucosa (p < 0.01). The expression of 65 genes was found to be significantly associated with prognosis (p < 0.01). Specifically, we found that up-regulation of the IL17RA, IGF2BP2 and ABCC2 genes, and of genes acting in the mTOR and cytokine receptor pathways, were strongly associated with a poor survival. Subsequent integrated analyses revealed that increased expression levels of the MMP9, BMP7, UBE2C, I-CAM, NOTCH3, NOTCH1, PTGES2, HMGB1 and ERBB3 genes were associated with copy number gains, whereas decreased expression levels of the MUC1, E2F2, HRAS and SIRT3 genes were associated with copy number losses. Pathways related to cell cycle progression, eicosanoid metabolism, and

  6. Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material.

    PubMed

    Deprez, Liesbet; Corbisier, Philippe; Kortekaas, Anne-Marie; Mazoua, Stéphane; Beaz Hidalgo, Roxana; Trapmann, Stefanie; Emons, Hendrik

    2016-09-01

    Digital PCR has become the emerging technique for the sequence-specific detection and quantification of nucleic acids for various applications. During the past years, numerous reports on the development of new digital PCR methods have been published. Maturation of these developments into reliable analytical methods suitable for diagnostic or other routine testing purposes requires their validation for the intended use. Here, the results of an in-house validation of a droplet digital PCR method are presented. This method is intended for the quantification of the absolute copy number concentration of a purified linearized plasmid in solution with a nucleic acid background. It has been investigated which factors within the measurement process have a significant effect on the measurement results, and the contribution to the overall measurement uncertainty has been estimated. A comprehensive overview is provided on all the aspects that should be investigated when performing an in-house method validation of a digital PCR method.

  7. Identification of Novel Breast Cancer Subtype-Specific Biomarkers by Integrating Genomics Analysis of DNA Copy Number Aberrations and miRNA-mRNA Dual Expression Profiling.

    PubMed

    Li, Dongguo; Xia, Hong; Li, Zhen-ya; Hua, Lin; Li, Lin

    2015-01-01

    Breast cancer is a heterogeneous disease with well-defined molecular subtypes. Currently, comparative genomic hybridization arrays (aCGH) techniques have been developed rapidly, and recent evidences in studies of breast cancer suggest that tumors within gene expression subtypes share similar DNA copy number aberrations (CNA) which can be used to further subdivide subtypes. Moreover, subtype-specific miRNA expression profiles are also proposed as novel signatures for breast cancer classification. The identification of mRNA or miRNA expression-based breast cancer subtypes is considered an instructive means of prognosis. Here, we conducted an integrated analysis based on copy number aberrations data and miRNA-mRNA dual expression profiling data to identify breast cancer subtype-specific biomarkers. Interestingly, we found a group of genes residing in subtype-specific CNA regions that also display the corresponding changes in mRNAs levels and their target miRNAs' expression. Among them, the predicted direct correlation of BRCA1-miR-143-miR-145 pairs was selected for experimental validation. The study results indicated that BRCA1 positively regulates miR-143-miR-145 expression and miR-143-miR-145 can serve as promising novel biomarkers for breast cancer subtyping. In our integrated genomics analysis and experimental validation, a new frame to predict candidate biomarkers of breast cancer subtype is provided and offers assistance in order to understand the potential disease etiology of the breast cancer subtypes.

  8. Performance Evaluation of NIPT in Detection of Chromosomal Copy Number Variants Using Low-Coverage Whole-Genome Sequencing of Plasma DNA

    PubMed Central

    Lin, Linhua; Yin, Xuyang; Wang, Jun; Chen, Dayang; Chen, Fang; Jiang, Hui; Ren, Jinghui; Wang, Wei

    2016-01-01

    Objectives The aim of this study was to assess the performance of noninvasively prenatal testing (NIPT) for fetal copy number variants (CNVs) in clinical samples, using a whole-genome sequencing method. Method A total of 919 archived maternal plasma samples with karyotyping/microarray results, including 33 CNVs samples and 886 normal samples from September 1, 2011 to May 31, 2013, were enrolled in this study. The samples were randomly rearranged and blindly sequenced by low-coverage (about 7M reads) whole-genome sequencing of plasma DNA. Fetal CNVs were detected by Fetal Copy-number Analysis through Maternal Plasma Sequencing (FCAPS) to compare to the karyotyping/microarray results. Sensitivity, specificity and were evaluated. Results 33 samples with deletions/duplications ranging from 1 to 129 Mb were detected with the consistent CNV size and location to karyotyping/microarray results in the study. Ten false positive results and two false negative results were obtained. The sensitivity and specificity of detection deletions/duplications were 84.21% and 98.42%, respectively. Conclusion Whole-genome sequencing-based NIPT has high performance in detecting genome-wide CNVs, in particular >10Mb CNVs using the current FCAPS algorithm. It is possible to implement the current method in NIPT to prenatally screening for fetal CNVs. PMID:27415003

  9. A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K.

    PubMed Central

    Inuzuka, M; Wada, Y

    1985-01-01

    A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division. Images Fig. 2. Fig. 5. PMID:3000771

  10. Potential use of low-copy nuclear genes in DNA barcoding: a comparison with plastid genes in two Hawaiian plant radiations

    PubMed Central

    2013-01-01

    Background DNA barcoding of land plants has relied traditionally on a small number of markers from the plastid genome. In contrast, low-copy nuclear genes have received little attention as DNA barcodes because of the absence of universal primers for PCR amplification. Results From pooled-species 454 transcriptome data we identified two variable intron-less nuclear loci for each of two species-rich genera of the Hawaiian flora: Clermontia (Campanulaceae) and Cyrtandra (Gesneriaceae) and compared their utility as DNA barcodes with that of plastid genes. We found that nuclear genes showed an overall greater variability, but also displayed a high level of heterozygosity, intraspecific variation, and retention of ancient alleles. Thus, nuclear genes displayed fewer species-diagnostic haplotypes compared to plastid genes and no interspecies gaps. Conclusions The apparently greater coalescence times of nuclear genes are likely to limit their utility as barcodes, as only a small proportion of their alleles were fixed and unique to individual species. In both groups, species-diagnostic markers from either genome were scarce on the youngest island; a minimum age of ca. two million years may be needed for a species flock to be barcoded. For young plant groups, nuclear genes may not be a superior alternative to slowly evolving plastid genes. PMID:23394592

  11. Potential use of low-copy nuclear genes in DNA barcoding: a comparison with plastid genes in two Hawaiian plant radiations.

    PubMed

    Pillon, Yohan; Johansen, Jennifer; Sakishima, Tomoko; Chamala, Srikar; Barbazuk, W Brad; Roalson, Eric H; Price, Donald K; Stacy, Elizabeth A

    2013-02-09

    DNA barcoding of land plants has relied traditionally on a small number of markers from the plastid genome. In contrast, low-copy nuclear genes have received little attention as DNA barcodes because of the absence of universal primers for PCR amplification. From pooled-species 454 transcriptome data we identified two variable intron-less nuclear loci for each of two species-rich genera of the Hawaiian flora: Clermontia (Campanulaceae) and Cyrtandra (Gesneriaceae) and compared their utility as DNA barcodes with that of plastid genes. We found that nuclear genes showed an overall greater variability, but also displayed a high level of heterozygosity, intraspecific variation, and retention of ancient alleles. Thus, nuclear genes displayed fewer species-diagnostic haplotypes compared to plastid genes and no interspecies gaps. The apparently greater coalescence times of nuclear genes are likely to limit their utility as barcodes, as only a small proportion of their alleles were fixed and unique to individual species. In both groups, species-diagnostic markers from either genome were scarce on the youngest island; a minimum age of ca. two million years may be needed for a species flock to be barcoded. For young plant groups, nuclear genes may not be a superior alternative to slowly evolving plastid genes.

  12. B chromosomes in the grasshopper Eyprepocnemis plorans are present in all body parts analyzed and show extensive variation for rDNA copy number.

    PubMed

    Ruiz-Estévez, Mercedes; Cabrero, Josefa; Camacho, Juan Pedro M; López-León, María Dolores

    2014-01-01

    B chromosomes in the grasshopper Eyprepocnemis plorans are considered to be mitotically stable, because all meiotic (primary spermatocytes and oocytes) or mitotic (embryos, ovarioles, and gastric caecum) cells analyzed within the same individual show the same B chromosome number. Nothing is known, however, about body parts with somatic tissues with no mitotic activity in adult individuals, constituting the immense majority of their body. Therefore, we investigated whether B chromosomes are present in 8 non-mitotically active somatic body parts from both sexes in addition to ovarioles and testes by PCR analysis of 2 B-specific molecular markers. We also elucidated the number of B chromosomes that an individual carried through quantifying the B-located rDNA copy number by qPCR. Our results indicated the amplification of both B-specific markers in all analyzed body parts. However, we found high variation between males for the estimated number of rDNA units in the B chromosomes. These results demonstrate the presence of B chromosomes in all body parts from the same individual and suggest a high variation in the rDNA content of the B chromosomes carried by different individuals from the same population, presumably due to unequal crossovers during meiosis. © 2014 S. Karger AG, Basel.

  13. Validation and development of interpretation guidelines for low copy number (LCN) DNA profiling in New Zealand using the AmpFlSTR SGM Plus multiplex.

    PubMed

    Petricevic, Sue; Whitaker, Jonathan; Buckleton, John; Vintiner, Sue; Patel, Jayshree; Simon, Pauline; Ferraby, Helen; Hermiz, Waseem; Russell, Amanda

    2010-10-01

    The characteristics of STR profiles produced from approximately 1 ng starting template using the AMPFlSTR SGM Plus multiplex and 28 PCR cycles, are well documented. However, the analysis of samples perceived as low in starting template (less than 100 pg), and referred to as low template DNA (LTDNA), can require a test of higher sensitivity in order to achieve successful results. One way of increasing this sensitivity is to increase the number of PCR amplification cycles from 28 to 34. This type of analysis has become known as low copy number, or LCN, DNA profiling. Amplification of LTDNA under LCN conditions can result in increased incidents of profile characteristics such as allelic 'drop-in' and allelic 'drop-out'. Adopting a testing regime which includes duplicate analysis, and maintaining a laboratory environment of stringent and monitored cleanliness, enables the scientist to identify and control these phenomena for a reliable interpretation of the DNA profiling results. A recent court ruling has questioned the reliability of LCN analysis and commented on the paucity of publications surrounding the validation of the technique. We present data for the LCN validation undertaken in our laboratory, and describe the guidelines and working practices we have developed for the analysis and interpretation of profiles generated after LCN profiling. This study augments the published record relating to LCN validation and should act as a useful guide for other laboratories who are considering implementing LCN profiling.

  14. A multi-locus analysis of phylogenetic relationships within grass subfamily Pooideae (Poaceae) inferred from sequences of nuclear single copy gene regions compared with plastid DNA.

    PubMed

    Hochbach, Anne; Schneider, Julia; Röser, Martin

    2015-06-01

    To investigate phylogenetic relationships within the grass subfamily Pooideae we studied about 50 taxa covering all recognized tribes, using one plastid DNA (cpDNA) marker (matK gene-3'trnK exon) and for the first time four nuclear single copy gene loci. DNA sequence information from two parts of the nuclear genes topoisomerase 6 (Topo6) spanning the exons 8-13 and 17-19, the exons 9-13 encoding plastid acetyl-CoA-carboxylase (Acc1) and the partial exon 1 of phytochrome B (PhyB) were generated. Individual and nuclear combined data were evaluated using maximum parsimony, maximum likelihood and Bayesian methods. All of the phylogenetic results show Brachyelytrum and the tribe Nardeae as earliest diverging lineages within the subfamily. The 'core' Pooideae (Hordeeae and the Aveneae/Poeae tribe complex) are also strongly supported, as well as the monophyly of the tribes Brachypodieae, Meliceae and Stipeae (except PhyB). The beak grass tribe Diarrheneae and the tribe Duthieeae are not monophyletic in some of the analyses. However, the combined nuclear DNA (nDNA) tree yields the highest resolution and the best delimitation of the tribes, and provides the following evolutionary hypothesis for the tribes: Brachyelytrum, Nardeae, Duthieeae, Meliceae, Stipeae, Diarrheneae, Brachypodieae and the 'core' Pooideae. Within the individual datasets, the phylogenetic trees obtained from Topo6 exon 8-13 shows the most interesting results. The divergent positions of some clone sequences of Ampelodesmos mauritanicus and Trikeraia pappiformis, for instance, may indicate a hybrid origin of these stipoid taxa. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Analysis of DNA Copy Number Alterations in Ovarian Serous Tumors Identifies New Molecular Genetic Changes in Low-grade and High-grade Carcinomas

    PubMed Central

    Kuo, Kuan-Ting; Guan, Bin; Feng, Yuanjian; Mao, Tsui-Lien; Chen, Xu; Jinawath, Natini; Wang, Yue; Kurman, Robert J.; Shih, Ie-Ming; Wang, Tian-Li

    2009-01-01

    Ovarian serous carcinoma, the most common and lethal type of ovarian cancer, was thought to develop from two distinct molecular pathways. High-grade (HG) serous carcinomas contain frequent TP53 mutations while low-grade (LG) carcinomas arise from serous borderline tumors (SBT) and harbor mutations in KRAS/BRAF/ERBB2 pathway. However, the molecular alterations involved in the progression from SBT to LG carcinoma remain largely unknown. As well, the extent of deletion of tumor suppressors in ovarian serous carcinomas has not been well-studied. To further address these two issues, we assessed DNA copy number changes among affinity-purified tumor cells from 37 ovarian serous neoplasms including SBT, LG and HG tumors using high density 250K SNP arrays. Chromosomal instability index as measured by changes in DNA copy number was significantly higher in HG than in LG serous carcinomas. Hemizygous ch1p36 deletion was common in LG serous carcinomas but was rarely seen in SBT. This region contains several candidate tumor suppressors including miR-34a. In contrast, in HG serous carcinomas, significant numbers of amplifications and deletions including homozygous deletions were identified. Among homozygous deletions, loci containing Rb1, CDKN2A/B, CSMD1, and DOCK4 were most common, being present in 10.6%, 6.4%, 6.4% and 4.3%, respectively, in independent 47 affinity-purified HG serous carcinomas. Except the CDKN2A/B region, these homozygous deletions were not present in either SBT or LG tumors. Our study provides a genome-wide homozygous deletion profiles in HG serous carcinomas, serving as a molecular foundation to study tumor suppressors in ovarian cancer. PMID:19383911

  16. Effects of ginseng on peripheral blood mitochondrial DNA copy number and hormones in men with metabolic syndrome: A randomized clinical and pilot study.

    PubMed

    Jung, Dong-Hyuk; Lee, Yong-Jae; Kim, Chun-Bae; Kim, Jang-Young; Shin, Seung-Hun; Park, Jong-Ku

    2016-02-01

    It has been observed that mitochondrial dysfunction is associated with an increased risk of metabolic syndrome. There is growing evidence that hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis and hormone (testosterone and growth hormone) deficiency may lead to metabolic syndrome. Recent studies have reported that ginseng treatment improves mitochondrial and HPA-axis function and increases anabolic hormone secretion. The objective of this study was to investigate the effect of red ginseng (RG) on metabolic syndrome, hormones, and mitochondrial function using leukocyte mitochondrial DNA copy number in men with metabolic syndrome. We performed a randomized, double-blind, placebo-controlled study in 62 subjects who were not taking drugs that could affect their metabolic function. A total of 62 men with metabolic syndrome were randomly assigned to either an RG group (3.0g/day) or a placebo group for 4 weeks. We analyzed changes in metabolic syndrome components, leukocyte mitochondrial DNA copy number, hormones (total testosterone, IGF-1, cortisol, and DHEAS) and inflammatory markers (C-reactive protein, ferritin) from baseline to 4 weeks. Significant improvement in mitochondrial function (95% CI -44.9 to -1.3) and an increase in total testosterone (95% CI -70.1 to -1.0) and IGF-1(P=0.01) levels were observed in the RG group when compared with the placebo group. Diastolic blood pressure (95% CI 2.0-9.4) and serum cortisol (95% CI 1.1-5.5) significantly decreased in the RG group. We found evidence that RG had a favorable effect on mitochondrial function and hormones in men with metabolic syndrome. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Personal exposure to fine particulate matter and benzo[a]pyrene from indoor air pollution and leukocyte mitochondrial DNA copy number in rural China.

    PubMed

    Wong, Jason Y Y; Hu, Wei; Downward, George S; Seow, Wei Jie; Bassig, Bryan A; Ji, Bu-Tian; Wei, Fusheng; Wu, Guoping; Li, Jihua; He, Jun; Liu, Chin-San; Cheng, Wen-Ling; Huang, Yunchao; Yang, Kaiyun; Chen, Ying; Rothman, Nathaniel; Vermeulen, Roel C; Lan, Qing

    2017-09-01

    Households in Xuanwei and Fuyuan, China, possess hazardous levels of fine particulate matter with an aerodynamic diameter <2.5 microns (PM2.5) and polycyclic aromatic hydrocarbons (PAHs) from coal combustion. Previous studies found that increased exposure to PM2.5 and benzo[a]pyrene (BaP; a PAH) were associated with decreased mitochondrial DNA copy number (mtDNAcn), a marker of oxidative stress. We further evaluated these associations in a cross-sectional study of 148 healthy non-smoking women from Xuanwei and Fuyuan. Personal exposure to PM2.5 and BaP was measured using portable devices. MtDNAcn was measured using qPCR amplification of leukocyte DNA that was collected after air measurements. Linear regression models were used to estimate the associations between personal exposure to PM2.5 and BaP, and mtDNAcn adjusted for age, body mass index (BMI) and fuel type. We found inverse associations between exposure to PM2.5 and BaP, and mtDNAcn. Each incremental log-μg/m3 increase in PM2.5 was associated with a significant decrease in mtDNAcn of -10.3 copies per cell [95% confidence interval (95% CI): -18.6, -2.0; P = 0.02]. Additionally, each log-ng/m3 increase in BaP was associated with a significant decrease in mtDNAcn of -5.4 copies per cell (95% CI: -9.9, -0.8, P = 0.02). Age, BMI, fuel type and coal mine type were not significantly associated with mtDNAcn. Exposure to PM2.5 and BaP may alter mitochondrial dynamics in non-smoking Chinese women. MtDNAcn may be a potential mediator of indoor air pollution on chronic disease development. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Multiple Components of the VHL Tumor Suppressor Complex Are Frequently Affected by DNA Copy Number Loss in Pheochromocytoma.

    PubMed

    Rowbotham, David A; Enfield, Katey S S; Martinez, Victor D; Thu, Kelsie L; Vucic, Emily A; Stewart, Greg L; Bennewith, Kevin L; Lam, Wan L

    2014-01-01

    Pheochromocytomas (PCC) are rare tumors that arise in chromaffin tissue of the adrenal gland. PCC are frequently inherited through predisposing mutations in genes such as the von Hippel-Lindau (VHL) tumor suppressor. VHL is part of the VHL elongin BC protein complex that also includes CUL2/5, TCEB1, TCEB2, and RBX1; in normoxic conditions this complex targets hypoxia-inducible factor 1 alpha (HIF1A) for degradation, thus preventing a hypoxic response. VHL inactivation by genetic mechanisms, such as mutation and loss of heterozygosity, inhibits HIF1A degradation, even in the presence of oxygen, and induces a pseudohypoxic response. However, the described <10% VHL mutation rate cannot account for the high frequency of hypoxic response observed. Indeed, little is known about genetic mechanisms disrupting other complex component genes. Here, we show that, in a panel of 171 PCC tumors, 59.6% harbored gene copy number loss (CNL) of at least one complex component. CNL significantly reduced gene expression and was associated with enrichment of gene targets controlled by HIF1. Interestingly, we show that VHL-related renal clear cell carcinoma harbored disruption of VHL alone. Our results indicate that VHL elongin BC protein complex components other than VHL could be important for PCC tumorigenesis and merit further investigation.

  19. A DNA target-enrichment approach to detect mutations, copy number changes and immunoglobulin translocations in multiple myeloma

    PubMed Central

    Bolli, N; Li, Y; Sathiaseelan, V; Raine, K; Jones, D; Ganly, P; Cocito, F; Bignell, G; Chapman, M A; Sperling, A S; Anderson, K C; Avet-Loiseau, H; Minvielle, S; Campbell, P J; Munshi, N C

    2016-01-01

    Genomic lesions are not investigated during routine diagnostic workup for multiple myeloma (MM). Cytogenetic studies are performed to assess prognosis but with limited impact on therapeutic decisions. Recently, several recurrently mutated genes have been described, but their clinical value remains to be defined. Therefore, clinical-grade strategies to investigate the genomic landscape of myeloma samples are needed to integrate new and old prognostic markers. We developed a target-enrichment strategy followed by next-generation sequencing (NGS) to streamline simultaneous analysis of gene mutations, copy number changes and immunoglobulin heavy chain (IGH) translocations in MM in a high-throughput manner, and validated it in a panel of cell lines. We identified 548 likely oncogenic mutations in 182 genes. By integrating published data sets of NGS in MM, we retrieved a list of genes with significant relevance to myeloma and found that the mutational spectrum of primary samples and MM cell lines is partially overlapping. Gains and losses of chromosomes, chromosomal segments and gene loci were identified with accuracy comparable to conventional arrays, allowing identification of lesions with known prognostic significance. Furthermore, we identified IGH translocations with high positive and negative predictive value. Our approach could allow the identification of novel biomarkers with clinical relevance in myeloma. PMID:27588520

  20. Multiple Components of the VHL Tumor Suppressor Complex Are Frequently Affected by DNA Copy Number Loss in Pheochromocytoma

    PubMed Central

    Rowbotham, David A.; Enfield, Katey S. S.; Martinez, Victor D.; Thu, Kelsie L.; Vucic, Emily A.; Stewart, Greg L.; Bennewith, Kevin L.; Lam, Wan L.

    2014-01-01

    Pheochromocytomas (PCC) are rare tumors that arise in chromaffin tissue of the adrenal gland. PCC are frequently inherited through predisposing mutations in genes such as the von Hippel-Lindau (VHL) tumor suppressor. VHL is part of the VHL elongin BC protein complex that also includes CUL2/5, TCEB1, TCEB2, and RBX1; in normoxic conditions this complex targets hypoxia-inducible factor 1 alpha (HIF1A) for degradation, thus preventing a hypoxic response. VHL inactivation by genetic mechanisms, such as mutation and loss of heterozygosity, inhibits HIF1A degradation, even in the presence of oxygen, and induces a pseudohypoxic response. However, the described <10% VHL mutation rate cannot account for the high frequency of hypoxic response observed. Indeed, little is known about genetic mechanisms disrupting other complex component genes. Here, we show that, in a panel of 171 PCC tumors, 59.6% harbored gene copy number loss (CNL) of at least one complex component. CNL significantly reduced gene expression and was associated with enrichment of gene targets controlled by HIF1. Interestingly, we show that VHL-related renal clear cell carcinoma harbored disruption of VHL alone. Our results indicate that VHL elongin BC protein complex components other than VHL could be important for PCC tumorigenesis and merit further investigation. PMID:25298778

  1. Integrated immunohistochemical and DNA copy number profiling analysis provides insight into the molecular pathogenesis of canine follicular lymphoma.

    PubMed

    Thomas, R; Demeter, Z; Kennedy, K A; Borst, L; Singh, K; Valli, V E; Le Boedec, K; Breen, M

    2017-09-01

    Follicular lymphomas (FLs) typically exhibit a chromosome translocation that induces constitutive expression of the anti-apoptotic bcl2 protein and accumulation of additional molecular defects. This rearrangement offers a promising therapeutic target, but its nature as a fundamental driver of FL pathogenesis remains unclear as 15% of cases lack the translocation. We performed an integrated immunohistochemical and genomic investigation of 10 naturally occurring FL cases from domestic dogs, showing that, as with human tumours, they exhibit marked heterogeneity in the frequency and intensity of bcl2 protein expression. Genomic copy number aberrations were infrequent and broadly consistent with those of other canine B-cell lymphoma subtypes. None of the canine FL specimens exhibited a rearrangement consistent with the hallmark translocation of human FL, despite their remarkable histomorphologic similarity. Parallel exploration of canine and human cases may reveal alternative tumour-initiating mechanisms other than BCL2 disruption, yielding a more complete definition of the molecular pathogenesis of FL. © 2016 John Wiley & Sons Ltd.

  2. Types, levels and patterns of low-copy DNA sequence divergence, and phylogenetic implications, for Gossypium genome types

    PubMed Central

    Rong, J; Wang, X; Schulze, S R; Compton, R O; Williams-Coplin, T D; Goff, V; Chee, P W; Paterson, A H

    2012-01-01

    To explore types, levels and patterns of genetic divergence among diploid Gossypium (cotton) genomes, 780 cDNA, genomic DNA and simple sequence repeat (SSR) loci were re-sequenced in Gossypium herbaceum (A1 genome), G. arboreum (A2), G. raimondii (D5), G. trilobum (D8), G. sturtianum (C1) and an outgroup, Gossypioides kirkii. Divergence among these genomes ranged from 7.32 polymorphic base pairs per 100 between G. kirkii and G. herbaceum (A1) to only 1.44 between G. herbaceum (A1) and G. arboreum (A2). SSR loci are least conserved with 12.71 polymorphic base pairs and 3.77 polymorphic sites per 100 base pairs, whereas expressed sequence tags are most conserved with 3.96 polymorphic base pairs and 2.06 sites. SSR loci also exhibit the highest percentage of ‘extended polymorphisms' (spanning multiple consecutive nucleotides). The A genome lineage was particularly rapidly evolving, with the D genome also showing accelerated evolution relative to the C genome. Unexpected asymmetry in mutation rates was found, with much more transition than transversion mutation in the D genome after its divergence from a common ancestor shared with the A genome. This large quantity of orthologous DNA sequence strongly supports a phylogeny in which A–C divergence is more recent than A–D divergence, a subject that is of much importance in view of A–D polyploid formation being key to the evolution of the most productive and finest-quality cottons. Loci that are monomorphic within A or D genome types, but polymorphic between genome types, may be of practical importance for identifying locus-specific DNA markers in tetraploid cottons including leading cultivars. PMID:22511177

  3. Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number.

    PubMed

    Thompson, Kyle; Majd, Homa; Dallabona, Christina; Reinson, Karit; King, Martin S; Alston, Charlotte L; He, Langping; Lodi, Tiziana; Jones, Simon A; Fattal-Valevski, Aviva; Fraenkel, Nitay D; Saada, Ann; Haham, Alon; Isohanni, Pirjo; Vara, Roshni; Barbosa, Inês A; Simpson, Michael A; Deshpande, Charu; Puusepp, Sanna; Bonnen, Penelope E; Rodenburg, Richard J; Suomalainen, Anu; Õunap, Katrin; Elpeleg, Orly; Ferrero, Ileana; McFarland, Robert; Kunji, Edmund R S; Taylor, Robert W

    2016-10-06

    Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. Episomal High Copy Number Maintenance of Hairpin-capped DNA Bearing a Replication Initiation Region in Human Cells*

    PubMed Central

    Harada, Seiyu; Uchida, Masafumi; Shimizu, Noriaki

    2009-01-01

    We previously found that a plasmid bearing a replication initiation region efficiently initiates gene amplification in mammalian cells and that it generates extrachromosomal double minutes and/or chromosomal homogeneously staining regions. During analysis of the underlying mechanism, we serendipitously found that hairpin-capped linear DNA was stably maintained as numerous extrachromosomal tiny episomes for more than a few months in a human cancer cell line. Generation of such episomes depended on the presence of the replication initiation region in the original plasmid. Despite extrachromosomal maintenance, episomal gene expression was epigenetically suppressed. The Southern blot analysis of the DNA of cloned cells revealed that the region around the hairpin end was diversified between the clones. Furthermore, the bisulfite-modified PCR and the sequencing analyses revealed that the palindrome sequence that derived from the original hairpin end or its end-resected structure were well preserved during clonal long term growth. From these data, we propose a model that explains the formation and maintenance of these episomes, in which replication of the hairpin-capped DNA and cruciform formation and its resolution play central roles. Our findings may be relevant for the dissection of mammalian replicator sequences. PMID:19617622

  5. High-resolution copy number profiling by array CGH using DNA isolated from formalin-fixed, paraffin-embedded tissues.

    PubMed

    van Essen, Hendrik F; Ylstra, Bauke

    2012-01-01

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

  6. Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

    PubMed Central

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; De Spiegelaere, Ward

    2014-01-01

    Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation. PMID:25502524

  7. Alterations of telomere length and mtDNA copy number are associated with overall survival in hepatocellular carcinoma patients treated with transarterial chemoembolization.

    PubMed

    Bao, Dengke; Ba, Yanna; Zhou, Feng; Zhao, Jing; Yang, Qi; Ge, Naijian; Guo, Xu; Wu, Zhenbiao; Zhang, Hongxin; Yang, Hushan; Wan, Shaogui; Xing, Jinliang

    2016-10-01

    Increasing evidence suggests that alterations in mitochondrial DNA (mtDNA) copy number (mtDNAcn) and relative telomere length (RTL) may be implicated in the tumorigenesis of several malignancies. Alterations of both RTL and mtDNAcn are generally accepted as independent biomarkers for predicting risk and prognosis in various cancers. The aim of this study was to evaluate the prognostic value of combining leukocyte RTL with mtDNAcn (RTL-mtDNAcn) in hepatocellular carcinoma (HCC). RTL and mtDNAcn in peripheral blood leukocytes (PBLs) were measured using a real-time PCR-based method in a total of 250 HCC patients treated with transcatheter arterial chemoembolization (TACE). We evaluated the associations between RTL and/or mtDNAcn and HCC overall survival using Kaplan-Meier curve analysis and Cox proportional hazards regression model. We found that patients with longer leukocyte RTL or lower mtDNAcn had shorter overall survival time. The univariate analysis (HR 1.63, 95 % CI 1.23-2.17, P = 7.7 × 10(-4)) and multivariate analysis (HR 1.78, 95 % CI 1.31-2.42, P = 2.4 × 10(-4)) indicated that longer leukocyte RTL was significantly associated with poorer OS in HCC patients. Kaplan-Meier curve analysis showed that patients with longer RTL had shorter overall survival time than those with shorter RTL (log-rank P = 0.001). Patients with lower mtDNA copy number was significantly associated with poorer OS by Cox proportional hazards model using both univariate (HR 1.60, 95 % CI 1.21-2.13, P = 0.001) and multivariate analyses (HR 1.77, 95 % CI 1.30-2.41, P = 2.8 × 10(-4)). Kaplan-Meier curve analysis showed that patients with lower mtDNA content had significantly shorter overall survival time than those with higher mtDNA content (log-rank P = 0.001). Furthermore, combination of leukocyte RTL and mtDNAcn significantly improved the efficacy of predicting HCC prognosis. Patients with longer RTL and lower mtDNAcn exhibited a significantly poorer overall

  8. Genome-Wide Loss of Heterozygosity and DNA Copy Number Aberration in HPV-Negative Oral Squamous Cell Carcinoma and Their Associations with Disease-Specific Survival.

    PubMed

    Chen, Chu; Zhang, Yuzheng; Loomis, Melissa M; Upton, Melissa P; Lohavanichbutr, Pawadee; Houck, John R; Doody, David R; Mendez, Eduardo; Futran, Neal; Schwartz, Stephen M; Wang, Pei

    2015-01-01

    Oral squamous cell cancer of the oral cavity and oropharynx (OSCC) is associated with high case-fatality. For reasons that are largely unknown, patients with the same clinical and pathologic staging have heterogeneous response to treatment and different probability of recurrence and survival, with patients with Human Papillomavirus (HPV)-positive oropharyngeal tumors having the most favorable survival. To gain insight into the complexity of OSCC and to identify potential chromosomal changes that may be associated with OSCC mortality, we used Affymtrix 6.0 SNP arrays to examine paired DNA from peripheral blood and tumor cell populations isolated by laser capture microdissection to assess genome-wide loss of heterozygosity (LOH) and DNA copy number aberration (CNA) and their associations with risk factors, tumor characteristics, and oral cancer-specific mortality among 75 patients with HPV-negative OSCC. We found a highly heterogeneous and complex genomic landscape of HPV-negative tumors, and identified regions in 4q, 8p, 9p and 11q that seem to play an important role in oral cancer biology and survival from this disease. If confirmed, these findings could assist in designing personalized treatment or in the creation of models to predict survival in patients with HPV-negative OSCC.

  9. Loss of 6q or 8p23 is associated with the total number of DNA copy number aberrations in adenoid cystic carcinoma.

    PubMed

    Oga, Atsunori; Uchida, Kenichiro; Nakao, Motonao; Kawauchi, Shigeto; Furuya, Tomoko; Chochi, Yasuyo; Ikemoto, Kenzo; Okada, Takae; Ueyama, Yoshiya; Sasaki, Kohsuke; Yousefpour, Fatemeh

    2011-12-01

    We analyzed 10 adenoid cystic carcinomas (ACCs) of the salivary glands by array-based comparative genomic hybridization (a-CGH) using DNA chips spotted with 4,030 bacterial artificial chromosome clones. After the data smoothing procedure was applied, a total of 88 DNA copy number aberrations (DCNAs) were detected. The frequent (≥30%) DCNAs were loss of 6q23-27 and 8p23, and gains of 6p, 6q23, 8p23 and 22q13. High-level gains were detected on 12q15, including MDM2 in two cases. These two cases showed an immunohistochemically high-level (>50%) expression of MDM2 and a low-level expression of p53 (<20%). Furthermore, the total number of DCNAs was significantly greater in ACCs with loss of 6q compared to other ACCs, and in ACCs without the loss of 8p23 compared to other ACCs, respectively. Although limitations exist, a-CGH detected several candidate chromosomal imbalances associated with accumulation of DCNAs in ACCs.

  10. Distinct patterns of DNA copy number alteration are associated with different clinicopathological features and gene-expression subtypes of breast cancer.

    PubMed

    Bergamaschi, Anna; Kim, Young H; Wang, Pei; Sørlie, Therese; Hernandez-Boussard, Tina; Lonning, Per E; Tibshirani, Robert; Børresen-Dale, Anne-Lise; Pollack, Jonathan R

    2006-11-01

    Breast cancer is a leading cause of cancer-death among women, where the clinicopathological features of tumors are used to prognosticate and guide therapy. DNA copy number alterations (CNAs), which occur frequently in breast cancer and define key pathogenetic events, are also potentially useful prognostic or predictive factors. Here, we report a genome-wide array-based comparative genomic hybridization (array CGH) survey of CNAs in 89 breast tumors from a patient cohort with locally advanced disease. Statistical analysis links distinct cytoband loci harboring CNAs to specific clinicopathological parameters, including tumor grade, estrogen receptor status, presence of TP53 mutation, and overall survival. Notably, distinct spectra of CNAs also underlie the different subtypes of breast cancer recently defined by expression-profiling, implying these subtypes develop along distinct genetic pathways. In addition, higher numbers of gains/losses are associated with the "basal-like" tumor subtype, while high-level DNA amplification is more frequent in "luminal-B" subtype tumors, suggesting also that distinct mechanisms of genomic instability might underlie their pathogenesis. The identified CNAs may provide a basis for improved patient prognostication, as well as a starting point to define important genes to further our understanding of the pathobiology of breast cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat

  11. Genome-wide analysis of abdominal and pleural malignant mesothelioma with DNA arrays reveals both common and distinct regions of copy number alteration

    PubMed Central

    Borczuk, Alain C.; Pei, Jianming; Taub, Robert N.; Levy, Brynn; Nahum, Odelia; Chen, Jinli; Chen, Katherine; Testa, Joseph R.

    2016-01-01

    ABSTRACT Malignant mesothelioma (MM) is an aggressive tumor arising from mesothelial linings of the serosal cavities. Pleural space is the most common site, accounting for about 80% of cases, while peritoneum makes up the majority of the remaining 20%. While histologically similar, tumors from these sites are epidemiologically and clinically distinct and their attribution to asbestos exposure differs. We compared DNA array-based findings from 48 epithelioid peritoneal MMs and 41 epithelioid pleural MMs to identify similarities and differences in copy number alterations (CNAs). Losses in 3p (BAP1 gene), 9p (CDKN2A) and 22q (NF2) were seen in tumors from both tumor sites, although CDKN2A and NF2 losses were seen at a higher rate in pleural disease (p<0.01). Overall, regions of copy number gain were more common in peritoneal MM, whereas losses were more common in pleural MM, with regions of loss containing known tumor suppressor genes and regions of gain encompassing genes encoding receptor tyrosine kinase pathway members. Cases with known asbestos causation (n = 32 ) were compared with those linked to radiation exposure (n = 9 ). Deletions in 6q, 14q, 17p and 22q, and gain of 17q were seen in asbestos-associated but not radiation-related cases. As reported in post-radiation sarcoma, gains outnumbered losses in radiation-associated MM. The patterns of genomic imbalances suggest overlapping and distinct molecular pathways in MM of the pleura and peritoneum, and that differences in causation (i.e., asbestos vs. radiation) may account for some of these site-dependent differences PMID:26853494

  12. Molecular methods (digital PCR and real-time PCR) for the quantification of low copy DNA of Phytophthora nicotianae in environmental samples.

    PubMed

    Blaya, Josefa; Lloret, Eva; Santísima-Trinidad, Ana B; Ros, Margarita; Pascual, Jose A

    2016-04-01

    Currently, real-time polymerase chain reaction (qPCR) is the technique most often used to quantify pathogen presence. Digital PCR (dPCR) is a new technique with the potential to have a substantial impact on plant pathology research owing to its reproducibility, sensitivity and low susceptibility to inhibitors. In this study, we evaluated the feasibility of using dPCR and qPCR to quantify Phytophthora nicotianae in several background matrices, including host tissues (stems and roots) and soil samples. In spite of the low dynamic range of dPCR (3 logs compared with 7 logs for qPCR), this technique proved to have very high precision applicable at very low copy numbers. The dPCR was able to detect accurately the pathogen in all type of samples in a broad concentration range. Moreover, dPCR seems to be less susceptible to inhibitors than qPCR in plant samples. Linear regression analysis showed a high correlation between the results obtained with the two techniques in soil, stem and root samples, with R(2) = 0.873, 0.999 and 0.995 respectively. These results suggest that dPCR is a promising alternative for quantifying soil-borne pathogens in environmental samples, even in early stages of the disease. © 2015 Society of Chemical Industry.

  13. Haplotype Detection from Next-Generation Sequencing in High-Ploidy-Level Species: 45S rDNA Gene Copies in the Hexaploid Spartina maritima

    PubMed Central

    Boutte, Julien; Aliaga, Benoît; Lima, Oscar; Ferreira de Carvalho, Julie; Ainouche, Abdelkader; Macas, Jiri; Rousseau-Gueutin, Mathieu; Coriton, Olivier; Ainouche, Malika; Salmon, Armel

    2015-01-01

    Gene and whole-genome duplications are widespread in plant nuclear genomes, resulting in sequence heterogeneity. Identification of duplicated genes may be particularly challenging in highly redundant genomes, especially when there are no diploid parents as a reference. Here, we developed a pipeline to detect the different copies in the ribosomal RNA gene family in the hexaploid grass Spartina maritima from next-generation sequencing (Roche-454) reads. The heterogeneity of the different domains of the highly repeated 45S unit was explored by identifying single nucleotide polymorphisms (SNPs) and assembling reads based on shared polymorphisms. SNPs were validated using comparisons with Illumina sequence data sets and by cloning and Sanger (re)sequencing. Using this approach, 29 validated polymorphisms and 11 validated haplotypes were reported (out of 34 and 20, respectively, that were initially predicted by our program). The rDNA domains of S. maritima have similar lengths as those found in other Poaceae, apart from the 5′-ETS, which is approximately two-times longer in S. maritima. Sequence homogeneity was encountered in coding regions and both internal transcribed spacers (ITS), whereas high intragenomic variability was detected in the intergenic spacer (IGS) and the external transcribed spacer (ETS). Molecular cytogenetic analysis by fluorescent in situ hybridization (FISH) revealed the presence of one pair of 45S rDNA signals on the chromosomes of S. maritima instead of three expected pairs for a hexaploid genome, indicating loss of duplicated homeologous loci through the diploidization process. The procedure developed here may be used at any ploidy level and using different sequencing technologies. PMID:26530424

  14. In vitro replication of human mitochondrial DNA: accurate initiation at the origin of light-strand synthesis.

    PubMed

    Wong, T W; Clayton, D A

    1985-10-01

    Synthesis of human light-strand mitochondrial DNA was accomplished in vitro using DNA primase, DNA polymerase, and other accessory proteins isolated from human mitochondria. Replication begins with the synthesis of primer RNA on a T-rich sequence in the origin stem-loop structure of the template DNA and absolutely requires ATP. A transition from RNA synthesis to DNA synthesis occurs near the base of the stem-loop structure and a potential recognition site for signaling that transition has been identified. The start sites of the in vitro products were mapped at the nucleotide level and were found to be in excellent agreement with those of in vivo nascent light-strand DNA. Isolated human mitochondrial enzymes recognize and utilize the bovine, but not the mouse, origin of light-strand replication.

  15. [Characterization of recombinant single-stranded DNA-binding protein from Escherichia coli and its application in accurate pyrosequencing].

    PubMed

    Wang, Jianping; Zou, Bingjie; Chen, Zhiyao; Ma, Yinjiao; Xu, Shu; Zhou, Guohua

    2011-10-01

    We expressed recombinant single-stranded DNA-binding protein (r-SSBP) from Escherichia coli with the molecular weight of 24-kDa by using genetic engineering strategy, and demonstrated the single-stranded DNA (ssDNA)-binding activity of r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize r-SSBP, we studied the effects of r-SSBP on melting temperature (T(m)) of DNA. The results showed that r-SSBP could bind to ssDNA, and lower the T(m) of DNA, especially for single-base mismatched DNA. Therefore, r-SSBP significantly increased the T(m) difference between single-base mismatched DNA and perfect matched DNA. These results are very beneficial for single-nucleotide polymorphism detection. Moreover, we applied r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggest that the r-SSBP decreased non-specific signals, corrected the proportion of signal peak height and improved the performance of pyrosequencing.

  16. A Comprehensive Approach for Accurate Measurement of Proton-Proton Coupling Constants in the Sugar Ring of DNA

    SciTech Connect

    Yang, Jiping; McAteer, Kathleen; Silks, Louis A.; Wu, Ruilian; Isern, Nancy G.; Unkefer, Clifford J.; Kennedy, Michael A.

    2000-10-01

    Stereo-selective deuteration has been explored in the 12 base pair DNA Dickerson sequence [d(CGCGAATTCGCG)2] as an approach for improving the accuracy of NMR derived, three-bond vicinal proton-proton coupling constants. The coupling constants are useful for DNA structure determination in restrained molecular dynamics calculations.

  17. The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F.

    PubMed

    Chevaliez, Stéphane; Dauvillier, Claude; Dubernet, Fabienne; Poveda, Jean-Dominique; Laperche, Syria; Hézode, Christophe; Pawlotsky, Jean-Michel

    2017-04-01

    Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTime HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice. Copyright © 2017 American Society for Microbiology.

  18. Retrospective analysis of varicella zoster virus (VZV) copy DNA numbers in plasma of immunocompetent patients with herpes zoster, of immunocompromised patients with disseminated VZV disease, and of asymptomatic solid organ transplant recipients.

    PubMed

    Kronenberg, A; Bossart, W; Wuthrich, R P; Cao, C; Lautenschlager, S; Wiegand, N D; Mullhaupt, B; Noll, G; Mueller, N J; Speck, R F

    2005-01-01

    Varicella zoster virus (VZV) causes significant morbidity and mortality in immunocompromised patients. Subclinical reactivation has been described in solid organ recipients and has been associated with graft versus host disease in bone marrow transplantation. Newer studies assessing the prevalence and impact of subclinical VZV reactivation in solid organ transplant (SOT) recipients are lacking. In a first step we developed a highly sensitive quantitative polymerase chain reaction (qPCR) assay for VZV DNA with a detection limit of < or = 20 copies/mL. Using this assay, we retrospectively analyzed plasma samples of different patient groups for VZV DNA. VZV DNA was found in 10/10 plasma samples of immunocompetent patients with herpes zoster (VZV copy numbers/mL: mean+/-SEM 1710+/-1018), in 1/1 sample of a human immunodeficiency virus-infected patient with primary VZV disease (15,192 copies/mL) and in 4/4 plasma samples of immunocompromised patients with visceral VZV disease (mean of first value 214,214+/-178,572). All 108 plasma samples of asymptomatic SOT recipients off any antiviral therapy, randomly sampled over 1 year, were negative for VZV DNA. Our qPCR assay proved to be highly sensitive (100%) in symptomatic VZV disease. We did not detect subclinical reactivation in asymptomatic SOT recipients during the first post-transplant year. Thus, subclinical VZV reactivation is either a rare event or does not exist. These data need to be confirmed in larger prospective trials.

  19. Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

    PubMed Central

    Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

    2017-01-01

    Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a

  20. Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR.

    PubMed

    Plotka, Magdalena; Wozniak, Mateusz; Kaczorowski, Tadeusz

    2017-01-01

    Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within Escherichia coli DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a

  1. SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR.

    PubMed

    Stabley, Deborah L; Harris, Ashlee W; Holbrook, Jennifer; Chubbs, Nicholas J; Lozo, Kevin W; Crawford, Thomas O; Swoboda, Kathryn J; Funanage, Vicky L; Wang, Wenlan; Mackenzie, William; Scavina, Mena; Sol-Church, Katia; Butchbach, Matthew E R

    2015-07-01

    Proximal spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutation of survival motor neuron 1 (SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene (SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR (dPCR) system. This dPCR assay can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples.

  2. SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR

    PubMed Central

    Stabley, Deborah L; Harris, Ashlee W; Holbrook, Jennifer; Chubbs, Nicholas J; Lozo, Kevin W; Crawford, Thomas O; Swoboda, Kathryn J; Funanage, Vicky L; Wang, Wenlan; Mackenzie, William; Scavina, Mena; Sol-Church, Katia; Butchbach, Matthew E R

    2015-01-01

    Proximal spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutation of survival motor neuron 1 (SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene (SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR (dPCR) system. This dPCR assay can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples. PMID:26247043

  3. Measurement methods and accuracy in copy number variation: failure to replicate associations of beta-defensin copy number with Crohn's disease

    PubMed Central

    Aldhous, Marian C.; Abu Bakar, Suhaili; Prescott, Natalie J.; Palla, Raquel; Soo, Kimberley; Mansfield, John C.; Mathew, Christopher G.; Satsangi, Jack; Armour, John A.L.

    2010-01-01

    The copy number variation in beta-defensin genes on human chromosome 8 has been proposed to underlie susceptibility to inflammatory disorders, but presents considerable challenges for accurate typing on the scale required for adequately powered case–control studies. In this work, we have used accurate methods of copy number typing based on the paralogue ratio test (PRT) to assess beta-defensin copy number in more than 1500 UK DNA samples including more than 1000 cases of Crohn's disease. A subset of 625 samples was typed using both PRT-based methods and standard real-time PCR methods, from which direct comparisons highlight potentially serious shortcomings of a real-time PCR assay for typing this variant. Comparing our PRT-based results with two previous studies based only on real-time PCR, we find no evidence to support the reported association of Crohn's disease with either low or high beta-defensin copy number; furthermore, it is noteworthy that there are disagreements between different studies on the observed frequency distribution of copy number states among European controls. We suggest safeguards to be adopted in assessing and reporting the accuracy of copy number measurement, with particular emphasis on integer clustering of results, to avoid reporting of spurious associations in future case–control studies. PMID:20858604

  4. Improved methods of carnivore faecal sample preservation, DNA extraction and quantification for accurate genotyping of wild tigers.

    PubMed

    Reddy, Patlolla Anuradha; Bhavanishankar, Maradani; Bhagavatula, Jyotsna; Harika, Katakam; Mahla, Ranjeet Singh; Shivaji, Sisinthy

    2012-01-01

    Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively. Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols.

  5. Improved Methods of Carnivore Faecal Sample Preservation, DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

    PubMed Central

    Harika, Katakam; Mahla, Ranjeet Singh; Shivaji, Sisinthy

    2012-01-01

    Background Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. Methods We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Results Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively. Conclusions Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols. PMID:23071624

  6. Developmental exposure to second-hand smoke increases adult atherogenesis and alters mitochondrial DNA copy number and deletions in apoE(-/-) mice.

    PubMed

    Fetterman, Jessica L; Pompilius, Melissa; Westbrook, David G; Uyeminami, Dale; Brown, Jamelle; Pinkerton, Kent E; Ballinger, Scott W

    2013-01-01

    Cardiovascular disease is a major cause of morbidity and mortality in the United States. While many studies have focused upon the effects of adult second-hand smoke exposure on cardiovascular disease development, disease development occurs over decades and is likely influenced by childhood exposure. The impacts of in utero versus neonatal second-hand smoke exposure on adult atherosclerotic disease development are not known. The objective of the current study was to determine the effects of in utero versus neonatal exposure to a low dose (1 mg/m(3) total suspended particulate) of second-hand smoke on adult atherosclerotic lesion development using the apolipoprotein E null mouse model. Consequently, apolipoprotein E null mice were exposed to either filtered air or second-hand smoke: (i) in utero from gestation days 1-19, or (ii) from birth until 3 weeks of age (neonatal). Subsequently, all animals were exposed to filtered air and sacrificed at 12-14 weeks of age. Oil red-O staining of whole aortas, measures of mitochondrial damage, and oxidative stress were performed. Results show that both in utero and neonatal second-hand smoke exposure significantly increased adult atherogenesis in mice compared to filtered air controls. These changes were associated with changes in aconitase and mitochondrial superoxide dismutase activities consistent with increased oxidative stress in the aorta, changes in mitochondrial DNA copy number and deletion levels. These studies show that in utero or neonatal exposure to second-hand smoke significantly influences adult atherosclerotic lesion development and results in significant alterations to the mitochondrion and its genome that may contribute to atherogenesis.

  7. Low-copy nuclear DNA, phylogeny and the evolution of dichogamy in the betel nut palms and their relatives (Arecinae; Arecaceae).

    PubMed

    Loo, Adrian H B; Dransfield, John; Chase, Mark W; Baker, William J

    2006-06-01

    For the betel nut palm genus Areca and the other seven genera in subtribe Arecinae (Areceae; Arecoideae; Arecaceae) we collected DNA sequences from two low-copy nuclear genes, phosphoribulokinase (PRK) and the second largest subunit of RNA polymerase II (RPB2). The data were used to evaluate monophyly of the subtribe and its component genera, explore the radiation of the group across its range, and examine evolution of protandry and protogyny, which is particularly diverse in Arecinae. The subtribe and some genera are not monophyletic. Three lineages of Arecinae are recovered: one widespread, but centered on the Sunda Shelf, another endemic to the islands east of Wallace's line and a third, comprising the Sri Lanka endemic Loxococcus, that is most closely related to genera from outside subtribe Arecinae. Strong support is obtained for broadening the circumscription of the genus Hydriastele to include Gronophyllum, Gulubia and Siphokentia. In clarifying phylogenetic relationships, we have demonstrated that a perceived bimodal distribution of the subtribe across Wallace's line does not in fact exist. Character optimizations indicate that the evolution of protogyny, an unusual condition in palms, is potentially correlated with a large radiation in the genus Pinanga and possibly also to dramatic diversification in pollen morphology and genome size. The evolution of dichogamy in the clade endemic to the east of Wallace's line is complex and reveals a pattern of numerous transformations between protandry and protogyny that is in marked contrast with other Arecinae. We suggest that this contrast is most likely a reflection of differing geological histories and pollinator spectra in each region.

  8. Translesion DNA Synthesis and Mutagenesis in Eukaryotes

    PubMed Central

    Sale, Julian E.

    2013-01-01

    The structural features that enable replicative DNA polymerases to synthesize DNA rapidly and accurately also limit their ability to copy damaged DNA. Direct replication of DNA damage is termed translesion synthesis (TLS), a mechanism conserved from bacteria to mammals and executed by an array of specialized DNA polymerases. This chapter examines how these translesion polymerases replicate damaged DNA and how they are regulated to balance their ability to replicate DNA lesions with the risk of undesirable mutagenesis. It also discusses how TLS is co-opted to increase the diversity of the immunoglobulin gene hypermutation and the contribution it makes to the mutations that sculpt the genome of cancer cells. PMID:23457261

  9. Highly Informative Single-Copy Nuclear Microsatellite DNA Markers Developed Using an AFLP-SSR Approach in Black Spruce (Picea mariana) and Red Spruce (P. rubens)

    PubMed Central

    Shi, Yong-Zhong; Forneris, Natascha; Rajora, Om P.

    2014-01-01

    Background Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies. Principal Findings A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies. Significance The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce

  10. DNA extraction techniques compared for accurate detection of genetically modified organisms (GMOs) in maize food and feed products.

    PubMed

    Turkec, Aydin; Kazan, Hande; Karacanli, Burçin; Lucas, Stuart J

    2015-08-01

    In this paper, DNA extraction methods have been evaluated to detect the presence of genetically modified organisms (GMOs) in maize food and feed products commercialised in Turkey. All the extraction methods tested performed well for the majority of maize foods and feed products analysed. However, the highest DNA content was achieved by the Wizard, Genespin or the CTAB method, all of which produced optimal DNA yield and purity for different maize food and feed products. The samples were then screened for the presence of GM elements, along with certified reference materials. Of the food and feed samples, 8 % tested positive for the presence of one GM element (NOS terminator), of which half (4 % of the total) also contained a second element (the Cauliflower Mosaic Virus 35S promoter). The results obtained herein clearly demonstrate the presence of GM maize in the Turkish market, and that the Foodproof GMO Screening Kit provides reliable screening of maize food and feed products.

  11. Rapid and accurate detection of RMP- and INH- resistant Mycobacterium tuberculosis in spinal tuberculosis specimens by CapitalBio™ DNA microarray: a prospective validation study.

    PubMed

    Zhang, Zehua; Li, Litao; Luo, Fei; Cheng, Peng; Wu, Feng; Wu, Zheng; Hou, Tianyong; Zhong, Min; Xu, Jianzhong

    2012-11-14

    DNA microarrays can detect tuberculosis and its multi-drug resistant form in M. tuberculosis isolates and sputum specimens with high sensitivity and specificity. However, no performance data currently exists for its use in spinal tuberculosis specimens. This study was aimed to assess the performance of the CapitalBio™ DNA microarray in the detection of isoniazid (INH) and rifampicin (RMP) resistance in spinal tuberculosis compared with the BACT/MGIT 960 system. From March 2009 to December 2011, 153 consecutive patients from Southwest Hospital, Chongqing with clinically and pathologically diagnosed spinal tuberculosis were enrolled into this study. Specimens collected during surgery from the tuberculosis patients were subjected to M. tuberculosis species identification and drug-resistance detection by the CapitalBio™ DNA microarray, and results were compared with those obtained from the absolute concentration drug susceptibility testing. The CapitalBio™ DNA microarray achieved 93.55% sensitivity for the correct M. tuberculosis species identification of the 93 specimens that tested positive for spinal tuberculosis through culture. In addition, twenty-seven additional patients (45.0%) were detected by the DNA microarray to be positive for M. tuberculosis among sixty spinal tuberculosis patients who were culture negative. Moreover, the DNA microarray had a sensitivity of 88.9% and a specificity of 90.7% for RMP resistance, and the microarray had a sensitivity of 80.0% and a specificity of 91.0% for INH resistance. The mean turn-around time of M. tuberculosis species identification and drug resistance detection using the DNA microarray was 5.8 (range, 4-9) hours. The CapitalBio™ DNA microarray is a feasible and accurate tool for the species identification of M. tuberculosis and for directly detecting RMP and INH resistance from spinal tuberculosis specimens in fewer than 9 hours.

  12. MBRidge: an accurate and cost-effective method for profiling DNA methylome at single-base resolution.

    PubMed

    Cai, Wanshi; Mao, Fengbiao; Teng, Huajing; Cai, Tao; Zhao, Fangqing; Wu, Jinyu; Sun, Zhong Sheng

    2015-08-01

    Organisms and cells, in response to environmental influences or during development, undergo considerable changes in DNA methylation on a genome-wide scale, which are linked to a variety of biological processes. Using MethylC-seq to decipher DNA methylome at single-base resolution is prohibitively costly. In this study, we develop a novel approach, named MBRidge, to detect the methylation levels of repertoire CpGs, by innovatively introducing C-hydroxylmethylated adapters and bisulfate treatment into the MeDIP-seq protocol and employing ridge regression in data analysis. A systematic evaluation of DNA methylome in a human ovarian cell line T29 showed that MBRidge achieved high correlation (R > 0.90) with much less cost (∼10%) in comparison with MethylC-seq. We further applied MBRidge to profiling DNA methylome in T29H, an oncogenic counterpart of T29's. By comparing methylomes of T29H and T29, we identified 131790 differential methylation regions (DMRs), which are mainly enriched in carcinogenesis-related pathways. These are substantially different from 7567 DMRs that were obtained by RRBS and related with cell development or differentiation. The integrated analysis of DMRs in the promoter and expression of DMR-corresponding genes revealed that DNA methylation enforced reverse regulation of gene expression, depending on the distance from the proximal DMR to transcription starting sites in both mRNA and lncRNA. Taken together, our results demonstrate that MBRidge is an efficient and cost-effective method that can be widely applied to profiling DNA methylomes.

  13. Roles of the Y-family DNA polymerase Dbh in accurate replication of the Sulfolobus genome at high temperature.

    PubMed

    Sakofsky, Cynthia J; Foster, Patricia L; Grogan, Dennis W

    2012-04-01

    The intrinsically thermostable Y-family DNA polymerases of Sulfolobus spp. have revealed detailed three-dimensional structure and catalytic mechanisms of trans-lesion DNA polymerases, yet their functions in maintaining their native genomes remain largely unexplored. To identify functions of the Y-family DNA polymerase Dbh in replicating the Sulfolobus genome under extreme conditions, we disrupted the dbh gene in Sulfolobus acidocaldarius and characterized the resulting mutant strains phenotypically. Disruption of dbh did not cause any obvious growth defect, sensitivity to any of several DNA-damaging agents, or change in overall rate of spontaneous mutation at a well-characterized target gene. Loss of dbh did, however, cause significant changes in the spectrum of spontaneous forward mutation in each of two orthologous target genes of different sequence. Relative to wild-type strains, dbh(-) constructs exhibited fewer frame-shift and other small insertion-deletion mutations, but exhibited more base-pair substitutions that converted G:C base pairs to T:A base pairs. These changes, which were confirmed to be statistically significant, indicate two distinct activities of the Dbh polymerase in Sulfolobus cells growing under nearly optimal culture conditions (78-80°C and pH 3). The first activity promotes slipped-strand events within simple repetitive motifs, such as mononucleotide runs or triplet repeats, and the second promotes insertion of C opposite a potentially miscoding form of G, thereby avoiding G:C to T:A transversions.

  14. Stochastic and reversible assembly of a multiprotein DNA repair complex ensures accurate target site recognition and efficient repair

    PubMed Central

    Luijsterburg, Martijn S.; von Bornstaedt, Gesa; Gourdin, Audrey M.; Politi, Antonio Z.; Moné, Martijn J.; Warmerdam, Daniël O.; Goedhart, Joachim; Vermeulen, Wim

    2010-01-01

    To understand how multiprotein complexes assemble and function on chromatin, we combined quantitative analysis of the mammalian nucleotide excision DNA repair (NER) machinery in living cells with computational modeling. We found that individual NER components exchange within tens of seconds between the bound state in repair complexes and the diffusive state in the nucleoplasm, whereas their net accumulation at repair sites evolves over several hours. Based on these in vivo data, we developed a predictive kinetic model for the assembly and function of repair complexes. DNA repair is orchestrated by the interplay of reversible protein-binding events and progressive enzymatic modifications of the chromatin substrate. We demonstrate that faithful recognition of DNA lesions is time consuming, whereas subsequently, repair complexes form rapidly through random and reversible assembly of NER proteins. Our kinetic analysis of the NER system reveals a fundamental conflict between specificity and efficiency of chromatin-associated protein machineries and shows how a trade off is negotiated through reversibility of protein binding. PMID:20439997

  15. High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature

    SciTech Connect

    Mathew, M.K.; Smith, C.L.; Cantor, C.R. )

    1988-12-27

    Pulsed-field gel electrophoresis (PGF) subjects DNA alternately to two electrical fields to resolve DNA ranging from 10,000 base pairs (10 kb) to 10,000 kb in size. The separations are quite sensitive to a variety of experimental variables. This makes it critical to have a wide range of reliable size standards. A technique is described for preparing mixtures of bacteriophage DNA oligomers that span a size range from monomer to more than 30-mer. The relationship between size and mobility of oligomers of different bacteriophage DNA monomers is generally self-consistent. Thus, these samples can serve as primary length standards for DNAs ranging from 10 kb to more than 1,500 kb. They have been used to estimate the size of the chromosomal DNAs from various Saccharomyces cerevisiae strains and to test the effect of gel concentration and temperature on PFG. DNA resolution during PFG is slightly improved in agarose gels with small pore sizes, in contrast to continuous electrophoresis where the opposite is observed. PFG mobility is surprisingly sensitive to changes in the running temperature.

  16. Evolution of ribosomal RNA gene copy number on the sex chromosomes of Drosophila melanogaster.

    PubMed

    Lyckegaard, E M; Clark, A G

    1991-07-01

    A diverse array of cellular and evolutionary forces--including unequal crossing-over, magnification, compensation, and natural selection--is at play modulating the number of copies of ribosomal RNA (rRNA) genes on the X and Y chromosomes of Drosophila. Accurate estimates of naturally occurring distributions of copy numbers on both the X and Y chromosomes are needed in order to explore the evolutionary end result of these forces. Estimates of relative copy numbers of the ribosomal DNA repeat, as well as of the type I and type II inserts, were obtained for a series of 96 X chromosomes and 144 Y chromosomes by using densitometric measurements of slot blots of genomic DNA from adult D. melanogaster bearing appropriate deficiencies that reveal chromosome-specific copy numbers. Estimates of copy number were put on an absolute scale with slot blots having serial dilutions both of the repeat and of genomic DNA from nonpolytene larval brain and imaginal discs. The distributions of rRNA copy number are decidedly skewed, with a long tail toward higher copy numbers. These distributions were fitted by a population genetic model that posits three different types of exchange events--sister-chromatid exchange, intrachromatid exchange, and interchromosomal crossing-over. In addition, the model incorporates natural selection, because experimental evidence shows that there is a minimum number of functional elements necessary for survival. Adequate fits of the model were found, indicating that either natural selection also eliminates chromosomes with high copy number or that the rate of intrachromatid exchange exceeds the rate of interchromosomal exchange.

  17. Characterization of three active transposable elements recently inserted in three independent DFR-A alleles and one high-copy DNA transposon isolated from the Pink allele of the ANS gene in onion (Allium cepa L.).

    PubMed

    Kim, Sunggil; Park, Jee Young; Yang, Tae-Jin

    2015-06-01

    Intact retrotransposon and DNA transposons inserted in a single gene were characterized in onions (Allium cepa) and their transcription and copy numbers were estimated in this study. While analyzing diverse onion germplasm, large insertions in the DFR-A gene encoding dihydroflavonol 4-reductase (DFR) involved in the anthocyanin biosynthesis pathway were found in two accessions. A 5,070-bp long terminal repeat (LTR) retrotransposon inserted in the active DFR-A (R4) allele was identified from one of the large insertions and designated AcCOPIA1. An intact ORF encoded typical domains of copia-like LTR retrotransposons. However, AcCOPIA1 contained atypical 'TG' and 'TA' dinucleotides at the ends of the LTRs. A 4,615-bp DNA transposon was identified in the other large insertion. This DNA transposon, designated AcCACTA1, contained an ORF coding for a transposase showing homology with the CACTA superfamily transposable elements (TEs). Another 5,073-bp DNA transposon was identified from the DFR-A (TRN) allele. This DNA transposon, designated AchAT1, belonged to the hAT superfamily with short 4-bp terminal inverted repeats (TIRs). Finally, a 6,258-bp non-autonomous DNA transposon, designated AcPINK, was identified in the ANS-p allele encoding anthocyanidin synthase, the next downstream enzyme to DFR in the anthocyanin biosynthesis pathway. AcPINK also possessed very short 3-bp TIRs. Active transcription of AcCOPIA1, AcCACTA1, and AchAT1 was observed through RNA-Seq analysis and RT-PCR. The copy numbers of AcPINK estimated by mapping the genomic DNA reads produced by NextSeq 500 were predominantly high compared with the other TEs. A series of evidence indicated that these TEs might have transposed in these onion genes very recently, providing a stepping stone for elucidation of enormously large-sized onion genome structure.

  18. IrisPlex: a sensitive DNA tool for accurate prediction of blue and brown eye colour in the absence of ancestry information.

    PubMed

    Walsh, Susan; Liu, Fan; Ballantyne, Kaye N; van Oven, Mannis; Lao, Oscar; Kayser, Manfred

    2011-06-01

    A new era of 'DNA intelligence' is arriving in forensic biology, due to the impending ability to predict externally visible characteristics (EVCs) from biological material such as those found at crime scenes. EVC prediction from forensic samples, or from body parts, is expected to help concentrate police investigations towards finding unknown individuals, at times when conventional DNA profiling fails to provide informative leads. Here we present a robust and sensitive tool, termed IrisPlex, for the accurate prediction of blue and brown eye colour from DNA in future forensic applications. We used the six currently most eye colour-informative single nucleotide polymorphisms (SNPs) that previously revealed prevalence-adjusted prediction accuracies of over 90% for blue and brown eye colour in 6168 Dutch Europeans. The single multiplex assay, based on SNaPshot chemistry and capillary electrophoresis, both widely used in forensic laboratories, displays high levels of genotyping sensitivity with complete profiles generated from as little as 31pg of DNA, approximately six human diploid cell equivalents. We also present a prediction model to correctly classify an individual's eye colour, via probability estimation solely based on DNA data, and illustrate the accuracy of the developed prediction test on 40 individuals from various geographic origins. Moreover, we obtained insights into the worldwide allele distribution of these six SNPs using the HGDP-CEPH samples of 51 populations. Eye colour prediction analyses from HGDP-CEPH samples provide evidence that the test and model presented here perform reliably without prior ancestry information, although future worldwide genotype and phenotype data shall confirm this notion. As our IrisPlex eye colour prediction test is capable of immediate implementation in forensic casework, it represents one of the first steps forward in the creation of a fully individualised EVC prediction system for future use in forensic DNA intelligence.

  19. Use of competitive PCR to assay copy number of repetitive elements in banana.

    PubMed

    Baurens, F C; Noyer, J L; Lanaud, C; Lagoda, P J

    1996-11-27

    Banana is one of the most important subtropical fruit crops. Genetic improvement by traditional breeding strategies is difficult and better knowledge of genomic structure is needed. Repeated sequences are powerful markers for genetic fingerprinting. The method proposed here to determine the copy number of nuclear repetitive elements is based on competitive reverse transcription-polymerase chain reaction and can also be used for quantifying cytosolic sequences. The reliability of this method was investigated on crude preparations of total DNA. Variations due to the heterogeneity of crude DNA extracts showed that a single locus reference is needed for accurate quantification. A mapped microsatellite locus was used to normalize copy number measurements. Copy number assay of repetitive elements using this method clearly distinguishes between the two banana subspecies investigated: Musa acuminata spp. banskii and M. acuminata spp. malaccensis. Two repetitive sequence families, pMaCIR1115 and pA9-26, were assayed that cover up to 1% of the M. acuminata genome. Their copy number varied up to six fold between the two subspecies. Furthermore, sequence quantification showed that mitochondrial genomes are present in crude leaf-extracted banana DNA at up to 40 copies per cell.

  20. Quantitative analysis of waterfowl parvoviruses in geese and Muscovy ducks by real-time polymerase chain reaction: correlation between age, clinical symptoms and DNA copy number of waterfowl parvoviruses.

    PubMed

    Woźniakowski, Grzegorz; Samorek-Salamonowicz, Elżbieta; Kozdruń, Wojciech

    2012-03-15

    Waterfowl parvoviruses cause serious loss in geese and ducks production. Goose parvovirus (GPV) is infectious for geese and ducks while Muscovy duck parvovirus (MDPV) infects Muscovy ducks only. So far, for these viruses' sensitive detection polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were applied. However, there was no molecular biology method for both waterfowl parvoviruses detection and quantification which could unify the laboratory procedures. The level of GPV and MDPV replication and distribution plays a significant role in the parvoviral infection progress and is strictly correlated to clinical symptoms. Meanwhile, experiments conducted previously on GPV distribution in geese, performed as animal trial, did not involve epidemiological data from the disease field cases. The study on the correlation between age, clinical symptoms and viral DNA copy number may be benefitable in understanding the GPV and MDPV infection. Such data may also aid in determination of the stage and severity of the infection with parvoviruses. Therefore the aim of this study was to develop quantitative real-time PCR for parallel detection of GPV and MDPV in geese and Muscovy ducks and to determine the correlation between the age of the infected birds, clinical symptoms and DNA copy number for the estimation of the disease stage or severity. In order to develop quantitative real-time PCR the viral material was collected from 13 farms of geese and 3 farms of Muscovy ducks. The designed primers and Taqman probe for real-time PCR were complementary to GPV and MDPV inverted terminal repeats region. The pITR plasmid was constructed, purified and used to prepare dilutions for standard curve preparation and DNA quantification. The applied method detected both GPV and MDPV in all the examined samples extracted from the heart and liver of the infected birds. The conducted correlation tests have shown relationship between age, clinical symptoms during

  1. Visual System Homeobox 1 (VSX1) Gene Analysis in Keratoconus: Design of Specific Primers and DNA Amplification Protocols for Accurate Molecular Characterization.

    PubMed

    Ng, Jun B; Poh, Rozaida Y Y; Lee, Kenneth R; Subrayan, Visvaraja; Deva, Jenny P; Lau, Amy Y L; Tan, Jin A M A

    2016-09-01

    Keratoconus is an ocular degeneration characterized by the thinning of corneal stroma that may lead to varying degrees of myopia and visual impairment. Genetic factors have been reported in the pathology of keratoconus where Asians have a higher incidence, earlier onset, and undergo earlier corneal grafts compared to Caucasians. The visual system homeobox 1 (VSX1) gene forms part of a paired-like homeodomain transcription factor which is responsible for ocular development. The gene was marked as a candidate in genetic studies of keratoconus in various populations. Single nucleotide polymorphisms (SNPs) in the VSX1 gene have been reported to be associated with keratoconus. The detection of the SNPs involves DNA amplification of the VSX1 gene followed by genomic sequencing. Thus, the objective of this study aims to establish sensitive and accurate screening protocols for the molecular characterization of VSX1 polymorphisms. Keratoconic (n = 74) and control subjects (n = 96) were recruited based on clinical diagnostic tests and selection criteria. DNA extracted from the blood samples was used to genotype VSX1 polymorphisms. In-house designed primers and optimization of PCR conditions were carried out to amplify exons 1 and 3 of the VSX1 gene. PCR conditions including percentage GC content, melting temperatures, and differences in melting temperatures of primers were evaluated to produce sensitive and specific DNA amplifications. Genotyping was successfully carried out in 4 exons of the VSX1 gene. Primer annealing temperatures were observed to be crucial in enhancing PCR sensitivity and specificity. Annealing temperatures were carefully evaluated to produce increased specificity, yet not allowing sensitivity to be compromised. In addition, exon 1 of the VSX1 gene was amplified using 2 different sets of primers to produce 2 smaller amplified products with absence of non-specific bands. DNA amplification of exons 1 and 3 consistently showed single band products which

  2. Nucleic Acids Research Group (NRG): The Importance of DNA Extraction in Metagenomics: The Gatekeeper to Accurate Results!

    PubMed Central

    Carmical, R.; Nadella, V.; Herbert, Z.; Beckloff, N.; Chittur, S.; Rosato, C.; Perera, A.; Auer, H.; Robinson, M.; Tighe, S.; Holbrook, Jennifer

    2013-01-01

    It is well recognized that the field of metagenomics is becoming a critical tool for studying previously unobtainable population dynamics at both an identification of species level and a functional or transcriptional level. Because the power to resolve microbial information is so important for identifying the components in an mixed sample, metagenomics can be used to study nearly any possible environment or system including clinical, environmental, and industrial, to name a few. Clinically, it may be used to determine sub-populations colonizing regions of the body or determining a rare infection to assist in treatment strategies. Environmentally it may be used to identify microbial populations within a soil, water or air sample, or within a bioreactor to characterize a population- based functional process. The possibilities are endless. However, the accuracy of a metagenomics dataset relies on three important “gatekeepers” including 1) The ability to effectively extract all DNA or RNA from every cell within a sample, 2) The reliability of the methods used for deep or high-throughput sequencing, and 3) The software used to analyze the data. Since DNA extraction is the first step in the technical process of metagenomics, the Nucleic Acid Research Group (NARG) conducted a study to evaluate extraction methods using a synthetic microbial sample. The synthetic microbial sample was prepared from 10 known bacteria at specific concentrations and ranging in diversity. Samples were extracted in duplicate using various popular kit based methods as well as several homebrew protocols then analyzed by NextGen sequencing on an Illumina HiSeq. Results of the study include determining the percent recovery of those organisms by comparing to the known quantity in the original synthetic mix.

  3. Selection of suitable endogenous reference genes for relative copy number detection in sugarcane.

    PubMed

    Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

    2014-05-19

    Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential "single copy" genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3--high copy number group, TST-1 and PRR-1--medium copy number group, P4H-1, APRT-2 and CYC-2--low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

  4. Evaluation of gene expression and DNA copy number profiles of adipose tissue-derived stromal cells and consecutive neurosphere-like cells generated from dogs with naturally occurring spinal cord injury.

    PubMed

    Lim, Ji-Hey; Koh, Sehwon; Thomas, Rachael; Breen, Matthew; Olby, Natasha J

    2017-03-01

    OBJECTIVE To evaluate gene expression and DNA copy number in adipose tissue-derived stromal cells (ADSCs) and in ADSC-derived neurosphere-like cell clusters (ADSC-NSCs) generated from tissues of chronically paraplegic dogs. ANIMALS 14 client-owned paraplegic dogs. PROCEDURES Dorsal subcutaneous adipose tissue (< 1 cm(3)) was collected under general anesthesia; ADSCs were isolated and cultured. Third-passage ADSCs were cultured in neural cell induction medium to generate ADSC-NSCs. Relative gene expression of mesenchymal cell surface marker CD90 and neural progenitor marker nestin was assessed in ADSCs and ADSC-NSCs from 3 dogs by quantitative real-time PCR assay; expression of these and various neural lineage genes was evaluated for the same dogs by reverse transcription PCR assay. Percentages of cells expressing CD90, nestin, glial fibrillary acidic protein (GFAP), and tubulin β 3 class III (TUJ1) proteins were determined by flow cytometry for all dogs. The DNA copy number stability (in samples from 6 dogs) and neural cell differentiation (14 dogs) were assessed with array-comparative genomic hybridization analysis and immunocytochemical evaluation, respectively. RESULTS ADSCs and ADSC-NSCs expressed neural cell progenitor and differentiation markers; GFAP and microtubule-associated protein 2 were expressed by ADSC-NSCs but not ADSCs. Relative gene expression of CD90 and nestin was subjectively higher in ADSC-NSCs than in ADSCs. Percentages of ADSC-NSCs expressing nestin, GFAP, and TUJ1 proteins were substantially higher than those of ADSCs. Cells expressing neuronal and glial markers were generated from ADSC-NSCs and had no DNA copy number instability detectable by the methods used. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested ADSCs can potentially be a safe and clinically relevant autologous source for canine neural progenitor cells. Further research is needed to verify these findings.

  5. Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli.

    PubMed Central

    Firshein, W; Strumph, P; Benjamin, P; Burnstein, K; Kornacki, J

    1982-01-01

    A DNA-membrane complex was extracted from minicells of an Escherichia coli mutant harboring a "miniplasmid" derivative (11.2 kilobases) of the low-copynumber plasmid RK2 (56 kilobases). The complex contained various species of supercoiled and intermediate forms of plasmid DNA, of which approximately 20% was bound firmly to the membrane after centrifugation in a CsCl density gradient. The plasmid DNA-membrane complex synthesized new plasmid DNA without the addition of exogenous template, enzymes, or other proteins. DNA synthesis appeared to proceed semi-conservatively, was dependent on the four deoxynucleoside triphosphates, partially dependent on ribonucleoside triphosphates, and was sensitive to rifampin, an antibiotic known to inhibit initiation of replication. Novobiocin and nalidixic acid also inhibited synthesis, as did the omission of ATP, N-Ethylmaleimide, an inhibitor of DNA polymerase II and III activity, but not DNA polymerase I activity, also partially inhibited the synthetic reaction, as did chloramphenicol. The plasmid DNA synthetic product was analyzed by alkaline sucrose and dye-CsCl gradient centrifugation, as well as by agarose gel electrophoresis. In each case, the product consisted of parental and intermediate forms of plasmid DNA. Some chromosomal DNA was also synthesized by a contaminating bacterial DNA-membrane complex, but this synthesis was rifampin insensitive and could be separated from plasmid DNA synthesis. PMID:7042688

  6. Breaking and Entering: Copying and Copy Protection.

    ERIC Educational Resources Information Center

    Westlake, Wayne; And Others

    1985-01-01

    Describes several commercially-available computer programs which allow users to make copies of "protected" software. Current costs, program features, and ordering information are provided for these "encryption" programs. Also describes a monthly journal (The HARDCORE Computist) which focuses on unlocking copy-protected…

  7. Breaking and Entering: Copying and Copy Protection.

    ERIC Educational Resources Information Center

    Westlake, Wayne; And Others

    1985-01-01

    Describes several commercially-available computer programs which allow users to make copies of "protected" software. Current costs, program features, and ordering information are provided for these "encryption" programs. Also describes a monthly journal (The HARDCORE Computist) which focuses on unlocking copy-protected…

  8. Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[a]pyrene dG adduct.

    PubMed

    Liu, Yang; Yang, Yeran; Tang, Tie-Shan; Zhang, Hui; Wang, Zhifeng; Friedberg, Errol; Yang, Wei; Guo, Caixia

    2014-02-04

    DNA polymerase κ (Polκ) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide (BPDE)-N(2)-deoxyguanine adducts efficiently and accurately. The unique features of Polκ, a large structure gap between the catalytic core and little finger domain and a 90-residue addition at the N terminus known as the N-clasp, may give rise to its special translesion capability. We designed and constructed two mouse Polκ variants, which have reduced gap size on both sides [Polκ Gap Mutant (PGM) 1] or one side flanking the template base (PGM2). These Polκ variants are nearly as efficient as WT in normal DNA synthesis, albeit with reduced accuracy. However, PGM1 is strongly blocked by the 10S (+)-trans-anti-BPDE-N(2)-dG lesion. Steady-state kinetic measurements reveal a significant reduction in efficiency of dCTP incorporation opposite the lesion by PGM1 and a moderate reduction by PGM2. Consistently, Polκ-deficient cells stably complemented with PGM1 GFP-Polκ remained hypersensitive to BPDE treatment, and complementation with WT or PGM2 GFP-Polκ restored BPDE resistance. Furthermore, deletion of the first 51 residues of the N-clasp in mouse Polκ (mPolκ(52-516)) leads to reduced polymerization activity, and the mutant PGM2(52-516) but not PGM1(52-516) can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPolκ(52-516) retains BPDE bypass activity. We conclude that the structural gap physically accommodates the bulky aromatic adduct and the N-clasp is essential for the structural integrity and flexibility of Polκ during translesion synthesis.

  9. Phytochip: development of a DNA-microarray for rapid and accurate identification of Pseudo-nitzschia spp and other harmful algal species.

    PubMed

    Noyer, Charlotte; Abot, Anne; Trouilh, Lidwine; Leberre, Véronique Anton; Dreanno, Catherine

    2015-05-01

    Detection of harmful algal blooms has become a challenging concern because of the direct impacts on public health and economy. The identification of toxic dinoflagellates and diatoms in monitoring programs requires an extensive taxonomic expertise and is time consuming. Advances in molecular biology have allowed the development of new approaches, more rapid, accurate and cost-effective for detecting these microorganisms. In this context, we developed a new DNA microarray (called, Phytochip) for the simultaneous detection of multiple HAB species with a particular emphasis on Pseudo-nitzschia species. Oligonucleotide probes were designed along the rRNA operon. After DNA extraction, the target rDNA genes were amplified and labeled using an asymmetric PCR; then, the amplicons were hybridized to the oligonucleotide probes present on the chips. The total assay from seawater sampling to data acquisition can be performed within a working day. Specificity and sensitivity were assessed by using monoclonal cultures, mixtures of species and field samples spiked with a known amount of cultured cells. The Phytochip with its 81 validated oligonucleotide probes was able to detect 12 species of Pseudo-nitzschia and 11 species of dinoflagellates among which were 3 species of Karenia and 3 species of Alexandrium. The Phytochip was applied to environmental samples already characterized by light microscopy and cloned into DNA libraries. The hybridizations on the Phytochip were in good agreement with the sequences retrieved from the clone libraries and the microscopic observations. The Phytochip enables a reliable multiplex detection of phytoplankton and can assist a water quality monitoring program as well as more general ecological research.

  10. Accurate Chromosome Segregation at First Meiotic Division Requires AGO4, a Protein Involved in RNA-Dependent DNA Methylation in Arabidopsis thaliana.

    PubMed

    Oliver, Cecilia; Santos, Juan Luis; Pradillo, Mónica

    2016-10-01

    The RNA-directed DNA methylation (RdDM) pathway is important for the transcriptional repression of transposable elements and for heterochromatin formation. Small RNAs are key players in this process by regulating both DNA and histone methylation. Taking into account that methylation underlies gene silencing and that there are genes with meiosis-specific expression profiles, we have wondered whether genes involved in RdDM could play a role during this specialized cell division. To address this issue, we have characterized meiosis progression in pollen mother cells from Arabidopsis thaliana mutant plants defective for several proteins related to RdDM. The most relevant results were obtained for ago4-1 In this mutant, meiocytes display a slight reduction in chiasma frequency, alterations in chromatin conformation around centromeric regions, lagging chromosomes at anaphase I, and defects in spindle organization. These abnormalities lead to the formation of polyads instead of tetrads at the end of meiosis, and might be responsible for the fertility defects observed in this mutant. Findings reported here highlight an involvement of AGO4 during meiosis by ensuring accurate chromosome segregation at anaphase I.

  11. Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

    PubMed Central

    Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

    2014-01-01

    Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane. PMID:24857916

  12. 37 CFR 1.13 - Copies and certified copies.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Copies and certified copies... Patent and Trademark Office § 1.13 Copies and certified copies. (a) Non-certified copies of patents, and... States Patent and Trademark Office to any person, and copies of other records or papers will be furnished...

  13. BCRgt: a Bayesian cluster regression-based genotyping algorithm for the samples with copy number alterations

    PubMed Central

    2014-01-01

    Background Accurate genotype calling is a pre-requisite of a successful Genome-Wide Association Study (GWAS). Although most genotyping algorithms can achieve an accuracy rate greater than 99% for genotyping DNA samples without copy number alterations (CNAs), almost all of these algorithms are not designed for genotyping tumor samples that are known to have large regions of CNAs. Results This study aims to develop a statistical method that can accurately genotype tumor samples with CNAs. The proposed method adds a Bayesian layer to a cluster regression model and is termed a Bayesian Cluster Regression-based genotyping algorithm (BCRgt). We demonstrate that high concordance rates with HapMap calls can be achieved without using reference/training samples, when CNAs do not exist. By adding a training step, we have obtained higher genotyping concordance rates, without requiring large sample sizes. When CNAs exist in the samples, accuracy can be dramatically improved in regions with DNA copy loss and slightly improved in regions with copy number gain, comparing with the Bayesian Robust Linear Model with Mahalanobis distance classifier (BRLMM). Conclusions In conclusion, we have demonstrated that BCRgt can provide accurate genotyping calls for tumor samples with CNAs. PMID:24629125

  14. Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers

    PubMed Central

    2011-01-01

    Background The transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The Sleeping Beauty (SB) system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI) method is able to determine SB transposon copy numbers regardless of the genetic cargo. Results We designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene RPPH1 and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle. Conclusions We have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy. PMID:21371313

  15. A Continental-Wide Perspective: The Genepool of Nuclear Encoded Ribosomal DNA and Single-Copy Gene Sequences in North American Boechera (Brassicaceae)

    PubMed Central

    Kiefer, Christiane; Koch, Marcus A.

    2012-01-01

    74 of the currently accepted 111 taxa of the North American genus Boechera (Brassicaceae) were subject to pyhlogenetic reconstruction and network analysis. The dataset comprised 911 accessions for which ITS sequences were analyzed. Phylogenetic analyses yielded largely unresolved trees. Together with the network analysis confirming this result this can be interpreted as an indication for multiple, independent, and rapid diversification events. Network analyses were superimposed with datasets describing i) geographical distribution, ii) taxonomy, iii) reproductive mode, and iv) distribution history based on phylogeographic evidence. Our results provide first direct evidence for enormous reticulate evolution in the entire genus and give further insights into the evolutionary history of this complex genus on a continental scale. In addition two novel single-copy gene markers, orthologues of the Arabidopsis thaliana genes At2g25920 and At3g18900, were analyzed for subsets of taxa and confirmed the findings obtained through the ITS data. PMID:22606266

  16. Confirmation of hybridity using GISH and determination of 18S rDNA copy number using FISH in interspecific F(1) hybrids of Tecoma (Bignoniaceae).

    PubMed

    Contreras, Ryan N; Ruter, John M; Conner, Joann; Zeng, Yajuan; Ozias-Akins, Peggy

    2012-06-01

    Interspecific hybridization in Tecoma Juss. was conducted to develop novel forms for the nursery industry. We report fertile hybrids from the cross T. garrocha Hieron. (pistillate parent) × T. stans (L.) Juss. ex Kunth. Leaf morphology of the F(1) hybrids of T. garrocha × T. stans was intermediate between the parents. GISH also confirmed hybridity. The F(1) hybrids were successfully backcrossed to both parents and self-pollinated to produce BC and F(2) progeny. Tecoma garrocha , T. stans, and T. guarume A. DC. 'Tangelo' were self-fertile. The F(1) hybrids also were crossed with T. capensis (Thunb.) Lindl. and T. guarume 'Tangelo', resulting in three-species hybrids. FISH conducted on F(1) hybrids identified four copies of the 18S internal transcribed spacer region. Further analysis using FISH has the potential to provide information on the evolution of Bignoniaceae and the potential role of polyploidy.

  17. Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

    PubMed Central

    Chen, Sharon C.-A.; Wang, He; Zhang, Li; Fan, Xin; Xu, Zhi-Peng; Cheng, Jing-Wei; Kong, Fanrong; Zhao, Yu-Pei; Xu, Ying-Chun

    2016-01-01

    Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species. PMID:27105313

  18. Copy-left and Copy-right

    NASA Astrophysics Data System (ADS)

    VanderPlas, Jacob

    2015-01-01

    Any discussion of open licensing almost invariably devolves into a debate between copy-left licenses and permissive licenses, both sides defending their views with a nearly religious fervor. Copy-left licenses, typified by the GPL family of licenses, require all derived products to maintain the open, GPL license. Permissive licenses, typified by the BSD family of licenses, do not impose such requirements. I'll briefly explore the common arguments put forth in favor of either approach, and discuss some concrete examples of where these approaches have helped or hindered the software packages that used them.

  19. Real-time polymerase chain reaction to determine the prevalence and copy number of epstein-barr virus and cytomegalovirus DNA in subgingival plaque at individual healthy and periodontal disease sites.

    PubMed

    Dawson, Dolphus R; Wang, Chunmei; Danaher, Robert J; Lin, Yushun; Kryscio, Richard J; Jacob, Robert J; Miller, Craig S

    2009-07-01

    Detection of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in plaque from patients with periodontal disease provides support for the theory that these viruses play a role in the pathogenesis of periodontitis. This study sought to further define this relationship by determining the prevalence of these viruses at individual disease and healthy sites of patients with periodontal disease and to determine whether the presence and amount of viral DNA correlate with disease severity. Subgingival plaque from three healthy and three disease sites of 65 patients who had chronic periodontitis were evaluated for the presence and amount of EBV, CMV, and Fusobacterium nucleatum DNA using real-time polymerase chain reaction. Patient serum was evaluated for antibodies against EBV and CMV using enzyme-linked immunosorbent assays. EBV DNA was detected in 18.5% of subgingival plaque samples (72/390) and in at least one of the six plaque samples in 44.6% (29/65) of the patients. CMV DNA was detected in one plaque sample (0.3%). EBV was significantly more prevalent in disease sites (28.2%; 55/195) than in healthy sites (8.7%; 17/195; P = 0.002). However, neither EBV prevalence nor its amount correlated with increased probing depth >5 mm or attachment loss >2 mm, whereas the amount of F. nucleatum DNA did. Sites positive for EBV had a median copy number of eight. Antibodies against EBV and CMV were detected in 85.7% and 78.6% of persons evaluated, respectively. EBV was infrequent and CMV was rarely present in individual subgingival sites affected by chronic periodontitis.

  20. Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

    NASA Technical Reports Server (NTRS)

    Fouladi, B.; Waldren, C. A.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Lobrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

  1. Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

    NASA Technical Reports Server (NTRS)

    Fouladi, B.; Waldren, C. A.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Lobrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

  2. Functional domains of chicken mitochondrial transcription factor A for the maintenance of mitochondrial DNA copy number in lymphoma cell line DT40.

    PubMed

    Matsushima, Yuichi; Matsumura, Kiyoshi; Ishii, Shoji; Inagaki, Hidetoshi; Suzuki, Tomohiro; Matsuda, Yoichi; Beck, Konrad; Kitagawa, Yasuo

    2003-08-15

    Nuclear and mitochondrial (mt) forms of chicken mt transcription factor A (c-TFAM) generated by alternative splicing of a gene (c-tfam) were cloned. c-tfam mapped at 6q1.1-q1.2 has similar exon/intron organization as mouse tfam except that the first exons encoding the nuclear and the mt form-specific sequences were positioned oppositely. When cDNA encoding the nuclear form was transiently expressed in chicken lymphoma DT40 cells after tagging at the C terminus with c-Myc, the product was localized into nucleus, whereas the only endogenous mt form of DT40 cells was immunostained exclusively within mitochondria. c-TFAM is most similar to Xenopus (xl-) TFAM in having extended C-terminal regions in addition to two high mobility group (HMG) boxes, a linker region between them, and a C-terminal tail, also found in human and mouse TFAM. Similarities between c- and xl-TFAM are higher in linker and C-terminal regions than in HMG boxes. Disruption of both tfam alleles in DT40 cells prevented proliferation. The tfam+/tfam- cells showed a 50 and 40-60% reduction of mtDNA and its transcripts, respectively. Expression of exogenous wild type c-tfam cDNA in the tfam+/tfam- cells increased mtDNA up to 4-fold in a dose-dependent manner, whereas its transcripts increased only marginally. A deletion mutant lacking the first HMG box lost this activity, whereas only marginal reduction of the activity was observed in a deletion mutant at the second HMG box. Despite the essential role of the C-terminal tail in mtDNA transcription demonstrated in vitro, deletion of c-TFAM at this region reduced the activity of maintenance of the mtDNA level only by 50%. A series of deletion mutant at the tail region suggested stimulatory and suppressive sequences in this region for the maintenance of mtDNA level.

  3. A Single-Array-Based Method for Detecting Copy Number Variants Using Affymetrix High Density SNP Arrays and its Application to Breast Cancer.

    PubMed

    Li, Ming; Wen, Yalu; Fu, Wenjiang

    2014-01-01

    Cumulative evidence has shown that structural variations, due to insertions, deletions, and inversions of DNA, may contribute considerably to the development of complex human diseases, such as breast cancer. High-throughput genotyping technologies, such as Affymetrix high density single-nucleotide polymorphism (SNP) arrays, have produced large amounts of genetic data for genome-wide SNP genotype calling and copy number estimation. Meanwhile, there is a great need for accurate and efficient statistical methods to detect copy number variants. In this article, we introduce a hidden-Markov-model (HMM)-based method, referred to as the PICR-CNV, for copy number inference. The proposed method first estimates copy number abundance for each single SNP on a single array based on the raw fluorescence values, and then standardizes the estimated copy number abundance to achieve equal footing among multiple arrays. This method requires no between-array normalization, and thus, maintains data integrity and independence of samples among individual subjects. In addition to our efforts to apply new statistical technology to raw fluorescence values, the HMM has been applied to the standardized copy number abundance in order to reduce experimental noise. Through simulations, we show our refined method is able to infer copy number variants accurately. Application of the proposed method to a breast cancer dataset helps to identify genomic regions significantly associated with the disease.

  4. A Single-Array-Based Method for Detecting Copy Number Variants Using Affymetrix High Density SNP Arrays and its Application to Breast Cancer

    PubMed Central

    Li, Ming; Wen, Yalu; Fu, Wenjiang

    2014-01-01

    Cumulative evidence has shown that structural variations, due to insertions, deletions, and inversions of DNA, may contribute considerably to the development of complex human diseases, such as breast cancer. High-throughput genotyping technologies, such as Affymetrix high density single-nucleotide polymorphism (SNP) arrays, have produced large amounts of genetic data for genome-wide SNP genotype calling and copy number estimation. Meanwhile, there is a great need for accurate and efficient statistical methods to detect copy number variants. In this article, we introduce a hidden-Markov-model (HMM)-based method, referred to as the PICR-CNV, for copy number inference. The proposed method first estimates copy number abundance for each single SNP on a single array based on the raw fluorescence values, and then standardizes the estimated copy number abundance to achieve equal footing among multiple arrays. This method requires no between-array normalization, and thus, maintains data integrity and independence of samples among individual subjects. In addition to our efforts to apply new statistical technology to raw fluorescence values, the HMM has been applied to the standardized copy number abundance in order to reduce experimental noise. Through simulations, we show our refined method is able to infer copy number variants accurately. Application of the proposed method to a breast cancer dataset helps to identify genomic regions significantly associated with the disease. PMID:26279618

  5. Integrative genomics identifies distinct molecular classes of neuroblastoma and shows that multiple genes are targeted by regional alterations in DNA copy number.

    PubMed

    Wang, Qun; Diskin, Sharon; Rappaport, Eric; Attiyeh, Edward; Mosse, Yael; Shue, Daniel; Seiser, Eric; Jagannathan, Jayanti; Shusterman, Suzanne; Bansal, Manisha; Khazi, Deepa; Winter, Cynthia; Okawa, Erin; Grant, Gregory; Cnaan, Avital; Zhao, Huaqing; Cheung, Nai-Kong; Gerald, William; London, Wendy; Matthay, Katherine K; Brodeur, Garrett M; Maris, John M

    2006-06-15

    Neuroblastoma is remarkable for its clinical heterogeneity and is characterized by genomic alterations that are strongly correlated with tumor behavior. The specific genes that influence neuroblastoma biology and are targeted by genomic alterations remain largely unknown. We quantified mRNA expression in a highly annotated series of 101 prospectively collected diagnostic neuroblastoma primary tumors using an oligonucleotide-based microarray. Genomic copy number status at the prognostically relevant loci 1p36, 2p24 (MYCN), 11q23, and 17q23 was determined by PCR and was aberrant in 26, 20, 40, and 38 cases, respectively. In addition, 72 diagnostic neuroblastoma primary tumors assayed in a different laboratory were used as an independent validation set. Unsupervised hierarchical clustering showed that gene expression was highly correlated with genomic alterations and clinical markers of tumor behavior. The vast majority of samples with MYCN amplification and 1p36 loss of heterozygosity (LOH) clustered together on a terminal node of the sample dendrogram, whereas the majority of samples with 11q deletion clustered separately and both of these were largely distinct from the copy number neutral group of tumors. Genes involved in neurodevelopment were broadly overrepresented in the more benign tumors, whereas genes involved in RNA processing and cellular proliferation were highly represented in the most malignant cases. By combining transcriptomic and genomic data, we showed that LOH at 1p and 11q was associated with significantly decreased expression of 122 (61%) and 88 (27%) of the genes mapping to 1p35-36 and all of 11q, respectively, suggesting that multiple genes may be targeted by LOH events. A total of 71 of the 1p35-36 genes were also differentially expressed in the independent validation data set, providing a prioritized list of candidate neuroblastoma suppressor genes. Taken together, these data are consistent with the hypotheses that the neuroblastoma

  6. Differences in aggressive behavior and DNA copy number variants between BALB/cJ and BALB/cByJ substrains.

    PubMed

    Velez, Lady; Sokoloff, Greta; Miczek, Klaus A; Palmer, Abraham A; Dulawa, Stephanie C

    2010-03-01

    Some BALB/c substrains exhibit different levels of aggression. We compared aggression levels between male BALB/cJ and BALB/cByJ substrains using the resident intruder paradigm. These substrains were also assessed in other tests of emotionality and information processing including the open field, forced swim, fear conditioning, and prepulse inhibition tests. We also evaluated single nucleotide polymorphisms (SNPs) previously reported between these BALB/c substrains. Finally, we compared BALB/cJ and BALB/cByJ mice for genomic deletions or duplications, collectively termed copy number variants (CNVs), to identify candidate genes that might underlie the observed behavioral differences. BALB/cJ mice showed substantially higher aggression levels than BALB/cByJ mice; however, only minor differences in other behaviors were observed. None of the previously reported SNPs were verified. Eleven CNV regions were identified between the two BALB/c substrains. Our findings identify a robust difference in aggressive behavior between BALB/cJ and BALB/cByJ substrains, which could be the result of the identified CNVs.

  7. 7 CFR 97.179 - Copies and certified copies.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Copies and certified copies. 97.179 Section 97.179... PLANT VARIETY AND PROTECTION Fees and Charges § 97.179 Copies and certified copies. (a) Upon request, copies of applications, certificates, or of any records, books, papers, drawings, or photographs in the...

  8. Evaluating quantitative methods for measuring plasmid copy numbers in single cells

    PubMed Central

    Tal, Shay; Paulsson, Johan

    2013-01-01

    The life of plasmids is a constant battle against fluctuations: failing to correct copy number fluctuations can increase the plasmid loss rate by many orders of magnitude, as can a failure to more evenly divide the copies between daughters at cell division. Plasmids are therefore long-standing model systems for stochastic processes in cells, much thanks to the efforts of Kurt Nordström to whose memory this issue is dedicated. Here we analyze a range of experimental methods for measuring plasmid copy numbers in single cells, focusing on challenges, trade-offs and necessary experimental controls. In particular we analyze published and unpublished strategies to infer copy numbers from expression of plasmid-encoded reporters, direct labeling of plasmids with fluorescent probes or DNA binding proteins fused to fluorescent reporters, PCR based methods applied to single cell lysates, and plasmid-specific replication arrest. We conclude that no method currently exists to measure plasmid copy numbers in single cells, and that most methods instead inadvertently measure various types of experimental noise. We also discuss how accurate methods can be developed. PMID:22305922

  9. Sparse representation and Bayesian detection of genome copy number alterations from microarray data

    PubMed Central

    Pique-Regi, Roger; Monso-Varona, Jordi; Ortega, Antonio; Seeger, Robert C.; Triche, Timothy J.; Asgharzadeh, Shahab

    2008-01-01

    Motivation: Genomic instability in cancer leads to abnormal genome copy number alterations (CNA) that are associated with the development and behavior of tumors. Advances in microarray technology have allowed for greater resolution in detection of DNA copy number changes (amplifications or deletions) across the genome. However, the increase in number of measured signals and accompanying noise from the array probes present a challenge in accurate and fast identification of breakpoints that define CNA. This article proposes a novel detection technique that exploits the use of piece wise constant (PWC) vectors to represent genome copy number and sparse Bayesian learning (SBL) to detect CNA breakpoints. Methods: First, a compact linear algebra representation for the genome copy number is developed from normalized probe intensities. Second, SBL is applied and optimized to infer locations where copy number changes occur. Third, a backward elimination (BE) procedure is used to rank the inferred breakpoints; and a cut-off point can be efficiently adjusted in this procedure to control for the false discovery rate (FDR). Results: The performance of our algorithm is evaluated using simulated and real genome datasets and compared to other existing techniques. Our approach achieves the highest accuracy and lowest FDR while improving computational speed by several orders of magnitude. The proposed algorithm has been developed into a free standing software application (GADA, Genome Alteration Detection Algorithm). Availability: http://biron.usc.edu/~piquereg/GADA Contact: jpei@chop.swmed.edu and rpique@ieee.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:18203770

  10. Kinetic versus Energetic Discrimination in Biological Copying

    NASA Astrophysics Data System (ADS)

    Sartori, Pablo; Pigolotti, Simone

    2013-05-01

    We study stochastic copying schemes in which discrimination between a right and a wrong match is achieved via different kinetic barriers or different binding energies of the two matches. We demonstrate that, in single-step reactions, the two discrimination mechanisms are strictly alternative and cannot be mixed to further reduce the error fraction. Close to the lowest error limit, kinetic discrimination results in a diverging copying velocity and dissipation per copied bit. On the other hand, energetic discrimination reaches its lowest error limit in an adiabatic regime where dissipation and velocity vanish. By analyzing experimentally measured kinetic rates of two DNA polymerases, T7 and Polγ, we argue that one of them operates in the kinetic and the other in the energetic regime. Finally, we show how the two mechanisms can be combined in copying schemes implementing error correction through a proofreading pathway.

  11. Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones.

    PubMed

    Wild, Jadwiga; Hradecna, Zdenka; Szybalski, Waclaw

    2002-09-01

    The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome

  12. Mechanisms of change in gene copy number.

    PubMed

    Hastings, P J; Lupski, James R; Rosenberg, Susan M; Ira, Grzegorz

    2009-08-01

    Deletions and duplications of chromosomal segments (copy number variants, CNVs) are a major source of variation between individual humans and are an underlying factor in human evolution and in many diseases, including mental illness, developmental disorders and cancer. CNVs form at a faster rate than other types of mutation, and seem to do so by similar mechanisms in bacteria, yeast and humans. Here we review current models of the mechanisms that cause copy number variation. Non-homologous end-joining mechanisms are well known, but recent models focus on perturbation of DNA replication and replication of non-contiguous DNA segments. For example, cellular stress might induce repair of broken replication forks to switch from high-fidelity homologous recombination to non-homologous repair, thus promoting copy number change.

  13. 32 CFR 1662.5 - Inspection, copying, and obtaining copies.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... SYSTEM FREEDOM OF INFORMATION ACT (FOIA) PROCEDURES § 1662.5 Inspection, copying, and obtaining copies... may make an appointment to inspect or copy the materials requested during regular business hours by...

  14. Long-term administration of valacyclovir reduces the number of Epstein-Barr virus (EBV)-infected B cells but not the number of EBV DNA copies per B cell in healthy volunteers.

    PubMed

    Hoshino, Yo; Katano, Harutaka; Zou, Ping; Hohman, Patricia; Marques, Adriana; Tyring, Stephen K; Follmann, Dean; Cohen, Jeffrey I

    2009-11-01

    Epstein-Barr virus (EBV) establishes a latent infection in B cells in the blood, and the latent EBV load in healthy individuals is generally stable over time, maintaining a "set point." It is unknown if the EBV load changes after long-term antiviral therapy in healthy individuals. We treated volunteers with either valacyclovir (valaciclovir) or no antiviral therapy for 1 year and measured the amount of EBV DNA in B cells every 3 months with a novel, highly sensitive assay. The number of EBV-infected B cells decreased in subjects receiving valacyclovir (half-life of 11 months; P = 0.02) but not in controls (half-life of 31 years; P = 0.86). The difference in the slopes of the lines for the number of EBV-infected B cells over time for the valacyclovir group versus the control group approached significance (P = 0.054). In contrast, the number of EBV DNA copies per B cell remained unchanged in both groups (P = 0.62 and P = 0.92 for the control and valacyclovir groups, respectively). Valacyclovir reduces the frequency of EBV-infected B cells when administered over a long period and, in theory, might allow eradication of EBV from the body if reinfection does not occur.

  15. Small single-copy region of plastid DNA in the non-photosynthetic angiosperm Epifagus virginiana contains only two genes. Differences among dicots, monocots and bryophytes in gene organization at a non-bioenergetic locus.

    PubMed

    Wolfe, K H; Morden, C W; Palmer, J D

    1992-01-05

    We have determined the nucleotide sequence of a 7 kb (1 kb = 10(3) base-pairs) region that includes the entire small single-copy region (SSC) of the plastid genome of Epifagus virginiana, a non-photosynthetic, parasitic flowering plant. The SSC (4.8 kb) is considerably smaller than those of photosynthetic plants due to the complete deletion of all photosynthetic, chlororespiratory and ribosomal protein genes. This leaves only two genes: a protein gene of 1738 codons whose product is unlikely to be involved in bioenergetic processes and a leucine tRNA gene (trn(LUAG)). Both genes span junctions between the inverted repeat and the SSC, with the consequence that the terminal 20 base-pairs of the repeat is transcribed in both directions and functions both as the 3' end of the tRNA gene and as an internal segment of orf1738. We find that the region of tobacco plastid DNA homologous to Epifagus orf1738 contains a single open reading frame (ORF) of 1901 codons rather than the three ORFs of 1244, 273 and 228 codons originally reported. However, we confirm that the equivalent region of the bryophyte Marchantia contains two genes (1068 and 464 codons) corresponding to the N and C-terminal portions of the dicot protein. In contrast, rice plastid DNA contains a severely truncated pseudogene at this locus.

  16. 37 CFR 1.217 - Publication of a redacted copy of an application.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... application that corresponds to the application for which a redacted copy is submitted; (2) A translation of... translation is accurate; (3) A marked-up copy of the application showing the redactions in brackets; and (4) A...

  17. Quadruplex MAPH: improvement of throughput in high-resolution copy number screening

    PubMed Central

    Tyson, Jess; Majerus, Tamsin MO; Walker, Susan; Armour, John AL

    2009-01-01

    Background Copy number variation (CNV) in the human genome is recognised as a widespread and important source of human genetic variation. Now the challenge is to screen for these CNVs at high resolution in a reliable, accurate and cost-effective way. Results Multiplex Amplifiable Probe Hybridisation (MAPH) is a sensitive, high-resolution technology appropriate for screening for CNVs in a defined region, for a targeted population. We have developed MAPH to a highly multiplexed format ("QuadMAPH") that allows the user a four-fold increase in the number of loci tested simultaneously. We have used this method to analyse a genomic region of 210 kb, including the MSH2 gene and 120 kb of flanking DNA. We show that the QuadMAPH probes report copy number with equivalent accuracy to simplex MAPH, reliably demonstrating diploid copy number in control samples and accurately detecting deletions in Hereditary Non-Polyposis Colorectal Cancer (HNPCC) samples. Conclusion QuadMAPH is an accurate, high-resolution method that allows targeted screening of large numbers of subjects without the expense of genome-wide approaches. Whilst we have applied this technique to a region of the human genome, it is equally applicable to the genomes of other organisms. PMID:19785739

  18. Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model.

    PubMed

    Snowdin, Jacob W; Hsiung, Chia-Heng; Kesterson, Daniel G; Kamath, Vasudeva G; McKee, Edward E

    2015-10-01

    The prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3'-azido-3'-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

  19. Spontaneous HBsAg loss in Korean patients: relevance of viral genotypes, S gene mutations, and covalently closed circular DNA copy numbers

    PubMed Central

    Chang, Hye-Young; Park, Jun Yong; Park, Eun-Sook; Park, Yong Kwang; Han, Kwang-Hyub

    2014-01-01

    Background/Aims Occult HBV infection can persist following HBsAg loss and be transmitted, but the virological features are not well defined. Methods Here we investigated 25 Korean patients who lost HBsAg during follow up, either spontaneously or subsequent to therapy. Results Whereas subtype adr (genotype C) was found in 96% of HBsAg positive patients, 75 % of patients who lost HBsAg spontaneously were seemed to be infected with the ayw subtype with sequence similar to genotype D. Mutations in the major hydrophilic region (MHR) of HBsAg were found in 7 patients who lost HBsAg spontaneously. The mutations include T123S, M125I/N, C139R, D144E, V177A, L192F, and W196L, some of which have not been reported before. Functional analysis via transfection experiments indicate that the C139R and D144E mutations drastically reduced HBsAg antigenicity, while the Y225del mutation found in one interferon-treated patient impaired HBsAg secretion. Conclusions Lack of detectable HBsAg in patient serum could be explained by low level of ccc DNA in liver tissue, low antigenicity of the surface protein, or its secretion defect. PMID:25320728

  20. Gene copy number and cell cycle arrest

    NASA Astrophysics Data System (ADS)

    Ghosh, Bhaswar; Bose, Indrani

    2006-03-01

    The cell cycle is an orderly sequence of events which ultimately lead to the division of a single cell into two daughter cells. In the case of DNA damage by radiation or chemicals, the damage checkpoints in the G1 and G2 phases of the cell cycle are activated. This results in an arrest of the cell cycle so that the DNA damage can be repaired. Once this is done, the cell continues with its usual cycle of activity. We study a mathematical model of the DNA damage checkpoint in the G2 phase which arrests the transition from the G2 to the M (mitotic) phase of the cell cycle. The tumor suppressor protein p53 plays a key role in activating the pathways leading to cell cycle arrest in mammalian systems. If the DNA damage is severe, the p53 proteins activate other pathways which bring about apoptosis, i.e., programmed cell death. Loss of the p53 gene results in the proliferation of cells containing damaged DNA, i.e., in the growth of tumors which may ultimately become cancerous. There is some recent experimental evidence which suggests that the mutation of a single copy of the p53 gene (in the normal cell each gene has two identical copies) is sufficient to trigger the formation of tumors. We study the effect of reducing the gene copy number of the p53 and two other genes on cell cycle arrest and obtain results consistent with experimental observations.

  1. DNA Dynamics.

    ERIC Educational Resources Information Center

    Warren, Michael D.

    1997-01-01

    Explains a method to enable students to understand DNA and protein synthesis using model-building and role-playing. Acquaints students with the triplet code and transcription. Includes copies of the charts used in this technique. (DDR)

  2. Identification of a differentiation-inducible promoter in the E7 open reading frame of human papillomavirus type 16 (HPV-16) in raft cultures of a new cell line containing high copy numbers of episomal HPV-16 DNA.

    PubMed

    Grassmann, K; Rapp, B; Maschek, H; Petry, K U; Iftner, T

    1996-04-01

    Gene expression of human papillomaviruses (HPV) is tightly linked to differentiation processes within the pluristratified epithelium. To analyze changes in the transcription pattern of HPV-16 during epithelial cell differentiation, we established a permanently growing HPV-16 positive cell line, designated KG, from a vulvar intraepithelial neoplasm. KG cells of early passages harbored multiple copies of the HPV-16 DNA as episomes and were able to form a stratified epithelium in an organotypic raft culture system. Analysis of viral gene expression revealed the known transcription pattern of the early region of HPV-16 with the exception of a so far undefined mRNA class with start sites in the E7 open reading frame. Quantitative analysis of primer extension experiments with RNA from KG cells grown in monolayer and raft culture showed a strong induction of this transcript in differentiated KG cells, whereas the level of the mRNAs initiated at the early promoter P97 remained almost constant. Primer extension analyses with four different primers and direct sequencing of the extension product revealed that the differentiation-inducible transcript initiated at a novel promoter with a major start site around nucleotide position 670 (P670) in the E7 open reading frame of HPV-16. Sequence analysis of cDNAs derived from RNA of KG cells grown in raft culture suggested that the transcripts initiated at P670 have a coding potential for an E1E4 fusion protein and for the E5 protein.

  3. Lake sediment multi-taxon DNA from North Greenland records early post-glacial appearance of vascular plants and accurately tracks environmental changes

    NASA Astrophysics Data System (ADS)

    Epp, L. S.; Gussarova, G.; Boessenkool, S.; Olsen, J.; Haile, J.; Schrøder-Nielsen, A.; Ludikova, A.; Hassel, K.; Stenøien, H. K.; Funder, S.; Willerslev, E.; Kjær, K.; Brochmann, C.

    2015-06-01

    High Arctic environments are particularly sensitive to climate changes, but retrieval of paleoecological data is challenging due to low productivity and biomass. At the same time, Arctic soils and sediments have proven exceptional for long-term DNA preservation due to their constantly low temperatures. Lake sediments contain DNA paleorecords of the surrounding ecosystems and can be used to retrieve a variety of organismal groups from a single sample. In this study, we analyzed vascular plant, bryophyte, algal (in particular diatom) and copepod DNA retrieved from a sediment core spanning the Holocene, taken from Bliss Lake on the northernmost coast of Greenland. A previous multi-proxy study including microscopic diatom analyses showed that this lake experienced changes between marine and lacustrine conditions. We inferred the same environmental changes from algal DNA preserved in the sediment core. Our DNA record was stratigraphically coherent, with no indication of leaching between layers, and our cross-taxon comparisons were in accordance with previously inferred local ecosystem changes. Authentic ancient plant DNA was retrieved from nearly all layers, both from the marine and the limnic phases, and distinct temporal changes in plant presence were recovered. The plant DNA was mostly in agreement with expected vegetation history, but very early occurrences of vascular plants, including the woody Empetrum nigrum, document terrestrial vegetation very shortly after glacial retreat. Our study shows that multi-taxon metabarcoding of sedimentary ancient DNA from lake cores is a valuable tool both for terrestrial and aquatic paleoecology, even in low-productivity ecosystems such as the High Arctic.

  4. Influence of storage time on DNA of Chlamydia trachomatis, Ureaplasma urealyticum, and Neisseria gonorrhoeae for accurate detection by quantitative real-time polymerase chain reaction.

    PubMed

    Lu, Y; Rong, C Z; Zhao, J Y; Lao, X J; Xie, L; Li, S; Qin, X

    2016-08-25

    The shipment and storage conditions of clinical samples pose a major challenge to the detection accuracy of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU) when using quantitative real-time polymerase chain reaction (qRT-PCR). The aim of the present study was to explore the influence of storage time at 4°C on the DNA of these pathogens and its effect on their detection by qRT-PCR. CT, NG, and UU positive genital swabs from 70 patients were collected, and DNA of all samples were extracted and divided into eight aliquots. One aliquot was immediately analyzed with qRT-PCR to assess the initial pathogen load, whereas the remaining samples were stored at 4°C and analyzed after 1, 2, 3, 7, 14, 21, and 28 days. No significant differences in CT, NG, and UU DNA loads were observed between baseline (day 0) and the subsequent time points (days 1, 2, 3, 7, 14, 21, and 28) in any of the 70 samples. Although a slight increase in DNA levels was observed at day 28 compared to day 0, paired sample t-test results revealed no significant differences between the mean DNA levels at different time points following storage at 4°C (all P>0.05). Overall, the CT, UU, and NG DNA loads from all genital swab samples were stable at 4°C over a 28-day period.

  5. The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment

    PubMed Central

    Harris, Leanne; McFarlane-Majeed, Laura; Campos-León, Karen; Roberts, Sally

    2016-01-01

    ABSTRACT In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2Y131A) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2Y131A showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2WT), the chromatin-bound pool of E2Y131A was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2Y131A occurred in mid-S phase. A similar alteration between the subcellular pools of the E2WT protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2Y131A genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. IMPORTANCE Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence

  6. The Cellular DNA Helicase ChlR1 Regulates Chromatin and Nuclear Matrix Attachment of the Human Papillomavirus 16 E2 Protein and High-Copy-Number Viral Genome Establishment.

    PubMed

    Harris, Leanne; McFarlane-Majeed, Laura; Campos-León, Karen; Roberts, Sally; Parish, Joanna L

    2017-01-01

    In papillomavirus infections, the viral genome is established as a double-stranded DNA episome. To segregate the episomes into daughter cells during mitosis, they are tethered to cellular chromatin by the viral E2 protein. We previously demonstrated that the E2 proteins of diverse papillomavirus types, including bovine papillomavirus (BPV) and human papillomavirus 16 (HPV16), associate with the cellular DNA helicase ChlR1. This virus-host interaction is important for the tethering of BPV E2 to mitotic chromatin and the stable maintenance of BPV episomes. The role of the association between E2 and ChlR1 in the HPV16 life cycle is unresolved. Here we show that an HPV16 E2 Y131A mutant (E2(Y131A)) had significantly reduced binding to ChlR1 but retained transcriptional activation and viral origin-dependent replication functions. Subcellular fractionation of keratinocytes expressing E2(Y131A) showed a marked change in the localization of the protein. Compared to that of wild-type E2 (E2(WT)), the chromatin-bound pool of E2(Y131A) was decreased, concomitant with an increase in nuclear matrix-associated protein. Cell cycle synchronization indicated that the shift in subcellular localization of E2(Y131A) occurred in mid-S phase. A similar alteration between the subcellular pools of the E2(WT) protein occurred upon ChlR1 silencing. Notably, in an HPV16 life cycle model in primary human keratinocytes, mutant E2(Y131A) genomes were established as episomes, but at a markedly lower copy number than that of wild-type HPV16 genomes, and they were not maintained upon cell passage. Our studies indicate that ChlR1 is an important regulator of the chromatin association of E2 and of the establishment and maintenance of HPV16 episomes. Infections with high-risk human papillomaviruses (HPVs) are a major cause of anogenital and oropharyngeal cancers. During infection, the circular DNA genome of HPV persists within the nucleus, independently of the host cell chromatin. Persistence of

  7. Copy Machine Art.

    ERIC Educational Resources Information Center

    Sommer, Jean

    1984-01-01

    Images created with copy machines make children feel successful, as their work acquires the authority of being printed. Students can learn advanced processes like electrostatic image-making and can get involved in projects like making collages. They acquire an appreciation of design and of two-dimensional composition. (CS)

  8. Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3' copy-back ambisense rabies virus.

    PubMed

    Finke, S; Conzelmann, K K

    1999-05-01

    Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.

  9. Utility of DNA barcoding for rapid and accurate assessment of bat diversity in Malaysia in the absence of formally described species.

    PubMed

    Wilson, J-J; Sing, K-W; Halim, M R A; Ramli, R; Hashim, R; Sofian-Azirun, M

    2014-02-19

    Bats are important flagship species for biodiversity research; however, diversity in Southeast Asia is considerably underestimated in the current checklists and field guides. Incorporation of DNA barcoding into surveys has revealed numerous species-level taxa overlooked by conventional methods. Inclusion of these taxa in inventories provides a more informative record of diversity, but is problematic as these species lack formal description. We investigated how frequently documented, but undescribed, bat taxa are encountered in Peninsular Malaysia. We discuss whether a barcode library provides a means of recognizing and recording these taxa across biodiversity inventories. Tissue was sampled from bats trapped at Pasir Raja, Dungun Terengganu, Peninsular Malaysia. The DNA was extracted and the COI barcode region amplified and sequenced. We identified 9 species-level taxa within our samples, based on analysis of the DNA barcodes. Six specimens matched to four previously documented taxa considered candidate species but currently lacking formal taxonomic status. This study confirms the high diversity of bats within Peninsular Malaysia (9 species in 13 samples) and demonstrates how DNA barcoding allows for inventory and documentation of known taxa lacking formal taxonomic status.

  10. The use of Taqman genotyping assays for rapid confirmation of β-thalassaemia mutations in the Malays: accurate diagnosis with low DNA concentrations.

    PubMed

    Teh, L-K; Lee, T-Y; Tan, J A M A; Lai, M-I; George, E

    2015-02-01

    In Malaysia, β-thalassaemia is a common inherited blood disorder in haemoglobin synthesis with a carrier rate of 4.5%. Currently, PCR-incorporating techniques such as amplification refractory mutation system (ARMS) or reverse dot blot hybridization (RDBH) are used in β-thalassaemia mutation detection. ARMS allows single-mutation identification using two reactions, one for wild type and another for mutant alleles. RDBH requires probe immobilization and optimization of hybridization and washing temperatures which is time consuming. The aim of our study was to investigate whether β-thalassaemia mutations can be identified in samples with low DNA concentrations. Genotype identification of common β-thalassaemia mutations in Malays was carried out using Taqman genotyping assays. Results show that the Taqman assays allow mutation detection with DNA template concentrations as low as 2-100 ng. In addition, consistent reproducibility was observed in the Taqman assays when repeated eight times and at different time intervals. The developed sensitive Taqman assays allow molecular characterization of β-thalassaemia mutations in samples with low DNA concentrations. The Taqman genotyping assays have potential as a diagnostic tool for foetal blood, chorionic villi or pre-implantation genetic diagnosis where DNA is limited and precious. © 2014 John Wiley & Sons Ltd.

  11. Eclipse period of R1 plasmids during downshift from elevated copy number: Nonrandom selection of copies for replication.

    PubMed

    Olsson, Jan A; Berg, Otto; Nordström, Kurt; Dasgupta, Santanu

    2012-03-01

    The classical Meselson-Stahl density-shift method was used to study replication of pOU71, a runaway-replication derivative of plasmid R1 in Escherichia coli. The miniplasmid maintained the normal low copy number of R1 during steady growth at 30°C, but as growth temperatures were raised above 34°C, the copy number of the plasmid increased to higher levels, and at 42°C, it replicated without control in a runaway replication mode with lethal consequences for the host. The eclipse periods (minimum time between successive replication of the same DNA) of the plasmid shortened with rising copy numbers at increasing growth temperatures (Olsson et al., 2003). In this work, eclipse periods were measured during downshifts in copy number of pOU71 after it had replicated at 39 and 42°C, resulting in 7- and 50-fold higher than normal plasmid copy number per cell, respectively. Eclipse periods for plasmid replication, measured during copy number downshift, suggested that plasmid R1, normally selected randomly for replication, showed a bias such that a newly replicated DNA had a higher probability of replication compared to the bulk of the R1 population. However, even the unexpected nonrandom replication followed the copy number kinetics such that every generation, the plasmids underwent the normal inherited number of replication, n, independent of the actual number of plasmid copies in a newborn cell.

  12. Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

    PubMed Central

    Zill, Oliver A.; Sebisanovic, Dragan; Lopez, Rene; Blau, Sibel; Collisson, Eric A.; Divers, Stephen G.; Hoon, Dave S. B.; Kopetz, E. Scott; Lee, Jeeyun; Nikolinakos, Petros G.; Baca, Arthur M.; Kermani, Bahram G.; Eltoukhy, Helmy; Talasaz, AmirAli

    2015-01-01

    Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing. PMID:26474073

  13. Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA.

    PubMed

    Lanman, Richard B; Mortimer, Stefanie A; Zill, Oliver A; Sebisanovic, Dragan; Lopez, Rene; Blau, Sibel; Collisson, Eric A; Divers, Stephen G; Hoon, Dave S B; Kopetz, E Scott; Lee, Jeeyun; Nikolinakos, Petros G; Baca, Arthur M; Kermani, Bahram G; Eltoukhy, Helmy; Talasaz, AmirAli

    2015-01-01

    Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

  14. Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

    PubMed

    Reid-Bayliss, Kate S; Loeb, Lawrence A

    2017-08-29

    Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

  15. CalMaTe: a method and software to improve allele-specific copy number of SNP arrays for downstream segmentation

    PubMed Central

    Ortiz-Estevez, Maria; Aramburu, Ander; Bengtsson, Henrik; Neuvial, Pierre; Rubio, Angel

    2012-01-01

    Summary: CalMaTe calibrates preprocessed allele-specific copy number estimates (ASCNs) from DNA microarrays by controlling for single-nucleotide polymorphism-specific allelic crosstalk. The resulting ASCNs are on average more accurate, which increases the power of segmentation methods for detecting changes between copy number states in tumor studies including copy neutral loss of heterozygosity. CalMaTe applies to any ASCNs regardless of preprocessing method and microarray technology, e.g. Affymetrix and Illumina. Availability: The method is available on CRAN (http://cran.r-project.org/) in the open-source R package calmate, which also includes an add-on to the Aroma Project framework (http://www.aroma-project.org/). Contact: arubio@ceit.es Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22576175

  16. Evolution vs the number of gene copies per primitive cell.

    PubMed

    Koch, A L

    1984-01-01

    Computer simulations are presented of the rate at which an advantageous mutant would displace the prototype in a replicating system without an accurate segregation mechanism. If the number of gene copies in the system is indefinitely large, Darwinian evolution is essentially stopped because there is no coupling of phenotype with genotype, i.e., there is no growth advantage to the advantageous gene relative to the prototype and therefore no "survival of the fittest." The inhibition of evolution due to a number of gene copies less than 100 would have been not insurmountable. Although the presence of multiple copies would have allowed replacement by an advantageous mutant, it provided a way for the primitive cell to conserve less immediately useful genes that could evolve into different or more effective genes. This possibility was lost as accurate segregation mechanisms evolved and cells with few copies of each gene, such as modern procaryotes, arose.

  17. Incorporating 16S gene copy number information improves estimates of microbial diversity and abundance.

    PubMed

    Kembel, Steven W; Wu, Martin; Eisen, Jonathan A; Green, Jessica L

    2012-01-01

    The abundance of different SSU rRNA ("16S") gene sequences in environmental samples is widely used in studies of microbial ecology as a measure of microbial community structure and diversity. However, the genomic copy number of the 16S gene varies greatly - from one in many species to up to 15 in some bacteria and to hundreds in some microbial eukaryotes. As a result of this variation the relative abundance of 16S genes in environmental samples can be attributed both to variation in the relative abundance of different organisms, and to variation in genomic 16S copy number among those organisms. Despite this fact, many studies assume that the abundance of 16S gene sequences is a surrogate measure of the relative abundance of the organisms containing those sequences. Here we present a method that uses data on sequences and genomic copy number of 16S genes along with phylogenetic placement and ancestral state estimation to estimate organismal abundances from environmental DNA sequence data. We use theory and simulations to demonstrate that 16S genomic copy number can be accurately estimated from the short reads typically obtained from high-throughput environmental sequencing of the 16S gene, and that organismal abundances in microbial communities are more strongly correlated with estimated abundances obtained from our method than with gene abundances. We re-analyze several published empirical data sets and demonstrate that the use of gene abundance versus estimated organismal abundance can lead to different inferences about community diversity and structure and the identity of the dominant taxa in microbial communities. Our approach will allow microbial ecologists to make more accurate inferences about microbial diversity and abundance based on 16S sequence data.

  18. Incorporating 16S Gene Copy Number Information Improves Estimates of Microbial Diversity and Abundance

    PubMed Central

    Kembel, Steven W.; Wu, Martin; Eisen, Jonathan A.; Green, Jessica L.

    2012-01-01

    The abundance of different SSU rRNA (“16S”) gene sequences in environmental samples is widely used in studies of microbial ecology as a measure of microbial community structure and diversity. However, the genomic copy number of the 16S gene varies greatly – from one in many species to up to 15 in some bacteria and to hundreds in some microbial eukaryotes. As a result of this variation the relative abundance of 16S genes in environmental samples can be attributed both to variation in the relative abundance of different organisms, and to variation in genomic 16S copy number among those organisms. Despite this fact, many studies assume that the abundance of 16S gene sequences is a surrogate measure of the relative abundance of the organisms containing those sequences. Here we present a method that uses data on sequences and genomic copy number of 16S genes along with phylogenetic placement and ancestral state estimation to estimate organismal abundances from environmental DNA sequence data. We use theory and simulations to demonstrate that 16S genomic copy number can be accurately estimated from the short reads typically obtained from high-throughput environmental sequencing of the 16S gene, and that organismal abundances in microbial communities are more strongly correlated with estimated abundances obtained from our method than with gene abundances. We re-analyze several published empirical data sets and demonstrate that the use of gene abundance versus estimated organismal abundance can lead to different inferences about community diversity and structure and the identity of the dominant taxa in microbial communities. Our approach will allow microbial ecologists to make more accurate inferences about microbial diversity and abundance based on 16S sequence data. PMID:23133348

  19. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  20. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  1. Capillary HPLC-Accurate Mass MS/MS Quantitation of N7-(2, 3, 4-trihydroxybut-1-yl)-guanine Adducts of 1,3-Butadiene in Human Leukocyte DNA

    PubMed Central

    Sangaraju, Dewakar; Villalta, Peter; Goggin, Melissa; Agunsoye, Maria O.; Campbell, Colin; Tretyakova, Natalia

    2013-01-01

    1,3-butadiene (BD) is a high volume industrial chemical commonly used in polymer and rubber production. It is also present in cigarette smoke, automobile exhaust, and urban air, leading to widespread exposure of human populations. Upon entering the body, BD is metabolized to electrophilic epoxides, 3,4-epoxy-1-butene (EB), diepoxybutane (DEB), and 3,4-epoxy-1,2-diol (EBD), which can alkylate DNA nucleobases. The most abundant BD epoxide, EBD, modifies the N7-guanine positions in DNA to form N7-(2, 3, 4-trihydroxybut-1-yl) guanine (N7-THBG) adducts, which can be useful as biomarkers of BD exposure and metabolic activation to DNA-reactive epoxides. In the present work, a capillary HPLC- high resolution ESI+-MS/MS (HPLC-ESI+-HRMS/MS) methodology was developed for accurate, sensitive, and reproducible quantification of N7-THBG in cell culture and in human white blood cells. In our approach, DNA is subjected to neutral thermal hydrolysis to release N7-guanine adducts from the DNA backbone, followed by ultrafiltration, solid phase extraction, and isotope dilution HPLC-ESI+-HRMS/MS analysis on an Orbitrap Velos mass spectrometer. Following method validation, N7-THBG was quantified in human fibrosarcoma (HT1080) cells treated with μmolar concentrations of DEB and in DNA isolated from blood of smokers, nonsmokers, individuals participating in a smoking cessation program, and occupationally exposed workers. N7-THBG concentrations increased linearly from 31.4μ± 4.84 to 966.55 ± 128.05 adducts per 109 nucleotides in HT1080 cells treated with 1 -100 μM DEB. N7-THBG amounts in leukocyte DNA of non-smokers, smokers, and occupationally exposed workers were 7.08 ± 5.29, 8.20 ± 5.12, and 9.72 ± 3.80 adducts per 109 nucleotides, respectively, suggesting the presence of an endogenous or environmental source for this adduct. The availability of sensitive HPLC-ESI+-HRMS/MS methodology for BD-induced DNA adducts in humans will enable future population studies of inter

  2. Structure and dynamics of near-threshold leptons driven by dipolar interactions: an accurate computational study for the DNA purinic bases

    NASA Astrophysics Data System (ADS)

    Carelli, Fabio; Gianturco, Francesco Antonio

    2016-06-01

    The interaction of low-energy scattering electrons/positrons with molecular targets characterized by a "supercritical" permanent dipole moment (≳2.0 D) presents special physical characteristics that affect their spatial distributions, around the nuclear network of the molecular partners, both above and below the energy thresholds. Such special states are described as either dipole scattering states (DSS) above thresholds or as dipole bound states (DBS) below thresholds. The details of their respective behaviour will be presented and discussed in this work in the case of the purinic DNA bases of adenine and guanine. The behavior of the additional electron, in particular, will be discussed in detail by providing new computational results that will be related to the findings from recent experiments on the same DNA bases, confirming the transient electron's behaviour surmised by them. This work is affectionately dedicated to Michael Allan on the occasion of his official retirement. We wish to this dear friend and outstanding scientist many years to come in the happy pursuit of his many scientific interests.Contribution to the Topical Issue "Advances in Positron and Electron Scattering", edited by Paulo Limao-Vieira, Gustavo Garcia, E. Krishnakumar, James Sullivan, Hajime Tanuma and Zoran Petrovic.

  3. CNARA: reliability assessment for genomic copy number profiles.

    PubMed

    Ai, Ni; Cai, Haoyang; Solovan, Caius; Baudis, Michael

    2016-10-12

    DNA copy number profiles from microarray and sequencing experiments sometimes contain wave artefacts which may be introduced during sample preparation and cannot be removed completely by existing preprocessing methods. Besides, large derivative log ratio spread (DLRS) of the probes correlating with poor DNA quality is sometimes observed in genome screening experiments and may lead to unreliable copy number profiles. Depending on the extent of these artefacts and the resulting misidentification of copy number alterations/variations (CNA/CNV), it may be desirable to exclude such samples from analyses or to adapt the downstream data analysis strategy accordingly. Here, we propose a method to distinguish reliable genomic copy number profiles from those containing heavy wave artefacts and/or large DLRS. We define four features that adequately summarize the copy number profiles for reliability assessment, and train a classifier on a dataset of 1522 copy number profiles from various microarray platforms. The method can be applied to predict the reliability of copy number profiles irrespective of the underlying microarray platform and may be adapted for those sequencing platforms from which copy number estimates could be computed as a piecewise constant signal. Further details can be found at https://github.com/baudisgroup/CNARA . We have developed a method for the assessment of genomic copy number profiling data, and suggest to apply the method in addition to and after other state-of-the-art noise correction and quality control procedures. CNARA could be instrumental in improving the assessment of data used for genomic data mining experiments and support the reliable functional attribution of copy number aberrations especially in cancer research.

  4. Barcode DNA high-resolution melting (Bar-HRM) analysis as a novel close-tubed and accurate tool for olive oil forensic use.

    PubMed

    Ganopoulos, Ioannis; Bazakos, Christos; Madesis, Panagiotis; Kalaitzis, Panagiotis; Tsaftaris, Athanasios

    2013-07-01

    The adulteration of high-priced olive oil with low-cost oils and the fraudulent labelling of oil products make the identification and traceability of vegetable oil species in the food chain very important. This paper describes a high-resolution melting analysis-based method using chloroplast barcoding regions as target (Bar-HRM) to obtain barcoding information for the major vegetable oil species and to quantitatively identify the botanical origin of plant oils. The detection of adulteration of olive oil with canola oil was used as a case study. The proposed method was capable of distinguishing among different vegetable oil species and detecting a level of 1% (w/w) of canola oil in olive oil. Bar-HRM analysis is a more accurate, faster and less costly alternative method to authenticate vegetable oils, including olive oil, and to detect mixtures of oils. © 2013 Society of Chemical Industry.

  5. Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing.

    PubMed

    Dilernia, Dario A; Chien, Jung-Ting; Monaco, Daniela C; Brown, Michael P S; Ende, Zachary; Deymier, Martin J; Yue, Ling; Paxinos, Ellen E; Allen, Susan; Tirado-Ramos, Alfredo; Hunter, Eric

    2015-11-16

    Single Molecule, Real-Time (SMRT) Sequencing (Pacific Biosciences, Menlo Park, CA, USA) provides the longest continuous DNA sequencing reads currently available. However, the relatively high error rate in the raw read data requires novel analysis methods to deconvolute sequences derived from complex samples. Here, we present a workflow of novel computer algorithms able to reconstruct viral variant genomes present in mixtures with an accuracy of >QV50. This approach relies exclusively on Continuous Long Reads (CLR), which are the raw reads generated during SMRT Sequencing. We successfully implement this workflow for simultaneous sequencing of mixtures containing up to forty different >9 kb HIV-1 full genomes. This was achieved using a single SMRT Cell for each mixture and desktop computing power. This novel approach opens the possibility of solving complex sequencing tasks that currently lack a solution.

  6. Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing

    PubMed Central

    Dilernia, Dario A.; Chien, Jung-Ting; Monaco, Daniela C.; Brown, Michael P.S.; Ende, Zachary; Deymier, Martin J.; Yue, Ling; Paxinos, Ellen E.; Allen, Susan; Tirado-Ramos, Alfredo; Hunter, Eric

    2015-01-01

    Single Molecule, Real-Time (SMRT®) Sequencing (Pacific Biosciences, Menlo Park, CA, USA) provides the longest continuous DNA sequencing reads currently available. However, the relatively high error rate in the raw read data requires novel analysis methods to deconvolute sequences derived from complex samples. Here, we present a workflow of novel computer algorithms able to reconstruct viral variant genomes present in mixtures with an accuracy of >QV50. This approach relies exclusively on Continuous Long Reads (CLR), which are the raw reads generated during SMRT Sequencing. We successfully implement this workflow for simultaneous sequencing of mixtures containing up to forty different >9 kb HIV-1 full genomes. This was achieved using a single SMRT Cell for each mixture and desktop computing power. This novel approach opens the possibility of solving complex sequencing tasks that currently lack a solution. PMID:26101252

  7. DNA studies are necessary for accurate patient diagnosis in compound heterozygosity for Hb Adana (HBA2:c.179>A) with deletional or nondeletional α-thalassaemia

    PubMed Central

    Tan, Jin Ai Mary Anne; Kho, Siew Leng; Ngim, Chin Fang; Chua, Kek Heng; Goh, Ai Sim; Yeoh, Seoh Leng; George, Elizabeth

    2016-01-01

    Haemoglobin (Hb) Adana (HBA2:c.179>A) interacts with deletional and nondeletional α-thalassaemia mutations to produce HbH disorders with varying clinical manifestations from asymptomatic to severe anaemia with significant hepatosplenomegaly. Hb Adana carriers are generally asymptomatic and haemoglobin subtyping is unable to detect this highly unstable α-haemoglobin variant. This study identified 13 patients with compound heterozygosity for Hb Adana with either the 3.7 kb gene deletion (-α3.7), Hb Constant Spring (HbCS) (HBA2:c.427T>C) or Hb Paksé (HBA2:429A>T). Multiplex Amplification Refractory Mutation System was used for the detection of five deletional and six nondeletional α-thalassaemia mutations. Duplex-PCR was used to confirm Hb Paksé and HbCS. Results showed 84.6% of the Hb Adana patients were Malays. Using DNA studies, compound heterozygosity for Hb Adana and HbCS (αcodon 59α/αCSα) was confirmed in 11 patients. A novel point in this investigation was that DNA studies confirmed Hb Paksé for the first time in a Malaysian patient (αcodon 59α/αPakséα) after nine years of being misdiagnosis with Hb Adana and HbCS (αcodon 59α/αCSα). Thus, the reliance on haematology studies and Hb subtyping to detect Hb variants is inadequate in countries where thalassaemia is prevalent and caused by a wide spectrum of mutations. PMID:27271331

  8. 11. Photographic copy of copy of Twin Lakes Outlet Works ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. Photographic copy of copy of Twin Lakes Outlet Works construction drawing dated January 15, 1951. Drawn by W.A. Doe for the Twin Lakes Reservoir and Canal Co. (copy in possession of Bureau of Reclamation, location of original unknown) 'AS CONSTRUCTED' PLANS OF 1949-1950, REHABILITATION OF TWIN LAKES RESERVOIR OUTLET WORKS, DETAILS OF UPSTREAM WING WALLS. - Twin Lakes Dam & Outlet Works, Beneath Twin Lakes Reservoir, T11S, R80W, S22, Twin Lakes, Lake County, CO

  9. 12. Photographic copy of copy of Twin Lakes Outlet Works ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. Photographic copy of copy of Twin Lakes Outlet Works construction drawing dated January 15, 1951. Drawn by W.A. Doe for the Twin Lakes Reservoir and Canal Co. (copy in possession of Bureau of Reclamation, location of original unknown) 'AS CONSTRUCTED' PLANS OF 1949-50, REHABILITATION OF TWIN LAKES RESERVOIR OUTLET WORKS, DETAILS OF DISCHARGE BASIN. - Twin Lakes Dam & Outlet Works, Beneath Twin Lakes Reservoir, T11S, R80W, S22, Twin Lakes, Lake County, CO

  10. Accurate, Direct, and High-Throughput Analyses of a Broad Spectrum of Endogenously Generated DNA Base Modifications with Isotope-Dilution Two-Dimensional Ultraperformance Liquid Chromatography with Tandem Mass Spectrometry: Possible Clinical Implication.

    PubMed

    Gackowski, Daniel; Starczak, Marta; Zarakowska, Ewelina; Modrzejewska, Martyna; Szpila, Anna; Banaszkiewicz, Zbigniew; Olinski, Ryszard

    2016-12-20

    Our hereby presented methodology is suitable for reliable assessment of the most common unavoidable DNA modifications which arise as a product of fundamental metabolic processes. 8-Oxoguanine, one of the oxidatively modified DNA bases, is a typical biomarker of oxidative stress. A noncanonical base, uracil, may be also present in small quantities in DNA. A set of ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can be also generated by TET enzymes. All of the aforementioned modifications seem to play some regulatory roles. We applied isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of the 5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, 2'-deoxyuridine, and 8-oxo-2'-deoxyguanosine. Analyses of DNA extracted from matched human samples showed that the 5-(hydroxymethyl)-2'-deoxycytidine level was 5-fold lower in colorectal carcinoma tumor in comparison with the normal one from the tumor's margin; also 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine were lower in colorectal carcinoma tissue (ca. 2.5- and 3.5-fold, respectively). No such differences was found for 2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxyuridine. The presented methodology is suitable for fast, accurate, and complex evaluation of an array of endogenously generated DNA deoxynucleosides modifications. This novel technique could be used for monitoring of cancer and other diseases related to oxidative stress, aberrant metabolism, and environmental exposure. Furthermore, the fully automated two-dimensional separation is extremely useful for analysis of material

  11. Accuracy and differential bias in copy number measurement of CCL3L1 in association studies with three auto-immune disorders

    PubMed Central

    2011-01-01

    Background Copy number variation (CNV) contributes to the variation observed between individuals and can influence human disease progression, but the accurate measurement of individual copy numbers is technically challenging. In the work presented here we describe a modification to a previously described paralogue ratio test (PRT) method for genotyping the CCL3L1/CCL4L1 copy variable region, which we use to ascertain CCL3L1/CCL4L1 copy number in 1581 European samples. As the products of CCL3L1 and CCL4L1 potentially play a role in autoimmunity we performed case control association studies with Crohn's disease, rheumatoid arthritis and psoriasis clinical cohorts. Results We evaluate the PRT methodology used, paying particular attention to accuracy and precision, and highlight the problems of differential bias in copy number measurements. Our PRT methods for measuring copy number were of sufficient precision to detect very slight but systematic differential bias between results from case and control DNA samples in one study. We find no evidence for an association between CCL3L1 copy number and Crohn's disease, rheumatoid arthritis or psoriasis. Conclusions Differential bias of this small magnitude, but applied systematically across large numbers of samples, would create a serious risk of false positive associations in copy number, if measured using methods of lower precision, or methods relying on single uncorroborated measurements. In this study the small differential bias detected by PRT in one sample set was resolved by a simple pre-treatment by restriction enzyme digestion. PMID:21851606

  12. High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq.

    PubMed

    Kondrashova, Olga; Love, Clare J; Lunke, Sebastian; Hsu, Arthur L; Waring, Paul M; Taylor, Graham R

    2015-01-01

    Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity.

  13. High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq

    PubMed Central

    Kondrashova, Olga; Love, Clare J.; Lunke, Sebastian; Hsu, Arthur L.; Waring, Paul M.; Taylor, Graham R.

    2015-01-01

    Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity. PMID:26569395

  14. An epigenetic biomarker combination of PCDH17 and POU4F2 detects bladder cancer accurately by methylation analyses of urine sediment DNA in Han Chinese

    PubMed Central

    Li, Qiaoling; An, Dan; Fang, Lu; Lin, Youcheng; Hou, Yong; Xu, Abai; Fu, Yu; Lu, Wei; Chen, Xin; Chen, Mingwei; Zhang, Meng; Jiang, Huiling; Zhang, Chuanxia; Dong, Pei; Li, Chong; Chen, Jun; Yang, Guosheng; Liu, Chunxiao; Cai, Zhiming; Zhou, Fangjian; Wu, Song

    2016-01-01

    To develop a routine and effectual procedure of detecting bladder cancer (BlCa), an optimized combination of epigenetic biomarkers that work synergistically with high sensitivity and specificity is necessary. In this study, methylation levels of seven biomarkers (EOMES, GDF15, NID2, PCDH17, POU4F2, TCF21, and ZNF154) in 148 individuals—which including 58 urothelial cell carcinoma (UCC) patients, 20 infected urinary calculi (IUC) patients, 20 kidney cancer (KC) patients,20 prostate cancer (PC) patients, and 30 healthy volunteers (HV)—were quantified by qMSP using the urine sediment DNA. Receiver operating characteristic (ROC) curves were generated for each biomarker. The combining predictors of possible combinations were calculated through logistic regression model. Subsequently, ROC curves of the three best performing combinations were constructed. Then, we validated the three best performing combinations and POU4F2 in another 72 UCC, 21 IUC, 26 KC and 22 PC, and 23 HV urine samples. The combination of POU4F2/PCDH17 has yielded the highest sensitivity and specificity of 90.00% and 93.96% in all the 312 individuals, showing the capability of detecting BlCa effectively among pathologically varied sample groups. PMID:26700620

  15. Digital Genotyping of Macrosatellites and Multicopy Genes Reveals Novel Biological Functions Associated with Copy Number Variation of Large Tandem Repeats

    PubMed Central

    Quilez, Javier; Hasson, Dan; Borel, Christelle; Warburton, Peter; Sharp, Andrew J.

    2014-01-01

    Tandem repeats are common in eukaryotic genomes, but due to difficulties in assaying them remain poorly studied. Here, we demonstrate the utility of Nanostring technology as a targeted approach to perform accurate measurement of tandem repeats even at extremely high copy number, and apply this technology to genotype 165 HapMap samples from three different populations and five species of non-human primates. We observed extreme variability in copy number of tandemly repeated genes, with many loci showing 5–10 fold variation in copy number among humans. Many of these loci show hallmarks of genome assembly errors, and the true copy number of many large tandem repeats is significantly under-represented even in the high quality ‘finished’ human reference assembly. Importantly, we demonstrate that most large tandem repeat variations are not tagged by nearby SNPs, and are therefore essentially invisible to SNP-based GWAS approaches. Using association analysis we identify many cis correlations of large tandem repeat variants with nearby gene expression and DNA methylation levels, indicating that variations of tandem repeat length are associated with functional effects on the local genomic environment. This includes an example where expansion of a macrosatellite repeat is associated with increased DNA methylation and suppression of nearby gene expression, suggesting a mechanism termed “repeat induced gene silencing”, which has previously been observed only in transgenic organisms. We also observed multiple signatures consistent with altered selective pressures at tandemly repeated loci, suggesting important biological functions. Our studies show that tandemly repeated loci represent a highly variable fraction of the genome that have been systematically ignored by most previous studies, copy number variation of which can exert functionally significant effects. We suggest that future studies of tandem repeat loci will lead to many novel insights into their role in

  16. Effect of sustained elevated temperature prior to amplification on template copy number estimation using digital polymerase chain reaction.

    PubMed

    Bhat, Somanath; McLaughlin, Jacob L H; Emslie, Kerry R

    2011-02-21

    Digital polymerase chain reaction (dPCR) has the potential to enable accurate quantification of target DNA copy number provided that all target DNA molecules are successfully amplified. Following duplex dPCR analysis from a linear DNA target sequence that contains single copies of two independent template sequences, we have observed that amplification of both templates in a single partition does not always occur. To investigate this finding, we heated the target DNA solution to 95 °C for increasing time intervals and then immediately chilled on ice prior to preparing the dPCR mix. We observed an exponential decline in estimated copy number (R(2)≥ 0.98) of the two template sequences when amplified from either a linearized plasmid or a 388 base pair (bp) amplicon containing the same two template sequences. The distribution of amplifiable templates and the final concentration (copies per µL) were both affected by heat treatment of the samples at 95 °C from 0 s to 30 min. The proportion of target sequences from which only one of the two templates was amplified in a single partition (either 1507 or hmg only) increased over time, while the proportion of target sequences where both templates were amplified (1507 and hmg) in each individual partition declined rapidly from 94% to 52% (plasmid) and 88% to 31% (388 bp amplicon) suggesting an increase in number of targets from which both templates no longer amplify. A 10 min incubation at 95 °C reduced the initial amplifiable template concentration of the plasmid and the 388 bp amplicon by 59% and 91%, respectively. To determine if a similar decrease in amplifiable target occurs during the default pre-activation step of typical PCR amplification protocol, we used mastermixes with a 20 s or 10 min hot-start. The choice of mastermix and consequent pre-activation time did not affect the estimated plasmid concentration. Therefore, we conclude that prolonged exposure of this DNA template to elevated temperatures could lead to

  17. 37 CFR 2.201 - Copies and certified copies.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Copies and certified copies. 2.201 Section 2.201 Patents, Trademarks, and Copyrights UNITED STATES PATENT AND TRADEMARK OFFICE, DEPARTMENT OF COMMERCE RULES OF PRACTICE IN TRADEMARK CASES Trademark Records and Files of the Patent and...

  18. 10. Photographic copy of copy of original construction drawing, dated ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. Photographic copy of copy of original construction drawing, dated 1899?. Original in possession of Twin Lakes Reservoir and Canal Company, Ordway, Colorado. PLAN OF DAM AND HEAD GATES FOR THE TWIN LAKES RESERVOIR. - Twin Lakes Dam & Outlet Works, Beneath Twin Lakes Reservoir, T11S, R80W, S22, Twin Lakes, Lake County, CO

  19. Photocopy of copy of 1922 map, revised in 1936. Copy ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Photocopy of copy of 1922 map, revised in 1936. Copy in the Fitzsimons Army Medical Center Directorate of Public Works, building 118. - Fitzsimons General Hospital, Bounded by East Colfax to south, Peoria Street to west, Denver City/County & Adams County Line to north, & U.S. Route 255 to east, Aurora, Adams County, CO

  20. Principles and Concepts of DNA Replication in Bacteria, Archaea, and Eukarya

    PubMed Central

    O’Donnell, Michael; Langston, Lance; Stillman, Bruce

    2013-01-01

    The accurate copying of genetic information in the double helix of DNA is essential for inheritance of traits that define the phenotype of cells and the organism. The core machineries that copy DNA are conserved in all three domains of life: bacteria, archaea, and eukaryotes. This article outlines the general nature of the DNA replication machinery, but also points out important and key differences. The most complex organisms, eukaryotes, have to coordinate the initiation of DNA replication from many origins in each genome and impose regulation that maintains genomic integrity, not only for the sake of each cell, but for the organism as a whole. In addition, DNA replication in eukaryotes needs to be coordinated with inheritance of chromatin, developmental patterning of tissues, and cell division to ensure that the genome replicates once per cell division cycle. PMID:23818497

  1. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  2. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  3. Mechanisms of COPI vesicle formation

    PubMed Central

    Hsu, Victor W.; Yang, Jia-Shu

    2009-01-01

    Coat Protein I (COPI) is one of the most intensely investigated coat complexes. Numerous studies have contributed to a general understanding of how coat proteins act to initiate intracellular vesicular transport. This review highlights key recent findings that have shaped our current understanding of how COPI vesicles are formed. PMID:19854177

  4. Counting copy number and calories.

    PubMed

    White, Stefan

    2015-08-01

    Copy number variation (CNV) at several genomic loci has been associated with different human traits and diseases, but in many cases the findings could not be replicated. A new study provides insights into the degree of variation present at the amylase locus and calls into question a previous association between amylase copy number and body mass index.

  5. Efficient and accurate calculations on the electronic structure of B-type poly(dG)•poly(dC) DNA by elongation method: First step toward the understanding of the biological properties of aperiodic DNA

    NASA Astrophysics Data System (ADS)

    Orimoto, Yuuichi; Gu, Feng Long; Imamura, Akira; Aoki, Yuriko

    2007-06-01

    Elongation method was applied to determine the electronic structures of B-type poly(dG)•poly(dC) DNA at the ab initio molecular orbital level as a first step toward the calculation of aperiodic DNA. The discrepancy in total energy between the elongation method and a conventional calculation was negligibly small in the order of 10-8hartree/at. for 14 G-C base pair model. The local density of states for 10 G-C base pair model estimated by the elongation method well reproduced the results by the conventional calculation. It was found that the band gap of the whole system is mainly due to the energy difference between the valence band of guanine and the conduction band of cytosine. Moreover, the electron transfer path through stacking G-C base pairs rather than sugar-phosphate backbones has been confirmed by the authors' calculations.

  6. Copy number variants calling for single cell sequencing data by multi-constrained optimization.

    PubMed

    Xu, Bo; Cai, Hongmin; Zhang, Changsheng; Yang, Xi; Han, Guoqiang

    2016-08-01

    Variations in DNA copy number carry important information on genome evolution and regulation of DNA replication in cancer cells. The rapid development of single-cell sequencing technology allows one to explore gene expression heterogeneity among single-cells, thus providing important cancer cell evolution information. Single-cell DNA/RNA sequencing data usually have low genome coverage, which requires an extra step of amplification to accumulate enough samples. However, such amplification will introduce large bias and makes bioinformatics analysis challenging. Accurately modeling the distribution of sequencing data and effectively suppressing the bias influence is the key to success variations analysis. Recent advances demonstrate the technical noises by amplification are more likely to follow negative binomial distribution, a special case of Poisson distribution. Thus, we tackle the problem CNV detection by formulating it into a quadratic optimization problem involving two constraints, in which the underling signals are corrupted by Poisson distributed noises. By imposing the constraints of sparsity and smoothness, the reconstructed read depth signals from single-cell sequencing data are anticipated to fit the CNVs patterns more accurately. An efficient numerical solution based on the classical alternating direction minimization method (ADMM) is tailored to solve the proposed model. We demonstrate the advantages of the proposed method using both synthetic and empirical single-cell sequencing data. Our experimental results demonstrate that the proposed method achieves excellent performance and high promise of success with single-cell sequencing data.

  7. Modeling genetic inheritance of copy number variations

    PubMed Central

    Wang, Kai; Chen, Zhen; Tadesse, Mahlet G.; Glessner, Joseph; Grant, Struan F. A.; Hakonarson, Hakon; Bucan, Maja

    2008-01-01

    Copy number variations (CNVs) are being used as genetic markers or functional candidates in gene-mapping studies. However, unlike single nucleotide polymorphism or microsatellite genotyping techniques, most CNV detection methods are limited to detecting total copy numbers, rather than copy number in each of the two homologous chromosomes. To address this issue, we developed a statistical framework for intensity-based CNV detection platforms using family data. Our algorithm identifies CNVs for a family simultaneously, thus avoiding the generation of calls with Mendelian inconsistency while maintaining the ability to detect de novo CNVs. Applications to simulated data and real data indicate that our method significantly improves both call rates and accuracy of boundary inference, compared to existing approaches. We further illustrate the use of Mendelian inheritance to infer SNP allele compositions in each of the two homologous chromosomes in CNV regions using real data. Finally, we applied our method to a set of families genotyped using both the Illumina HumanHap550 and Affymetrix genome-wide 5.0 arrays to demonstrate its performance on both inherited and de novo CNVs. In conclusion, our method produces accurate CNV calls, gives probabilistic estimates of CNV transmission and builds a solid foundation for the development of linkage and association tests utilizing CNVs. PMID:18832372

  8. Global variation in copy number in the human genome

    PubMed Central

    Redon, Richard; Ishikawa, Shumpei; Fitch, Karen R.; Feuk, Lars; Perry, George H.; Andrews, T. Daniel; Fiegler, Heike; Shapero, Michael H.; Carson, Andrew R.; Chen, Wenwei; Cho, Eun Kyung; Dallaire, Stephanie; Freeman, Jennifer L.; Gonzalez, Juan R.; Gratacos, Monica; Huang, Jing; Kalaitzopoulos, Dimitrios; Komura, Daisuke; MacDonald, Jeffrey R.; Marshall, Christian R.; Mei, Rui; Montgomery, Lyndal; Nishimura, Kunihiro; Okamura, Kohji; Shen, Fan; Somerville, Martin J.; Tchinda, Joelle; Valsesia, Armand; Woodwark, Cara; Yang, Fengtang; Zhang, Junjun; Zerjal, Tatiana; Zhang, Jane; Armengol, Lluis; Conrad, Donald F.; Estivill, Xavier; Tyler-Smith, Chris; Carter, Nigel P.; Aburatani, Hiroyuki; Lee, Charles; Jones, Keith W.; Scherer, Stephen W.; Hurles, Matthew E.

    2009-01-01

    Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. 1,447 copy number variable regions covering 360 megabases (12% of the genome) were i