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Sample records for accurate dna copy

  1. Hybridization modeling of oligonucleotide SNP arrays for accurate DNA copy number estimation

    PubMed Central

    Wan, Lin; Sun, Kelian; Ding, Qi; Cui, Yuehua; Li, Ming; Wen, Yalu; Elston, Robert C.; Qian, Minping; Fu, Wenjiang J

    2009-01-01

    Affymetrix SNP arrays have been widely used for single-nucleotide polymorphism (SNP) genotype calling and DNA copy number variation inference. Although numerous methods have achieved high accuracy in these fields, most studies have paid little attention to the modeling of hybridization of probes to off-target allele sequences, which can affect the accuracy greatly. In this study, we address this issue and demonstrate that hybridization with mismatch nucleotides (HWMMN) occurs in all SNP probe-sets and has a critical effect on the estimation of allelic concentrations (ACs). We study sequence binding through binding free energy and then binding affinity, and develop a probe intensity composite representation (PICR) model. The PICR model allows the estimation of ACs at a given SNP through statistical regression. Furthermore, we demonstrate with cell-line data of known true copy numbers that the PICR model can achieve reasonable accuracy in copy number estimation at a single SNP locus, by using the ratio of the estimated AC of each sample to that of the reference sample, and can reveal subtle genotype structure of SNPs at abnormal loci. We also demonstrate with HapMap data that the PICR model yields accurate SNP genotype calls consistently across samples, laboratories and even across array platforms. PMID:19586935

  2. 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

  3. Variable copy number DNA sequences in rice.

    PubMed

    Kikuchi, S; Takaiwa, F; Oono, K

    1987-12-01

    We have cloned two types of variable copy number DNA sequences from the rice embryo genome. One of these sequences, which was cloned in pRB301, was amplified about 50-fold during callus formation and diminished in copy number to the embryonic level during regeneration. The other clone, named pRB401, showed the reciprocal pattern. The copy numbers of both sequences were changed even in the early developmental stage and eliminated from nuclear DNA along with growth of the plant. Sequencing analysis of the pRB301 insert revealed some open reading frames and direct repeat structures, but corresponding sequences were not identified in the EMBL and LASL DNA databases. Sequencing of the nuclear genomic fragment cloned in pRB401 revealed the presence of the 3'rps12-rps7 region of rice chloroplast DNA. Our observations suggest that during callus formation (dedifferentiation), regeneration and the growth process the copy numbers of some DNA sequences are variable and that nuclear integrated chloroplast DNA acts as a variable copy number sequence in the rice genome. Based on data showing a common sequence in mitochondria and chloroplast DNA of maize (Stern and Lonsdale 1982) and that the rps12 gene of tobacco chloroplast DNA is a divided gene (Torazawa et al. 1986), it is suggested that the sequence on the inverted repeat structure of chloroplast DNA may have the character of a movable genetic element. PMID:3481021

  4. Accurate and reliable high-throughput detection of copy number variation in the human genome

    PubMed Central

    Fiegler, Heike; Redon, Richard; Andrews, Dan; Scott, Carol; Andrews, Robert; Carder, Carol; Clark, Richard; Dovey, Oliver; Ellis, Peter; Feuk, Lars; French, Lisa; Hunt, Paul; Kalaitzopoulos, Dimitrios; Larkin, James; Montgomery, Lyndal; Perry, George H.; Plumb, Bob W.; Porter, Keith; Rigby, Rachel E.; Rigler, Diane; Valsesia, Armand; Langford, Cordelia; Humphray, Sean J.; Scherer, Stephen W.; Lee, Charles; Hurles, Matthew E.; Carter, Nigel P.

    2006-01-01

    This study describes a new tool for accurate and reliable high-throughput detection of copy number variation in the human genome. We have constructed a large-insert clone DNA microarray covering the entire human genome in tiling path resolution that we have used to identify copy number variation in human populations. Crucial to this study has been the development of a robust array platform and analytic process for the automated identification of copy number variants (CNVs). The array consists of 26,574 clones covering 93.7% of euchromatic regions. Clones were selected primarily from the published “Golden Path,” and mapping was confirmed by fingerprinting and BAC-end sequencing. Array performance was extensively tested by a series of validation assays. These included determining the hybridization characteristics of each individual clone on the array by chromosome-specific add-in experiments. Estimation of data reproducibility and false-positive/negative rates was carried out using self–self hybridizations, replicate experiments, and independent validations of CNVs. Based on these studies, we developed a variance-based automatic copy number detection analysis process (CNVfinder) and have demonstrated its robustness by comparison with the SW-ARRAY method. PMID:17122085

  5. DNA copy number losses in human neoplasms.

    PubMed

    Knuutila, S; Aalto, Y; Autio, K; Björkqvist, A M; El-Rifai, W; Hemmer, S; Huhta, T; Kettunen, E; Kiuru-Kuhlefelt, S; Larramendy, M L; Lushnikova, T; Monni, O; Pere, H; Tapper, J; Tarkkanen, M; Varis, A; Wasenius, V M; Wolf, M; Zhu, Y

    1999-09-01

    This review summarizes reports of recurrent DNA sequence copy number losses in human neoplasms detected by comparative genomic hybridization. Recurrent losses that affect each of the chromosome arms in 73 tumor types are tabulated from 169 reports. The tables are available online at http://www.amjpathol.org and http://www. helsinki.fi/ approximately lglvwww/CMG.html. The genes relevant to the lost regions are discussed for each of the chromosomes. The review is supplemented also by a list of known and putative tumor suppressor genes and DNA repair genes (see Table 1, online). Losses are found in all chromosome arms, but they seem to be relatively rare at 1q, 2p, 3q, 5p, 6p, 7p, 7q, 8q, 12p, and 20q. Losses and their minimal common overlapping areas that were present in a great proportion of the 73 tumor entities reported in Table 2 (see online) are (in descending order of frequency): 9p23-p24 (48%), 13q21 (47%), 6q16 (44%), 6q26-q27 (44%), 8p23 (37%), 18q22-q23 (37%), 17p12-p13 (34%), 1p36.1 (34%), 11q23 (33%), 1p22 (32%), 4q32-qter (31%), 14q22-q23 (25%), 10q23 (25%), 10q25-qter (25%),15q21 (23%), 16q22 (23%), 5q21 (23%), 3p12-p14 (22%), 22q12 (22%), Xp21 (21%), Xq21 (21%), and 10p12 (20%). The frequency of losses at chromosomes 7 and 20 was less than 10% in all tumors. The chromosomal regions in which the most frequent losses are found implicate locations of essential tumor suppressor genes and DNA repair genes that may be involved in the pathogenesis of several tumor types. PMID:10487825

  6. 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. PMID:26258394

  7. Mitochondrial DNA Copy Number in Peripheral Blood and Melanoma Risk

    PubMed Central

    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). PMID:26110424

  8. RECONSTRUCTING DNA COPY NUMBER BY PENALIZED ESTIMATION AND IMPUTATION

    PubMed Central

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

    2011-01-01

    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. PMID:21572975

  9. Fast and accurate nonenzymatic copying of an RNA-like synthetic genetic polymer.

    PubMed

    Zhang, Shenglong; Blain, J Craig; Zielinska, Daria; Gryaznov, Sergei M; Szostak, Jack W

    2013-10-29

    Recent advances suggest that it may be possible to construct simple artificial cells from two subsystems: a self-replicating cell membrane and a self-replicating genetic polymer. Although multiple pathways for the growth and division of model protocell membranes have been characterized, no self-replicating genetic material is yet available. Nonenzymatic template-directed synthesis of RNA with activated ribonucleotide monomers has led to the copying of short RNA templates; however, these reactions are generally slow (taking days to weeks) and highly error prone. N3'-P5'-linked phosphoramidate DNA (3'-NP-DNA) is similar to RNA in its overall duplex structure, and is attractive as an alternative to RNA because the high reactivity of its corresponding monomers allows rapid and efficient copying of all four nucleobases on homopolymeric RNA and DNA templates. Here we show that both homopolymeric and mixed-sequence 3'-NP-DNA templates can be copied into complementary 3'-NP-DNA sequences. G:T and A:C wobble pairing leads to a high error rate, but the modified nucleoside 2-thiothymidine suppresses wobble pairing. We show that the 2-thiothymidine modification increases both polymerization rate and fidelity in the copying of a 3'-NP-DNA template into a complementary strand of 3'-NP-DNA. Our results suggest that 3'-NP-DNA has the potential to serve as the genetic material of artificial biological systems. PMID:24101473

  10. DNA sequence copy number analysis by Comparative Genomic Hybridization (CGH)

    SciTech Connect

    Pinkel, D.; Kallioniemi, A.; Kallioniemi, O.; Waldman, F.; Sudar, D.; Gray, I. ); Rutovitz, D.; Piper, I. )

    1993-01-01

    Comparative Genomic Hybridization (CGH) uses the kinetics of in situ hybridization to compare the copy numbers of different DNA sequences within the same genome and the copy numbers of the same sequences among different genomes. In a typical application genomic DNA from a tumor and from normal cells are differentially labeled and simultaneously hybridized to normal metaphase chromosomes, and detected with different fluorochromes. Properly registered images of each fluorochrome are obtained using a microscope equipped with multi-band filters and a CCD camera. Digital image analysis permits measurement of intensity ratio profiles along each of the target chromosomes. Studies of cells with known aberrations indicate that the intensity ratio at each position is proportional to the ratio of the copy numbers of the sequences that bind there in the tumor and normal genomes. Analytical challenges posed by the need to efficiently obtain copy number karyotypes are discussed.

  11. Accurate quantification of supercoiled DNA by digital PCR.

    PubMed

    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

  12. 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

  13. 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

  14. 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

  15. Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions.

    PubMed

    Orsztynowicz, M; Pawlak, P; Podstawski, Z; Nizanski, W; Partyka, A; Gotowiecka, M; Kosiniak-Kamysz, K; Lechniak, D

    2016-06-01

    Predicting male fertility on non-invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer-assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four age groups (3-5, 6-10, 11-14 and >15 years) and were subjected to a complex investigation including conventional analysis, CASA, flow cytometry and mtDNA content (real-time PCR). The mean (±SD) number of mtDNA copies equalled 14 ± 9 and varied from 3 to 64. Considering the great number of sperm parameters monitored in this study, only few of them were correlated with the mtDNA content: ejaculate volume (a positive correlation), the amplitude of lateral head displacement (ALH; a negative correlation) and the high mitochondrial activity index (a negative correlation). The stallion age was not correlated with the mtDNA copy number. This study provides the first set of data on mtDNA content in equine sperm and confirms phenomena previously described for humans and dog on associations between sperm mtDNA content and selected motility parameters monitored by the CASA. Basing our study on spermatozoa from fertile stallions could however limit the extent of detected associations. PMID:27037507

  16. Mitochondrial DNA copy number and replication in reprogramming and differentiation.

    PubMed

    St John, Justin C

    2016-04-01

    Until recently, it was thought that the role of the mitochondrial genome was confined to encoding key proteins that generate ATP through the process of oxidative phosphorylation in the electron transfer chain. However, with increasing new evidence, it is apparent that the mitochondrial genome has a major role to play in a number of diseases and phenotypes. For example, mitochondrial variants and copy number have been implicated in the processes of fertilisation outcome and development and the onset of tumorigenesis. On the other hand, mitochondrial DNA (mtDNA) haplotypes have been implicated in a variety of diseases and most likely account for the adaptation that our ancestors achieved in order that they were fit for their environments. The mechanisms, which enable the mitochondrial genome to either protect or promote the disease phenotype, require further elucidation. However, there appears to be significant 'crosstalk' between the chromosomal and mitochondrial genomes that enable this to take place. One such mechanism is the regulation of DNA methylation by mitochondrial DNA, which is often perturbed in reprogrammed cells that have undergone dedifferentiation and affects mitochondrial DNA copy number. Furthermore, it appears that the mitochondrial genome interacts with the chromosomal genome to regulate the transcription of key genes at certain stages during development. Additionally, the mitochondrial genome can accumulate a series of mtDNA variants, which can lead to diseases such as cancer. It is likely that a combination of certain mitochondrial variants and aberrant patterns of mtDNA copy number could indeed account for many diseases that have previously been unaccounted for. This review focuses on the role that the mitochondrial genome plays especially during early stages of development and in cancer. PMID:26827792

  17. 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

  18. PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

    PubMed

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

    2016-01-01

    Because of the role that 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

  19. 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

  20. 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

  1. PCR Based Determination of Mitochondrial DNA Copy Number in Multiple Species

    PubMed Central

    Rooney, JP; Ryde, IT; Sanders, LH; Howlett, EH; Colton, MD; Germ, KE; Mayer, GD; Greenamyre, JT; Meyer, JN

    2015-01-01

    Summary Mitochondrial DNA (mtDNA) copy number is a critical component of overall mitochondrial health. In this chapter we describe methods for isolation of both mtDNA and nuclear DNA (nucDNA), and measurement of their respective copy numbers using quantitative PCR. Methods differ depending on the species and cell type of the starting material, and availability of specific PCR reagents. PMID:25308485

  2. Accurate restoration of DNA sequences. Progress report

    SciTech Connect

    Churchill, G.A.

    1994-05-01

    The primary of this project are the development of (1) a general stochastic model for DNA sequencing errors (2) algorithms to restore the original DNA sequence and (3) statistical methods to assess the accuracy of this restoration. A secondary objective is to develop new algorithms for fragment assembly. Initially a stochastic model that assumes errors are independent and uniformly distributed will be developed. Generalizations of the basic model will be developed to account for (1) decay of accuracy along fragments, (2) variable error rates among fragments, (3) sequence dependent errors (e.g. homopolymeric, runs), and (4) strand--specific systematic errors (e.g. compressions). The emphasis of this project will be the development of a theoretical basis for determining sequence accuracy. However, new algorithms are proposed and these will be implemented as software (in the C programming language). This software will be tested using real and simulated data. It will be modular in design and will be made available for distribution to the scientific community.

  3. Improved Statistical Analysis for Array CGH-Based DNA Copy Number Aberrations

    PubMed Central

    Jiang, Hongmei; Zhu, Zhong-Zheng; Yu, Yue; Lin, Simon; Hou, Lifang

    2011-01-01

    Array-based comparative genomic hybridization (aCGH) allows measuring DNA copy number at the whole genome scale. In cancer studies, one may be interested in identifying DNA copy number aberrations (CNAs) associated with certain clinicopathological characteristics such as cancer metastasis. We proposed to define test regions based on copy number pattern profiles across multiple samples, using either smoothed log2-ratio or discrete data of copy number gain/loss calls. Association test performed on the refined test regions instead of the probes has improved power due to reduced number of tests. We also compared three types of measurement of copy number levels, normalized log2-ratio, smoothed log2-ratio, and copy number gain or loss calls in statistical hypothesis testing. The relative strengths and weaknesses of the proposed method were demonstrated using both simulation studies and real data analysis of a liver cancer study. PMID:22084565

  4. Reduced mitochondrial DNA copy number is a biomarker of Parkinson's disease

    PubMed Central

    Pyle, Angela; Anugrha, Haidyan; Kurzawa-Akanbi, Marzena; Yarnall, Alison; Burn, David; Hudson, Gavin

    2016-01-01

    Like any organ, the brain is susceptible to the march of time and a reduction in mitochondrial biogenesis is a hallmark of the aging process. In the largest investigation of mitochondrial copy number in Parkinson's disease (PD) to date and by using multiple tissues, we demonstrate that reduced Parkinson DNA (mitochondrial DNA mtDNA) copy number is a biomarker for the etiology of PD. We used established methods of mtDNA quantification to assess the copy number of mtDNA in n = 363 peripheral blood samples, n = 151 substantia nigra pars compacta tissue samples and n = 120 frontal cortex tissue samples from community-based PD cases fulfilling UK-PD Society brain bank criteria for the diagnosis of PD. Accepting technical limitations, our data show that PD patients suffer a significant reduction in mtDNA copy number in both peripheral blood and the vulnerable substantia nigra pars compacta when compared to matched controls. Our study indicates that reduced mtDNA copy number is restricted to the affected brain tissue, but is also reflected in the peripheral blood, suggesting that mtDNA copy number may be a viable diagnostic predictor of PD. PMID:26639155

  5. 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

  6. Leukocyte Mitochondrial DNA Copy Number Is Associated with Chronic Obstructive Pulmonary Disease

    PubMed Central

    Liu, Shih-Feng; Kuo, Ho-Chang; Tseng, Ching-Wan; Huang, Hung-Tu; Chen, Yung-Che; Tseng, Chia-Cheng; Lin, Meng-Chih

    2015-01-01

    Background Oxidative stress is known to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Evidence suggests that leukocytes mitochondria DNA (mtDNA) is susceptible to undergo mutations, insertions, or depletion in response to reactive oxidative stress (ROS). We hypothesize that mtDNA copy number is associated with the development of COPD. Methodology/Principal Findings Relative mtDNA copy number was measured by a quantitative real-time PCR assay using DNA extracted from peripheral leukocytes. MtDNA copy number of peripheral leukocytes in the COPD group (n = 86) is significantly decreased compared with non-smoker group (n = 77) (250.3± 21.5 VS. 464.2± 49.9, P<0.001). MtDNA copy number in the COPD group was less than that in the healthy smoking group, but P value nearly achieved significance (250.3± 21.5 VS. 404.0± 76.7, P = 0.08) MtDNA copy number has no significance with age, gender, body mass index, current smoking, and pack-years in COPD group, healthy smoker group and no smoker group, respectively. Serum glutathione level in the COPD group is significantly decreased compared with healthy smoker and non-smoker groups (4.5± 1.3 VS. 6.2± 1.9 and 4.5± 1.3 VS. 7.1±1.1 mU/mL; P<0.001 respectively). Pearson correlation test shows a significant liner correlation between mtDNA copy number and serum glutathione level (R = 0.2, P = 0.009). Conclusions/Significance COPD is associated with decreased leukocyte mtDNA copy number and serum glutathione. COPD is a regulatory disorder of leukocytes mitochondria. However, further studies are needed to determine the real mechanisms about the gene and the function of mitochondria. PMID:26394041

  7. 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. PMID:26679989

  8. Changes in DNA damage, molecular integrity, and copy number for plastid DNA and mitochondrial DNA during maize development.

    PubMed

    Kumar, Rachana A; Oldenburg, Delene J; Bendich, Arnold J

    2014-12-01

    The amount and structural integrity of organellar DNAs change during plant development, although the mechanisms of change are poorly understood. Using PCR-based methods, we quantified DNA damage, molecular integrity, and genome copy number for plastid and mitochondrial DNAs of maize seedlings. A DNA repair assay was also used to assess DNA impediments. During development, DNA damage increased and molecules with impediments that prevented amplification by Taq DNA polymerase increased, with light causing the greatest change. DNA copy number values depended on the assay method, with standard real-time quantitative PCR (qPCR) values exceeding those determined by long-PCR by 100- to 1000-fold. As the organelles develop, their DNAs may be damaged in oxidative environments created by photo-oxidative reactions and photosynthetic/respiratory electron transfer. Some molecules may be repaired, while molecules with unrepaired damage may be degraded to non-functional fragments measured by standard qPCR but not by long-PCR. PMID:25261192

  9. Changes in DNA damage, molecular integrity, and copy number for plastid DNA and mitochondrial DNA during maize development

    PubMed Central

    Kumar, Rachana A.; Oldenburg, Delene J.; Bendich, Arnold J.

    2014-01-01

    The amount and structural integrity of organellar DNAs change during plant development, although the mechanisms of change are poorly understood. Using PCR-based methods, we quantified DNA damage, molecular integrity, and genome copy number for plastid and mitochondrial DNAs of maize seedlings. A DNA repair assay was also used to assess DNA impediments. During development, DNA damage increased and molecules with impediments that prevented amplification by Taq DNA polymerase increased, with light causing the greatest change. DNA copy number values depended on the assay method, with standard real-time quantitative PCR (qPCR) values exceeding those determined by long-PCR by 100- to 1000-fold. As the organelles develop, their DNAs may be damaged in oxidative environments created by photo-oxidative reactions and photosynthetic/respiratory electron transfer. Some molecules may be repaired, while molecules with unrepaired damage may be degraded to non-functional fragments measured by standard qPCR but not by long-PCR. PMID:25261192

  10. Low Mitochondrial DNA Copy Number is Associated With Adverse Clinical Outcomes in Peritoneal Dialysis Patients.

    PubMed

    Yoon, Chang-Yun; Park, Jung Tak; Kee, Youn Kyung; Han, Seung Gyu; Han, In Mee; Kwon, Young Eun; Park, Kyoung Sook; Lee, Mi Jung; Han, Seung Hyeok; Kang, Shin-Wook; Yoo, Tae-Hyun

    2016-02-01

    Mitochondrial dysfunction may play an important role in abnormal glucose metabolism and systemic inflammation. We aimed to investigate the relationship between mitochondrial DNA (mtDNA) copy number and clinical outcomes in peritoneal dialysis (PD) patients. We recruited 120 prevalent PD patients and determined mtDNA copy number by PCR. Primary outcome was all-cause mortality, whereas secondary outcomes included cardiovascular events, technical PD failure, and incident malignancy. Cox proportional hazards analysis determined the independent association of mtDNA copy number with outcomes. The mean patient age was 52.3 years; 42.5% were men. The mean log mtDNA copy number was 3.30 ± 0.50. During a follow-up period of 35.4 ± 19.3 months, all-cause mortality and secondary outcomes were observed in 20.0% and 59.2% of patients, respectively. Secondary outcomes were significantly lower in the highest mtDNA copy number group than in the lower groups. In multiple Cox analysis, the mtDNA copy number was not associated with all-cause mortality (lower two vs highest tertile: hazard ratio [HR] = 1.208, 95% confidence interval [CI] = 0.477-3.061). However, the highest tertile group was significantly associated with lower incidences of secondary outcomes (lower two vs highest tertile: HR [95% CI] = 0.494 [0.277-0.882]) after adjusting for confounding factors. The decreased mtDNA copy number was significantly associated with adverse clinical outcomes in PD patients. PMID:26886611

  11. Low Mitochondrial DNA Copy Number is Associated With Adverse Clinical Outcomes in Peritoneal Dialysis Patients

    PubMed Central

    Yoon, Chang-Yun; Park, Jung Tak; Kee, Youn Kyung; Han, Seung Gyu; Han, In Mee; Kwon, Young Eun; Park, Kyoung Sook; Lee, Mi Jung; Han, Seung Hyeok; Kang, Shin-Wook; Yoo, Tae-Hyun

    2016-01-01

    Abstract Mitochondrial dysfunction may play an important role in abnormal glucose metabolism and systemic inflammation. We aimed to investigate the relationship between mitochondrial DNA (mtDNA) copy number and clinical outcomes in peritoneal dialysis (PD) patients. We recruited 120 prevalent PD patients and determined mtDNA copy number by PCR. Primary outcome was all-cause mortality, whereas secondary outcomes included cardiovascular events, technical PD failure, and incident malignancy. Cox proportional hazards analysis determined the independent association of mtDNA copy number with outcomes. The mean patient age was 52.3 years; 42.5% were men. The mean log mtDNA copy number was 3.30 ± 0.50. During a follow-up period of 35.4 ± 19.3 months, all-cause mortality and secondary outcomes were observed in 20.0% and 59.2% of patients, respectively. Secondary outcomes were significantly lower in the highest mtDNA copy number group than in the lower groups. In multiple Cox analysis, the mtDNA copy number was not associated with all-cause mortality (lower two vs highest tertile: hazard ratio [HR] = 1.208, 95% confidence interval [CI] = 0.477–3.061). However, the highest tertile group was significantly associated with lower incidences of secondary outcomes (lower two vs highest tertile: HR [95% CI] = 0.494 [0.277–0.882]) after adjusting for confounding factors. The decreased mtDNA copy number was significantly associated with adverse clinical outcomes in PD patients. PMID:26886611

  12. Retroelements contribute to the excess low-copy-number DNA in pine.

    PubMed

    Elsik, C G; Williams, C G

    2000-09-01

    Excess DNA in the single-copy component is rarely recognized as a contributor to the C-value paradox yet the single-copy component of the pine genome is reported to comprise over 3000 Mb of DNA, in large excess over the estimated 100 Mb required for gene expression. Two hypotheses regarding the factors that might contribute to the excess low-copy-number DNA were tested. The first hypothesis proposes that the excess low-copy kinetic component is actually overestimated by reassociation data analysis. To test this, a previously published C0t curve for Pinus strobus was reanalyzed using a new estimate of genome size based on laser flow cytometry. Part of the excess low-copy-number DNA in the pine genome could be attributed to the choice of parameters used in the analysis of the reassociation data. The second hypothesis holds that diverged retrotransposons contribute to the excess low-copy DNA. Sequences randomly sampled from single-copy and low-repetitive kinetic components of the P. taeda genome were characterized. Twelve of 46 fragments cloned from these fractions were found to show sequence similarity to retroelements: hence diverged retroelements contribute to the excess low-repetitive kinetic component in the pine genome. Similarity search was shown to be a conservative method for identifying retroelements, and thus the number of retroelements in the low-copy component was actually underestimated. Most of the retroelements in this fraction were nonfunctional. divergent from known retroelement families and previously reported only for flowering plants. Divergent retrotransposons are thus a major factor contributing to the expansion of the low-repetitive DNA component in higher plants. PMID:11016832

  13. Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags

    PubMed Central

    Wee, Eugene J.H.; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

    2016-01-01

    Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research. PMID:27446486

  14. Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA.

    PubMed

    Lowe, Brenda A; Shiva Prakash, N; Way, Melissa; Mann, Michael T; Spencer, T Michael; Boddupalli, Raghava S

    2009-12-01

    Transgene copy number is an important criterion for determining the utility of transgenic events. Single copy integration events are highly desirable when the objective is to produce marker free plants through segregation or when it is necessary to introgress different transgenes into commercial cultivars from different transgenic events. In contrast multi-copy events are advocated by several authors for higher expression of the transgene. Till recently, it was thought that employment of the particle gun for transformation results in the production of a high proportion of multi-copy events often with complex integration pattern when compared to Agrobacterium-mediated transformation. However, it has been demonstrated that usage of cassette DNA for bombardment in place of whole plasmids would result in simple insertion pattern of the transgenes. While investigating the effect of varying the cassette DNA amount on stable transformation, the frequency of occurrence of low copy events was observed to increase when lower doses of cassette DNA was employed for bombardment. Large scale experimentation with rigorous statistical analysis performed to verify the above observations employing Helium gun and the Electric discharge gun for gene delivery confirmed the above observations. Helium gun experiments involving production of more than 1,600 corn events consistently yielded single copy events at higher frequencies at lower cassette DNA load (46% at 2.5 ng/shot) as compared to higher cassette DNA load (29% at 25 ng/shot) across 18 independent experiments. Results were nearly identical with the Electric discharge particle gun device where single copy events were recovered at frequencies of 54% at 2.5 ng cassettes DNA per shot as compared to 18% at 25 ng cassette DNA per shot. The transformation frequency declined from 41 to 34% (Helium gun) and from 48 to 31% (Electric discharge gun) with reduction in cassette DNA quantity from 25 to 2.5 ng per shot. This reduction in the

  15. Accurate episomal HIV 2-LTR circles quantification using 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; Vandekerckhove, Linos; De Spiegelaere, Ward

    2014-01-01

    Introduction In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. Materials and Methods We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Results Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland–Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10). Conclusions 2-LTR circles

  16. Mitochondrial DNA copy number and lung cancer risk in a prospective cohort study

    PubMed Central

    Hosgood, H.Dean; Liu, Chin-San; Rothman, Nathaniel; Weinstein, Stephanie J.; Bonner, Matthew R.; Shen, Min; Lim, Unhee; Virtamo, Jarmo; Cheng, Wen-ling; Albanes, Demetrius; Lan, Qing

    2010-01-01

    Mitochondria are eukaryotic organelles responsible for energy production. Mitochondrial DNA (mtDNA) lack introns and protective histones, have limited DNA repair capacity and compensate for damage by increasing the number of mtDNA copies. As a consequence, mitochondria are more susceptible to reactive oxygen species, an important determinant of cancer risk, and it is hypothesized that increased mtDNA copy number may be associated with carcinogenesis. We assessed the association of mtDNA copy number and lung cancer risk in 227 prospectively collected cases and 227 matched controls from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for age at randomization, smoking years and number of cigarettes smoked per day. There was suggestion of a dose-dependent relationship between mtDNA copy number and subsequent risk of lung cancer, with a prominent effect observed in the highest mtDNA copy number quartile [ORs (95% CI) by quartile: 1.0 (reference), 1.3 (0.7–2.5), 1.1 (0.6–2.2) and 2.4 (1.1–5.1); Ptrend = 0.008]. This is the first report, to the best of our knowledge, to suggest that mtDNA copy number may be positively associated with subsequent risk of lung cancer in a prospective cohort study; however, replication is needed in other studies and populations. PMID:20176654

  17. 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

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

    PubMed Central

    Lyckegaard, E M; Clark, A G

    1989-01-01

    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. Images PMID:2494656

  19. Accurate Prediction of Binding Thermodynamics for DNA on Surfaces

    PubMed Central

    Vainrub, Arnold; Pettitt, B. Montgomery

    2011-01-01

    For DNA mounted on surfaces for microarrays, microbeads and nanoparticles, the nature of the random attachment of oligonucleotide probes to an amorphous surface gives rise to a locally inhomogeneous probe density. These fluctuations of the probe surface density are inherent to all common surface or bead platforms, regardless if they exploit either an attachment of pre-synthesized probes or probes synthesized in situ on the surface. Here, we demonstrate for the first time the crucial role of the probe surface density fluctuations in performance of DNA arrays. We account for the density fluctuations with a disordered two-dimensional surface model and derive the corresponding array hybridization isotherm that includes a counter-ion screened electrostatic repulsion between the assayed DNA and probe array. The calculated melting curves are in excellent agreement with published experimental results for arrays with both pre-synthesized and in-situ synthesized oligonucleotide probes. The approach developed allows one to accurately predict the melting curves of DNA arrays using only the known sequence dependent hybridization enthalpy and entropy in solution and the experimental macroscopic surface density of probes. This opens the way to high precision theoretical design and optimization of probes and primers in widely used DNA array-based high-throughput technologies for gene expression, genotyping, next-generation sequencing, and surface polymerase extension. PMID:21972932

  20. Urinary Mitochondrial DNA Copy Number Identifies Chronic Renal Injury in Hypertensive Patients.

    PubMed

    Eirin, Alfonso; Saad, Ahmed; Tang, Hui; Herrmann, Sandra M; Woollard, John R; Lerman, Amir; Textor, Stephen C; Lerman, Lilach O

    2016-08-01

    Mitochondrial injury contributes to renal dysfunction in several models of renal disease, but its involvement in human hypertension remains unknown. Fragments of the mitochondrial genome released from dying cells are considered surrogate markers of mitochondrial injury. We hypothesized that hypertension would be associated with increased urine mitochondrial DNA (mtDNA) copy numbers. We prospectively measured systemic and urinary copy number of the mtDNA genes cytochrome-c oxidase-3 and NADH dehydrogenase subunit-1 by quantitative polymerase chain reaction in essential (n=25) and renovascular (RVH, n=34) hypertensive patients and compared them with healthy volunteers (n=22). Urinary kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin served as indices of renal injury. Renal blood flow and oxygenation were assessed by multidetector computed tomography and blood oxygen level-dependent magnetic resonance imaging. Blood pressure, urinary neutrophil gelatinase-associated lipocalin, and kidney injury molecule-1 were similarly elevated in essential hypertension and RVH, and estimated glomerular filtration rate was lower in RVH versus healthy volunteers and essential hypertension. Renal blood flow was lower in RVH compared with essential hypertension. Urinary mtDNA copy number was higher in hypertension compared with healthy volunteers, directly correlated with urinary neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 and inversely with estimated glomerular filtration rate. In RVH, urinary mtDNA copy number correlated directly with intrarenal hypoxia. Furthermore, in an additional validation cohort, urinary mtDNA copy number was higher in RVH compared with healthy volunteers (n=10 each). The change in serum creatinine levels and estimated glomerular filtration rate 3 months after medical therapy without or with revascularization correlated with the change in urinary mtDNA. Therefore, elevated urinary mtDNA copy numbers in

  1. 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.

  2. High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

    PubMed Central

    2011-01-01

    Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics. PMID:22035192

  3. Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies.

    PubMed

    Alonso, Antonio; Martín, Pablo; Albarrán, Cristina; García, Pilar; García, Oscar; de Simón, Lourdes Fernández; García-Hirschfeld, Julia; Sancho, Manuel; de La Rúa, Concepción; Fernández-Piqueras, Jose

    2004-01-28

    We explore different designs to estimate both nuclear and mitochondrial human DNA (mtDNA) content based on the detection of the 5' nuclease activity of the Taq DNA polymerase using fluorogenic probes and a real-time quantitative PCR detection system. Human mtDNA quantification was accomplished by monitoring the real-time progress of the PCR-amplification of two different fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This mtDNA real-time PCR design has been used to assess the mtDNA preservation (copy number and degradation state) of DNA samples retrieved from 500 to 1500 years old human remains that showed low copy number and highly degraded mtDNA. The quantification of nuclear DNA was achieved by real-time PCR of a segment of the X-Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and a X-specific fragment (AMGX: 106 bp) making possible not only haploid or diploid DNA quantitation but also sex determination. The AMG real-time PCR design has been used to quantify a set of 57 DNA samples from 4-5 years old forensic bone remains with improved sensitivity compared with the slot-blot hybridization method. The potential utility of this technology to improve the quality of some PCR-based forensic and ancient DNA studies (microsatellite typing and mtDNA sequencing) is discussed. PMID:15040907

  4. 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

  5. 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

  6. Peripheral blood mitochondrial DNA copy number, length heteroplasmy and breast cancer risk: a replication study.

    PubMed

    Shen, Jie; Wan, Jie; Song, Renduo; Zhao, Hua

    2015-11-01

    Oxidative stress has consistently been linked to breast carcinogenesis, and mitochondria play a significant role in regulating reactive oxygen species generation. In our previous study, we found that increased levels of mitochondrial DNA (mtDNA) copy number and the presence of mitochondrial length heteroplasmies in the hypervariable (HV) regions 1 and 2 (HV1 and HV2) in peripheral blood are associated with increased risk of breast cancer. In current study with 1000 breast cancer cases and 1000 healthy controls, we intended to replicate our previous findings. Overall, levels of mtDNA copy number were significantly higher in breast cancer cases than healthy controls (mean: 1.17 versus 0.94, P < 0.001). In the multivariate linear regression analysis, increased mtDNA copy number levels were associated with a 1.32-fold increased risk of breast cancer [adjusted odds ratio (OR) = 1.32, 95% confidence interval (CI) = 1.15-1.67]. Breast cancer cases were more likely to have HV1 and HV2 region length heteroplasmies than healthy controls (P < 0.001, respectively). The existence of HV1 and HV2 length heteroplasmies was associated with 2.01- and 1.63-folds increased risk of breast cancer (for HV1: OR = 2.01, 95% CI = 1.66-2.42; for HV2: OR = 1.63, 95% CI = 1.34-1.92). Additionally, joint effects among mtDNA copy number, HV1 and HV2 length heteroplasmies were observed. Our results are consistent with our previous findings and further support the roles of mtDNA copy number and mtDNA length heteroplasmies that may play in the development of breast cancer. PMID:26363030

  7. 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

  8. The relationship between altered mitochondrial DNA copy number and cancer risk: a meta-analysis.

    PubMed

    Mi, Jia; Tian, Geng; Liu, Shuang; Li, Xianglin; Ni, Tianhui; Zhang, Liwei; Wang, Bin

    2015-01-01

    Currently, a comprehensive assessment between mitochondrial DNA (mtDNA) content and cancer risk is lacking. We designed this meta-analysis to test the hypothesis that altered mtDNA copy number might influence genetic susceptibility to some specific types of cancer. The processes of literature search, eligibility appraisal and data retrieval were independently completed in duplicate. The mtDNA copy number which was dichotomized or classified into tertiles was compared between cancer cases and controls. Twenty-six articles with 38 study groups were analyzed among 6682 cases and 9923 controls. When dichotomizing mtDNA copy number at the median value, there was an 11% increased cancer risk for carriers of high mtDNA content (P = 0.320). By cancer type, high mtDNA content was associated with an increased risk for lymphoma (OR = 1.76; P = 0.023) but a reduced risk for skeleton cancer (OR = 0.39; P = 0.001). Carriers of the 2(nd) and 3(rd) tertiles of mtDNA copy number had an 1.74-fold (P = 0.010) and 2.07-fold (P = 0.021) increased risk of lymphoma, respectively. By contrast, there was correspondingly a 56% (P < 0.001) and 80% (P < 0.001) reduced risk of skeleton cancer. Our findings suggested that elevated mtDNA content was associated with a higher risk for lymphoma, but a lower risk for skeleton cancer. PMID:25952580

  9. 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. PMID:25477383

  10. 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

  11. DNA Copy Number Profiles in Affinity-Purified Ovarian Clear Cell Carcinoma

    PubMed Central

    Kuo, Kuan-Ting; Mao, Tsui-Lien; Chen, Xu; Feng, Yuanjian; Nakayama, Kentaro; Wang, Yue; Glas, Ruth; Ma, M. Joe; Kurman, Robert J.; Shih, Ie-Ming; Wang, Tian-Li

    2010-01-01

    Purpose Advanced ovarian clear cell carcinoma (CCC) is one of the most malignant ovarian malignancies, in part because it tends to be resistant to platinum-based chemotherapy. At present, little is known about the molecular genetic alterations in CCCs except that there are frequent activating mutations in PIK3CA. The purpose of this study is to comprehensively define the genomic changes in CCC based on DNA copy number alterations. Experimental Design We performed 250K high-density SNP array analysis in 12 affinity-purified CCCs and 10 CCC cell lines. Discrete regions of amplification and deletion were also analyzed in additional 21 affinity-purified CCCs using quantitative real-time PCR. Results The level of chromosomal instability in CCC as defined by the extent of DNA copy number changes is similar to those previously reported in low-grade ovarian serous carcinoma but much less than those in high-grade serous carcinoma. The most remarkable region with DNA copy number gain is at chr20 which harbors a potential oncogene, ZNF217. This is observed in 36% of CCCs but rarely detected in serous carcinomas regardless of grade. In addition, homozygous deletions are detected at CDKN2A/2B and LZTS1 loci. Interestingly, the DNA copy number changes observed in fresh CCC tissues are rarely detected in the established CCC cell lines. Conclusions This study provides the first high resolution, genome-wide view of DNA copy number alterations in ovarian CCC. The findings provide a genomic landscape for future studies aimed at elucidating the pathogenesis and developing new target-based therapies for CCCs. PMID:20233889

  12. Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase

    PubMed Central

    Fukuoh, Atsushi; Cannino, Giuseppe; Gerards, Mike; Buckley, Suzanne; Kazancioglu, Selena; Scialo, Filippo; Lihavainen, Eero; Ribeiro, Andre; Dufour, Eric; Jacobs, Howard T

    2014-01-01

    The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number. PMID:24952591

  13. Extraction of single-copy nuclear DNA from forensic specimens with a variety of postmortem histories.

    PubMed

    Evison, M P; Smillie, D M; Chamberlain, A T

    1997-11-01

    Specimens of human bone, teeth and dried blood spots from 3 months to 91 years old, with a variety of postmortem histories, were used in a comparative study of recovery of single-copy nuclear DNA sequences from forensic material. Sequences of the amelogenin and HLA-DPB1 genes were chosen for their value in sexing and identification. Sequences of the mitochondrial non-coding region V were also amplified to compare the recovery of mitochondrial and single-copy nuclear DNA. A variation of the silica method for DNA extraction was refined for application to the forensic specimens in this sample. Single-copy nuclear DNA was amplified from 100% of recent postoperative bone specimens (n = 6), 80% of forensic teeth and bone specimens (n = 10), 78% of recently extracted teeth (n = 18), 78% of exhumed bone up to 91 years old (n = 37) and 69% of 15 year old bone specimens fixed in 10% formalin (n = 20). Amelogenin sexing was correct in 85% of cases (n = 74) in which the sex of the donor had been recorded. There was no correlation between the age of the specimen and the extent of DNA preservation. PMID:9397544

  14. 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

    2016-06-01

    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. PMID:27175474

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

    PubMed

    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

    2015-07-01

    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

  16. The copy number of rice CACTA DNA transposons carrying MIR820 does not correlate with MIR820 expression

    PubMed Central

    Nosaka, Misuzu; Ishiwata, Aiko; Shimizu-Sato, Sae; Ono, Akemi; Ishimoto, Kiyoe; Noda, Yusaku; Sato, Yutaka

    2013-01-01

    miR820 is a small RNA species (22 and 24 nucleotides), produced from transcripts originated from a region inside CACTA DNA transposons in rice. Because MIR820 is a transposon gene, its expression may depend on the transposon copy number. Here, we investigated the copy number of MIR820 and its expression levels in various cultivars and wild species of rice. We found no correlation between copy number and expression level, suggesting that MIR820 transcription is regulated not by the copy dosage but by the epigenetic state of each copy. PMID:23733074

  17. 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. PMID:26794963

  18. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus.

    PubMed

    Marx, Hans; Mecklenbräuker, Astrid; Gasser, Brigitte; Sauer, Michael; Mattanovich, Diethard

    2009-12-01

    The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants of P. pastoris are achievable only by screening of random events or by cloning of gene concatemers. Methods for rapid and reliable multicopy integration of the expression cassette are therefore desirable. Here we present such a method based on vector integration into the rDNA locus and post-transformational vector amplification by repeated selection on increased antibiotic concentrations. Data are presented for two exemplary products: human serum albumin, which is secreted into the supernatant, and human superoxide dismutase, which is accumulated in the cytoplasm of the cells. The striking picture evolving is that intracellular protein production is tightly correlated with gene copy number, while use of the secretory pathway introduces a high clonal variability and the correlation with gene copy number is valid only for low gene copy numbers. PMID:19799640

  19. Copy number of the transposon, Pokey, in rDNA is positively correlated with rDNA copy number in Daphnia obtuse [corrected].

    PubMed

    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

  20. 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

  1. 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. PMID:26103000

  2. 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

  3. 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. PMID:23390135

  4. DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials.

    PubMed

    Corbisier, Philippe; Pinheiro, Leonardo; Mazoua, Stéphane; Kortekaas, Anne-Marie; Chung, Pui Yan Jenny; Gerganova, Tsvetelina; Roebben, Gert; Emons, Hendrik; Emslie, Kerry

    2015-03-01

    The value assignment for properties of six certified reference materials (ERM-AD623a-f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per μL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (Bio-Rad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volume by optical microscopy, revealing an average droplet volume that is 8 % smaller than the droplet volume used as the defined parameter in the QuantaSoft software version 1.3.2.0 (Bio-Rad) to calculate the copy number concentration. This observation explains why copy number concentrations estimated with ddPCR and using an average droplet volume predefined in the QuantaSoft software were systematically lower than those measured by dPCR, creating a significant bias between the values obtained by these two techniques. The difference was not significant anymore when the measured droplet volume of 0.834 nL was used to estimate copy number concentrations. A new version of QuantaSoft software (version 1.6.6.0320), which has since been released with Bio-Rad's new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration. PMID:25600685

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

    PubMed

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

    2015-02-24

    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

  6. Accurate Measurement of the Relative Abundance of Different DNA Species in Complex DNA Mixtures

    PubMed Central

    Jeong, Sangkyun; Yu, Hyunjoo; Pfeifer, Karl

    2012-01-01

    A molecular tool that can compare the abundances of different DNA sequences is necessary for comparing intergenic or interspecific gene expression. We devised and verified such a tool using a quantitative competitive polymerase chain reaction approach. For this approach, we adapted a competitor array, an artificially made plasmid DNA in which all the competitor templates for the target DNAs are arranged with a defined ratio, and melting analysis for allele quantitation for accurate quantitation of the fractional ratios of competitively amplified DNAs. Assays on two sets of DNA mixtures with explicitly known compositional structures of the test sequences were performed. The resultant average relative errors of 0.059 and 0.021 emphasize the highly accurate nature of this method. Furthermore, the method's capability of obtaining biological data is demonstrated by the fact that it can illustrate the tissue-specific quantitative expression signatures of the three housekeeping genes G6pdx, Ubc, and Rps27 by using the forms of the relative abundances of their transcripts, and the differential preferences of Igf2 enhancers for each of the multiple Igf2 promoters for the transcription. PMID:22334570

  7. Genomic DNA Copy Number Aberrations, Histological Diagnosis, Oral Subsite and Aneuploidy in OPMDs/OSCCs

    PubMed Central

    Monticone, Massimiliano; Malacarne, Davide; Cirmena, Gabriella; Brown, David; Aiello, Cinzia; Maffei, Massimo; Marino, Roberto; Giaretti, Walter; Pentenero, Monica

    2015-01-01

    Oral potentially malignant disorders (OPMDs) characterized by the presence of dysplasia and DNA copy number aberrations (CNAs), may reflect chromosomal instability (CIN) and predispose to oral squamous cell carcinoma (OSCC). Early detection of OPMDs with such characteristics may play a crucial role in OSCC prevention. The aim of this study was to explore the relationship between CNAs, histological diagnosis, oral subsite and aneuploidy in OPMDs/OSCCs. Samples from OPMDs and OSCCs were processed by high-resolution DNA flow cytometry (hr DNA-FCM) to determine the relative nuclear DNA content. Additionally, CNAs were obtained for a subset of these samples by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. Our study shows that: i) aneuploidy, global genomic imbalance (measured as the total number of CNAs) and specific focal CNAs occur early in the development of oral cancer and become more frequent at later stages; ii) OPMDs limited to tongue (TNG) mucosa display a higher frequency of aneuploidy compared to OPMDs confined to buccal mucosa (BM) as measured by DNA-FCM; iii) TNG OPMDs/OSCCs show peculiar features of CIN compared to BM OPMDs/OSCCs given the preferential association with total broad and specific focal CNA gains. Follow-up studies are warranted to establish whether the presence of DNA aneuploidy and specific focal or broad CNAs may predict cancer development in non-dysplastic OPMDs. PMID:26540282

  8. 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

    2016-01-01

    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 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

  9. 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

  10. Increased mitochondrial DNA deletions and copy number in transfusion-dependent thalassemia

    PubMed Central

    Lal, Ashutosh; Gomez, Esteban; Calloway, Cassandra

    2016-01-01

    BACKGROUND Iron overload is the primary cause of morbidity in transfusion-dependent thalassemia. Increase in iron causes mitochondrial dysfunction under experimental conditions, but the occurrence and significance of mitochondrial damage is not understood in patients with thalassemia. METHODS Mitochondrial DNA (mtDNA) to nuclear DNA copy number (Mt/N) and frequency of the common 4977-bp mitochondrial deletion (ΔmtDNA4977) were quantified using a quantitative PCR assay on whole blood samples from 38 subjects with thalassemia who were receiving regular transfusions. RESULTS Compared with healthy controls, Mt/N and ΔmtDNA4977 frequency were elevated in thalassemia (P = 0.038 and P < 0.001, respectively). ΔmtDNA4977 was increased in the presence of either liver iron concentration > 15 mg/g dry-weight or splenectomy, with the highest levels observed in subjects who had both risk factors (P = 0.003). Myocardial iron (MRI T2* < 20 ms) was present in 0%, 22%, and 46% of subjects with ΔmtDNA4977 frequency < 20, 20–40, and > 40/1 × 107 mtDNA, respectively (P = 0.025). Subjects with Mt/N values below the group median had significantly lower Matsuda insulin sensitivity index (5.76 ± 0.53) compared with the high Mt/N group (9.11 ± 0.95, P = 0.008). CONCLUSION Individuals with transfusion-dependent thalassemia demonstrate age-related increase in mtDNA damage in leukocytes. These changes are markedly amplified by splenectomy and are associated with extrahepatic iron deposition. Elevated mtDNA damage in blood cells may predict the risk of iron-associated organ damage in thalassemia. PMID:27583305

  11. Focal DNA Copy Number Changes in Neuroblastoma Target MYCN Regulated Genes

    PubMed Central

    Mestdagh, Pieter; Menten, Björn; Lefever, Steve; Pattyn, Filip; De Brouwer, Sara; Sante, Tom; Schulte, Johannes Hubertus; Schramm, Alexander; Van Roy, Nadine; Van Maerken, Tom; Noguera, Rosa; Combaret, Valérie; Devalck, Christine; Westermann, Frank; Laureys, Geneviève; Eggert, Angelika; Vandesompele, Jo; De Preter, Katleen; Speleman, Frank

    2013-01-01

    Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17∼92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17∼92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17∼92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment. PMID:23308108

  12. Analysis of T-DNA/Host-Plant DNA Junction Sequences in Single-Copy Transgenic Barley Lines

    PubMed Central

    Bartlett, Joanne G.; Smedley, Mark A.; Harwood, Wendy A.

    2014-01-01

    Sequencing across the junction between an integrated transfer DNA (T-DNA) and a host plant genome provides two important pieces of information. The junctions themselves provide information regarding the proportion of T-DNA which has integrated into the host plant genome, whilst the transgene flanking sequences can be used to study the local genetic environment of the integrated transgene. In addition, this information is important in the safety assessment of GM crops and essential for GM traceability. In this study, a detailed analysis was carried out on the right-border T-DNA junction sequences of single-copy independent transgenic barley lines. T-DNA truncations at the right-border were found to be relatively common and affected 33.3% of the lines. In addition, 14.3% of lines had rearranged construct sequence after the right border break-point. An in depth analysis of the host-plant flanking sequences revealed that a significant proportion of the T-DNAs integrated into or close to known repetitive elements. However, this integration into repetitive DNA did not have a negative effect on transgene expression. PMID:24833334

  13. 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. PMID:25845980

  14. 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

  15. Array-based comparative genomic hybridization for the detection of DNA sequence copy number changes in Barrett's adenocarcinoma.

    PubMed

    Albrecht, Bettina; Hausmann, Michael; Zitzelsberger, Horst; Stein, Hubert; Siewert, Jörg Rüdiger; Hopt, Ulrich; Langer, Rupert; Höfler, Heinz; Werner, Martin; Walch, Axel

    2004-07-01

    Array-based comparative genomic hybridization (aCGH) allows the identification of DNA sequence copy number changes at high resolution by co-hybridizing differentially labelled test and control DNAs to a micro-array of genomic clones. The present study has analysed a series of 23 formalin-fixed, paraffin wax-embedded tissue samples of Barrett's adenocarcinoma (BCA, n = 18) and non-neoplastic squamous oesophageal (n = 2) and gastric cardia mucosa (n = 3) by aCGH. The micro-arrays used contained 287 genomic targets covering oncogenes, tumour suppressor genes, and DNA sequences localized within chromosomal regions previously reported to be altered in BCA. DNA sequence copy number changes for a panel of approximately 50 genes were identified, most of which have not been previously described in BCA. DNA sequence copy number gains (mean 41 +/- 25/BCA) were more frequent than DNA sequence copy number losses (mean 20 +/- 15/BCA). The highest frequencies for DNA sequence copy number gains were detected for SNRPN (61%); GNLY (44%); NME1 (44%); DDX15, ABCB1 (MDR), ATM, LAMA3, MYBL2, ZNF217, and TNFRSF6B (39% each); and MSH2, TERC, SERPINE1, AFM137XA11, IGF1R, and PTPN1 (33% each). DNA sequence copy number losses were identified for PDGFB (44%); D17S125 (39%); AKT3 (28%); and RASSFI, FHIT, CDKN2A (p16), and SAS (CDK4) (28% each). In all non-neoplastic tissue samples of squamous oesophageal and gastric cardia mucosa, the measured mean ratios were 1.00 (squamous oesophageal mucosa) or 1.01 (gastric mucosa), indicating that no DNA sequence copy number chances were present. For validation, the DNA sequence copy number changes of selected clones (SNRPN, CMYC, HER2, ZNF217) detected by aCGH were confirmed by fluorescence in situ hybridization (FISH). These data show the sensitivity of aCGH for the identification of DNA sequence copy number changes at high resolution in BCA. The newly identified genes may include so far unknown biomarkers in BCA and are therefore a starting point for

  16. Genomic profiling reveals extensive heterogeneity in somatic DNA copy number aberrations of canine hemangiosarcoma.

    PubMed

    Thomas, Rachael; Borst, Luke; Rotroff, Daniel; Motsinger-Reif, Alison; Lindblad-Toh, Kerstin; Modiano, Jaime F; Breen, Matthew

    2014-09-01

    Canine hemangiosarcoma is a highly aggressive vascular neoplasm associated with extensive clinical and anatomical heterogeneity and a grave prognosis. Comprehensive molecular characterization of hemangiosarcoma may identify novel therapeutic targets and advanced clinical management strategies, but there are no published reports of tumor-associated genome instability and disrupted gene dosage in this cancer. We performed genome-wide microarray-based somatic DNA copy number profiling of 75 primary intra-abdominal hemangiosarcomas from five popular dog breeds that are highly predisposed to this disease. The cohort exhibited limited global genomic instability, compared to other canine sarcomas studied to date, and DNA copy number aberrations (CNAs) were predominantly of low amplitude. Recurrent imbalances of several key cancer-associated genes were evident; however, the global penetrance of any single CNA was low and no distinct hallmark aberrations were evident. Copy number gains of dog chromosomes 13, 24, and 31, and loss of chromosome 16, were the most recurrent CNAs involving large chromosome regions, but their relative distribution within and between cases suggests they most likely represent passenger aberrations. CNAs involving CDKN2A, VEGFA, and the SKI oncogene were identified as potential driver aberrations of hemangiosarcoma development, highlighting potential targets for therapeutic modulation. CNA profiles were broadly conserved between the five breeds, although subregional variation was evident, including a near twofold lower incidence of VEGFA gain in Golden Retrievers versus other breeds (22 versus 40 %). These observations support prior transcriptional studies suggesting that the clinical heterogeneity of this cancer may reflect the existence of multiple, molecularly distinct subtypes of canine hemangiosarcoma. PMID:24599718

  17. 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. PMID:27622766

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

    PubMed

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

    2016-04-19

    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

  19. 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.

  20. 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

  1. Age-Related and Heteroplasmy-Related Variation in Human mtDNA Copy Number

    PubMed Central

    Li, Mingkun; Madea, Burkhard; Stoneking, Mark

    2016-01-01

    The mitochondrial (mt) genome is present in many copies in human cells, and intra-individual variation in mtDNA sequences is known as heteroplasmy. Recent studies found that heteroplasmies are highly tissue-specific, site-specific, and allele-specific, however the functional implications have not been explored. This study investigates variation in mtDNA copy numbers (mtCN) in 12 different tissues obtained at autopsy from 152 individuals (ranging in age from 3 days to 96 years). Three different methods to estimate mtCN were compared: shotgun sequencing (in 4 tissues), capture-enriched sequencing (in 12 tissues) and droplet digital PCR (ddPCR, in 2 tissues). The highest precision in mtCN estimation was achieved using shotgun sequencing data. However, capture-enrichment data provide reliable estimates of relative (albeit not absolute) mtCNs. Comparisons of mtCN from different tissues of the same individual revealed that mtCNs in different tissues are, with few exceptions, uncorrelated. Hence, each tissue of an individual seems to regulate mtCN in a tissue-related rather than an individual-dependent manner. Skeletal muscle (SM) samples showed an age-related decrease in mtCN that was especially pronounced in males, while there was an age-related increase in mtCN for liver (LIV) samples. MtCN in SM samples was significantly negatively correlated with both the total number of heteroplasmic sites and with minor allele frequency (MAF) at two heteroplasmic sites, 408 and 16327. Heteroplasmies at both sites are highly specific for SM, accumulate with aging and are part of functional elements that regulate mtDNA replication. These data support the hypothesis that selection acting on these heteroplasmic sites is reducing mtCN in SM of older individuals. PMID:26978189

  2. Accurate multiplex gene synthesis from programmable DNA microchips

    NASA Astrophysics Data System (ADS)

    Tian, Jingdong; Gong, Hui; Sheng, Nijing; Zhou, Xiaochuan; Gulari, Erdogan; Gao, Xiaolian; Church, George

    2004-12-01

    Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis. Here we describe a microchip-based technology for multiplex gene synthesis. Pools of thousands of `construction' oligonucleotides and tagged complementary `selection' oligonucleotides are synthesized on photo-programmable microfluidic chips, released, amplified and selected by hybridization to reduce synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple genes. This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general.

  3. 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. PMID:27316653

  4. 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. PMID:26670088

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Weitemier, Kevin; 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

  7. Invited review DNA copy number changes as diagnostic tools for lung cancer.

    PubMed

    Bowcock, Anne M

    2014-05-01

    Lung cancer usually presents as advanced stage disease and there is a need for early diagnosis so that appropriate treatments can be provided prior to tumour progression. Copy number variation is frequently detected in tumours and can contribute to tumour progression. This is because regions harbouring DNA imbalance can contain genes encoding critical proteins whose altered dosage contributes to the neoplastic process. Three copy number variations (CNVs) from chromosomes 3p26-p11.1 (loss), 3q26.2-29 (gain) and 6q25.3-24.3 (loss) have previously been described in individuals presenting with endobronchial squamous metaplasia. These CNVs were predictors of cancer diagnosed within 44 months with 97% accuracy. An evaluation of this CNV-based classifier with an independent set of 12 samples (10 men and 2 women), each with a carcinoma in situ or invasive carcinoma at the same site at follow-up demonstrated 92% prediction accuracy. The negative predictive value of this classifier was 89%. The gain at 3q26.2-q29 contributed the most to the classification, being present in virtually all lesions. This region harbours the PIK3CA gene and evaluation of the number of copies of this gene gave very similar results to those from array comparative genomic hybridisation. This type of test can be performed on sputum or bronchial brushings. Larger cohorts now need to be examined to confirm this finding and to possibly refine the regions of CNV. This type of approach paves the way for future molecular analyses to assist in selecting subjects with endobronchial squamous metaplastic or dysplastic lesions who might benefit from more aggressive therapeutic intervention or surveillance. PMID:24188925

  8. Is mitochondrial DNA copy number associated with clinical characteristics and prognosis in gastric cancer?

    PubMed

    Lee, Hyunsu; Lee, Jae-Ho; Kim, Dong-Choon; Hwang, IlSeon; Kang, Yu-Na; Gwon, Gi-Jeong; Choi, In-Jang; Kim, Shin

    2015-01-01

    Alterations in mitochondrial DNA (mtDNA) have been studied in various cancers. However, the clinical value of mtDNA copy number (mtCN) alterations in gastric cancer (GC) is poorly understood. In the present study, we investigated whether alterations in mtCNs might be associated with clinicopathological parameters in GC cases. mtCN was measured in 109 patients with GC by quantitative real-time PCR. Then, correlations with clinicopathological characteristics were analyzed. mtCN was elevated in 64.2% of GC tissues compared with paired, adjacent, non-cancerous tissue. However, the observed alterations in mtCN were not associated with any clinicopathological characteristics, including age, gender, TN stage, Lauren classification, lymph node metastasis, and depth of invasion. Moreover, Kaplan-Meier survival curves revealed that mtCN was not significantly associated with the survival of GC patients. In this study, we demonstrated that mtCN was not a significant marker for predicting clinical characteristics or prognosis in GC. PMID:25640396

  9. Estimation of correlations between copy-number variants in non-coding DNA.

    PubMed

    Stamoulis, Catherine

    2011-01-01

    Allelic DNA aberrations across our genome have been associated with normal human genetic heterogeneity as well as with a number of diseases and disorders. When copy-number variations (CNVs) occur in gene-coding regions, known relationships between genes may help us understand correlations between CNVs. However, a large number of these aberrations occur in non-coding, extragenic regions and their correlations may be characterized only quantitatively, e.g., probabilistically, but not functionally. Using a signal processing approach to CNV detection, we identified distributed CNVs in short, non-coding regions across chromosomes and investigated their potential correlations. We estimated predominantly local correlations between CNVs within the same chromosome, and a small number of apparently random long-distance correlations. PMID:22255599

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

    PubMed Central

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

    2016-01-01

    Abstract 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. PMID:27082579

  11. Prognostic impact of Epstein-Barr virus (EBV)-DNA copy number at diagnosis in chronic lymphocytic leukemia.

    PubMed

    Liang, Jin-Hua; Gao, Rui; Xia, Yi; Gale, Robert Peter; Chen, Rui-Ze; Yang, Yu-Qiong; Wang, Li; Qu, Xiao-Yan; Qiu, Hai-Rong; Cao, Lei; Hong, Min; Wang, Rong; Wang, Yan; Fan, Lei; Chen, Yao-Yu; Hu, Zhi-Bin; Li, Jian-Yong; Xu, Wei

    2016-01-12

    Epstein-Barr virus (EBV)-DNA is detected in the blood of some persons with chronic lymphocytic leukemia (CLL) at diagnosis. Whether this is important in the development or progression of CLL is controversial. We interrogated associations between blood EBV-DNA copy number and biological and clinical variables in 243 new-diagnosed consecutive subjects with CLL. Quantification of EBV-DNA copies was done by real-time quantitative PCR (RQ-PCR). All subjects had serological evidence of prior EBV-infection. However, only 24 subjects (10%) had a EBV-DNA-positive test at diagnosis. EBV-DNA-positive subjects at diagnosis had lower hemoglobin concentrations and platelet levels, higher thymidine kinase-1 and serum ferritin levels, un-mutated IGHV genes and a greater risk of Richter transformation compared with EBV-DNA-negative subjects. Percent CD20-, CD148- and ZAP70-positive cells and mean fluorescence intensity (MFI) of each cluster designation were also increased in EBV-DNA-positive subjects at diagnosis. EBV-DNA test positivity was associated with a briefer time-to-treatment interval (HR 1.85; [95% confidence interval, 1.13, 3.03]; P=0.014) and worse survival (HR 2.77; [1.18, 6.49]; P=0.019). Reduction in EBV copies was significantly associated with therapy-response. A positive blood EBV-DNA test at diagnosis and sequential testing of EBV copies during therapy were significantly associated with biological and clinical variables, time-to-treatment, therapy-response and survival. If validated these data may be added to CLL prognostic scoring systems. PMID:26539641

  12. Prognostic impact of Epstein-Barr virus (EBV)-DNA copy number at diagnosis in chronic lymphocytic leukemia

    PubMed Central

    Xia, Yi; Gale, Robert Peter; Chen, Rui-Ze; Yang, Yu-Qiong; Wang, Li; Qu, Xiao-Yan; Qiu, Hai-Rong; Cao, Lei; Hong, Min; Wang, Rong; Wang, Yan; Fan, Lei; Chen, Yao-Yu; Hu, Zhi-Bin; Li, Jian-Yong; Xu, Wei

    2016-01-01

    Epstein-Barr virus (EBV)-DNA is detected in the blood of some persons with chronic lymphocytic leukemia (CLL) at diagnosis. Whether this is important in the development or progression of CLL is controversial. We interrogated associations between blood EBV-DNA copy number and biological and clinical variables in 243 new-diagnosed consecutive subjects with CLL. Quantification of EBV-DNA copies was done by real-time quantitative PCR (RQ-PCR). All subjects had serological evidence of prior EBV-infection. However, only 24 subjects (10%) had a EBV-DNA-positive test at diagnosis. EBV-DNA-positive subjects at diagnosis had lower hemoglobin concentrations and platelet levels, higher thymidine kinase-1 and serum ferritin levels, un-mutated IGHV genes and a greater risk of Richter transformation compared with EBV-DNA-negative subjects. Percent CD20-, CD148- and ZAP70-positive cells and mean fluorescence intensity (MFI) of each cluster designation were also increased in EBV-DNA-positive subjects at diagnosis. EBV-DNA test positivity was associated with a briefer time-to-treatment interval (HR 1.85; [95% confidence interval, 1.13, 3.03]; P=0.014) and worse survival (HR 2.77; [1.18, 6.49]; P=0.019). Reduction in EBV copies was significantly associated with therapy-response. A positive blood EBV-DNA test at diagnosis and sequential testing of EBV copies during therapy were significantly associated with biological and clinical variables, time-to-treatment, therapy-response and survival. If validated these data may be added to CLL prognostic scoring systems. PMID:26539641

  13. 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

  14. 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. PMID:26195569

  15. LoComatioN: a software tool for the analysis of low copy number DNA profiles.

    PubMed

    Gill, Peter; Kirkham, Amanda; Curran, James

    2007-03-01

    Previously, the interpretation of low copy number (LCN) STR profiles has been carried out using the biological or 'consensus' method-essentially, alleles are not reported, unless duplicated in separate PCR analyses [P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forens. Sci. Int. 112 (2000) 17-40]. The method is now widely used throughout Europe. Although a probabilistic theory was simultaneously introduced, its time-consuming complexity meant that it could not be easily applied in practice. The 'consensus' method is not as efficient as the probabilistic approach, as the former wastes information in DNA profiles. However, the theory was subsequently extended to allow for DNA mixtures and population substructure in a programmed solution by Curran et al. [J.M. Curran, P. Gill, M.R. Bill, Interpretation of repeat measurement DNA evidence allowing for multiple contributors and population substructure, Forens. Sci. Int. 148 (2005) 47-53]. In this paper, we describe an expert interpretation system (LoComatioN) which removes this computational burden, and enables application of the full probabilistic method. This is the first expert system that can be used to rapidly evaluate numerous alternative explanations in a likelihood ratio approach, greatly facilitating court evaluation of the evidence. This would not be possible with manual calculation. Finally, the Gill et al. and Curran et al. papers both rely on the ability of the user to specify two quantities: the probability of allelic drop-out, and the probability of allelic contamination ("drop-in"). In this paper, we offer some guidelines on how these quantities may be specified. PMID:16759831

  16. 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

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

    PubMed

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

    1994-01-01

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

  18. Jagged1 DNA Copy Number Variation Is Associated with Poor Outcome in Liver Cancer.

    PubMed

    Kawaguchi, Kazunori; Honda, Masao; Yamashita, Taro; Okada, Hikari; Shirasaki, Takayoshi; Nishikawa, Masashi; Nio, Kouki; Arai, Kuniaki; Sakai, Yoshio; Yamashita, Tatsuya; Mizukoshi, Eishiro; Kaneko, Shuichi

    2016-08-01

    Notch signaling abnormalities are reported to be involved in the acceleration of malignancy in solid tumors and stem cell formation or regeneration in various organs. We analyzed specific genes for DNA copy number variations in liver cancer cells and investigated whether these factors relate to clinical outcome. Chromosome 20p, which includes the ligand for Notch pathways, Jagged1, was found to be amplified in several types of hepatoma cells, and its mRNA was up-regulated according to α-fetoprotein gene expression levels. Notch inhibition using Jagged1 shRNA and γ-secretase inhibitors produced significant suppression of cell growth in α-fetoprotein-producing cells with suppression of downstream genes. Using in vivo hepatoma models, the administration of γ-secretase inhibitors resulted in reduced tumor sizes and effective Notch inhibition with widespread apoptosis and necrosis of viable tumor cells. The γ-secretase inhibitors suppressed cell growth of the epithelial cell adhesion molecule-positive fraction in hepatoma cells, indicating that Notch inhibitors could suppress the stem cell features of liver cancer cells. Even in clinical liver cancer samples, the expression of α-fetoprotein and Jagged1 showed significant correlation, and amplification of the copy number of Jagged1 was associated with Jagged1 mRNA expression and poor survival after liver cancer surgical resection. In conclusion, amplification of Jagged1 contributed to mRNA expression that activates the Jagged1-Notch signaling pathway in liver cancer and led to poor outcome. PMID:27315779

  19. 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

  20. A Comparison of Assays for Accurate Copy Number Measurement of the Low-Affinity Fc Gamma Receptor Genes FCGR3A and FCGR3B

    PubMed Central

    Haridan, Umi Shakina; Mokhtar, Umairah; Machado, Lee R.; Abdul Aziz, Abu Thalhah; Shueb, Rafidah Hanim; Zaid, Masliza; Sim, Benedict; Mustafa, Mahiran; Nik Yusof, Nik Khairudin; Lee, Christopher K. C.; Abu Bakar, Suhaili; AbuBakar, Sazaly; Hollox, Edward J.; Boon Peng, Hoh

    2015-01-01

    The FCGR3 locus encoding the low affinity activating receptor FcγRIII, plays a vital role in immunity triggered by cellular effector and regulatory functions. Copy number of the genes FCGR3A and FCGR3B has previously been reported to affect susceptibility to several autoimmune diseases and chronic inflammatory conditions. However, such genetic association studies often yield inconsistent results; hence require assays that are robust with low error rate. We investigated the accuracy and efficiency in estimating FCGR3 CNV by comparing Sequenom MassARRAY and paralogue ratio test-restriction enzyme digest variant ratio (PRT-REDVR). In addition, since many genetic association studies of FCGR3B CNV were carried out using real-time quantitative PCR, we have also included the evaluation of that method’s performance in estimating the multi-allelic CNV of FCGR3B. The qPCR assay exhibited a considerably broader distribution of signal intensity, potentially introducing error in estimation of copy number and higher false positive rates. Both Sequenom and PRT-REDVR showed lesser systematic bias, but Sequenom skewed towards copy number normal (CN = 2). The discrepancy between Sequenom and PRT-REDVR might be attributed either to batch effects noise in individual measurements. Our study suggests that PRT-REDVR is more robust and accurate in genotyping the CNV of FCGR3, but highlights the needs of multiple independent assays for extensive validation when performing a genetic association study with multi-allelic CNVs. PMID:25594501

  1. Rapid and accurate identification of microorganisms contaminating cosmetic products based on DNA sequence homology.

    PubMed

    Fujita, Y; Shibayama, H; Suzuki, Y; Karita, S; Takamatsu, S

    2005-12-01

    The aim of this study was to develop rapid and accurate procedures to identify microorganisms contaminating cosmetic products, based on the identity of the nucleotide sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA coding DNA (rDNA). Five types of microorganisms were isolated from the inner portion of lotion bottle caps, skin care lotions, and cleansing gels. The rDNA ITS region of microorganisms was amplified through the use of colony-direct PCR or ordinal PCR using DNA extracts as templates. The nucleotide sequences of the amplified DNA were determined and subjected to homology search of a publicly available DNA database. Thereby, we obtained DNA sequences possessing high similarity with the query sequences from the databases of all the five organisms analyzed. The traditional identification procedure requires expert skills, and a time period of approximately 1 month to identify the microorganisms. On the contrary, 3-7 days were sufficient to complete all the procedures employed in the current method, including isolation and cultivation of organisms, DNA sequencing, and the database homology search. Moreover, it was possible to develop the skills necessary to perform the molecular techniques required for the identification procedures within 1 week. Consequently, the current method is useful for rapid and accurate identification of microorganisms, contaminating cosmetics. PMID:18492168

  2. 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

  3. 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

  4. Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples.

    PubMed Central

    Isola, J.; DeVries, S.; Chu, L.; Ghazvini, S.; Waldman, F.

    1994-01-01

    Analysis of previously unknown genetic aberrations in solid tumors has become possible through the use of comparative genomic hybridization (CGH), which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. CGH allows detection of DNA sequence copy number changes (deletions, gains, and amplifications) on a genome-wide scale in a single hybridization. We describe here an improved CGH technique, which enables reliable detection of copy number changes in archival formalin-fixed paraffin-embedded tumor samples. The technique includes a modified DNA extraction protocol, which produces high molecular weight DNA which is necessary for high quality CGH. The DNA extraction includes a 3-day digestion with proteinase K, which remarkably improves the yield of high molecular weight DNA. Labeling of the test DNA with a directly fluorescein-conjugated nucleotide (instead of biotin labeling) improved significantly the quality of hybridization. Using the paraffin-block technique, we could analyze 70 to 90% of paraffin blocks, including very old samples as well as samples taken at autopsy. CGH from paraffin blocks was highly concordant (95%) with analyses done from matched freshly frozen tumor samples (n = 5 sample pairs; kappa coefficient = 0.83). The method described here has wide applicability in tumor pathology, allowing large retrospective prognostic studies of genetic aberrations as well as studies on genetic pathogenesis of solid tumors, inasmuch as premalignant lesions and primary and metastatic tumors can be analyzed by using archival paraffin-embedded samples. Images Figure 1 Figure 3 PMID:7992835

  5. COPS: a sensitive and accurate tool for detecting somatic Copy Number Alterations using short-read sequence data from paired samples.

    PubMed

    Krishnan, Neeraja M; Gaur, Prakhar; Chaudhary, Rakshit; Rao, Arjun A; Panda, Binay

    2012-01-01

    Copy Number Alterations (CNAs) such as deletions and duplications; compose a larger percentage of genetic variations than single nucleotide polymorphisms or other structural variations in cancer genomes that undergo major chromosomal re-arrangements. It is, therefore, imperative to identify cancer-specific somatic copy number alterations (SCNAs), with respect to matched normal tissue, in order to understand their association with the disease. We have devised an accurate, sensitive, and easy-to-use tool, COPS, COpy number using Paired Samples, for detecting SCNAs. We rigorously tested the performance of COPS using short sequence simulated reads at various sizes and coverage of SCNAs, read depths, read lengths and also with real tumor:normal paired samples. We found COPS to perform better in comparison to other known SCNA detection tools for all evaluated parameters, namely, sensitivity (detection of true positives), specificity (detection of false positives) and size accuracy. COPS performed well for sequencing reads of all lengths when used with most upstream read alignment tools. Additionally, by incorporating a downstream boundary segmentation detection tool, the accuracy of SCNA boundaries was further improved. Here, we report an accurate, sensitive and easy to use tool in detecting cancer-specific SCNAs using short-read sequence data. In addition to cancer, COPS can be used for any disease as long as sequence reads from both disease and normal samples from the same individual are available. An added boundary segmentation detection module makes COPS detected SCNA boundaries more specific for the samples studied. COPS is available at ftp://115.119.160.213 with username "cops" and password "cops". PMID:23110103

  6. High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR.

    PubMed

    Miotke, Laura; Lau, Billy T; Rumma, Rowza T; Ji, Hanlee P

    2014-03-01

    In this study, we present a highly customizable method for quantifying copy number and point mutations utilizing a single-color, droplet digital PCR platform. Droplet digital polymerase chain reaction (ddPCR) is rapidly replacing real-time quantitative PCR (qRT-PCR) as an efficient method of independent DNA quantification. Compared to quantative PCR, ddPCR eliminates the needs for traditional standards; instead, it measures target and reference DNA within the same well. The applications for ddPCR are widespread including targeted quantitation of genetic aberrations, which is commonly achieved with a two-color fluorescent oligonucleotide probe (TaqMan) design. However, the overall cost and need for optimization can be greatly reduced with an alternative method of distinguishing between target and reference products using the nonspecific DNA binding properties of EvaGreen (EG) dye. By manipulating the length of the target and reference amplicons, we can distinguish between their fluorescent signals and quantify each independently. We demonstrate the effectiveness of this method by examining copy number in the proto-oncogene FLT3 and the common V600E point mutation in BRAF. Using a series of well-characterized control samples and cancer cell lines, we confirmed the accuracy of our method in quantifying mutation percentage and integer value copy number changes. As another novel feature, our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample, as well as copy number changes from cancers even in the context of significant dilution with normal DNA. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay. PMID:24483992

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. 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.

  9. 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

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

    PubMed

    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

  11. 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

  12. Gene copy number variations in the leukocyte genome of hepatocellular carcinoma patients with integrated hepatitis B virus DNA

    PubMed Central

    Xu, Guixia; Cheng, Kai; Cao, Guangwen; Wu, Mengchao; Cheng, Shuqun; Liu, Shanrong

    2016-01-01

    Integration of hepatitis B virus (HBV) DNA into the human liver cell genome is believed to promote HBV-related carcinogenesis. This study aimed to quantify the integration of HBV DNA into the leukocyte genome in hepatocellular carcinoma (HCC) patients in order to identify potential biomarkers for HBV-related diseases. Whole-genome comparative genomic hybridization (CGH) chip array analyses were performed to screen gene copy number variations (CNV) in the leukocyte genome, and the results were confirmed by quantitative polymerase chain reaction (qPCR). The commonly detected regions included chromosome arms 19p, 5q, 1q and 15p, where 200 copy number gain events and 270 copy number loss events were noted. In particular, gains were observed in 5q35.3 (OR4F3) and 19p13.3 (OR4F17) in 90% of the samples. Successful homologous recombination of OR4F3 and the HBV P gene was demonstrated, and the amplification at 5q35.3 is potentially associated with the integration of HBV P gene into natural killer cells isolated from peripheral blood mononuclear cells (PBMCs). Receiver operating characteristic (ROC) curve analysis indicated that the combination of OR4F3 and OR4F17 a novel potential biomarker of HBV-related diseases. PMID:26769853

  13. Multiple copies of Shope virus DNA are present in cells of benign and malignant non-virus-producing neoplasms.

    PubMed Central

    Stevens, J G; Wettstein, F O

    1979-01-01

    In an initial efforts to characterize the virological basis of neoplasia in the Shope papilloma-carcinoma system, the extent to which the viral genome is present in non-virus-producing benign and malignant tumors in domestic rabbits was established. Employing nick-translated radioactive viral DNA purified from productively infected papillomas on cotton tail rabbits as a probe, it was found that (i) papillomas, primary carcinomas, and metastatic carcinomas contain 10 to about 100 copies of the viral genome per diploid cell equivalent of DNA and (ii) viral DNA is present in detectable amounts in essentially all neoplastic cells. These results are consistent with the suggestion that continued presence of the viral genome is necessary for induction and maintenance of malignant as well as benign neoplasms. Images PMID:225548

  14. 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

  15. Effect of Maternal Age on the Ratio of Cleavage and Mitochondrial DNA Copy Number in Early Developmental Stage Bovine Embryos

    PubMed Central

    TAKEO, Shun; GOTO, Hiroya; KUWAYAMA, Takehito; MONJI, Yasunori; IWATA, Hisataka

    2012-01-01

    Abstract Age-associated deterioration in both the quality and quantity of mitochondria occurs in older women. The main aim of this study was to examine the effect of age on mitochondrial DNA copy number (mtDNA number) in early developmental stage bovine embryos as well as the dynamics of mtDNA number during early embryo development. Real-time PCR was used to determine mtDNA number. In vitro-produced embryos 48 h after insemination derived from Japanese black cows, ranging in age from 25 to 209 months were categorized based on their cleavage status. There was an overall negative relationship between the age of the cow and cleavage status, to the extent that the ratio of embryos cleaved over the 4-cell stage was greater in younger cows. The mtDNA number did not differ among the cleaved status of embryos. In the next experiment, oocytes collected from each donor cow were divided into 2 groups containing 10 oocytes each, in order to compare the mtDNA number of mature oocytes and early developmental stage embryos within individuals. Upon comparing the mtDNA number between oocytes at the M2 stage and early developmental stage 48 h post insemination, mtDNA number was found to decrease in most cows, but was found to increase in some cows. In conclusion, age affects the cleaving ability of oocytes, and very old cows (> 180 months) tend to have lower mtDNA numbers in their oocytes. The change in mtDNA number during early development varied among individual cows, although overall, it showed a tendency to decrease. PMID:23269452

  16. A nested case-control study of leukocyte mitochondrial DNA copy number and renal cell carcinoma in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

    PubMed

    Hofmann, Jonathan N; Hosgood, H Dean; Liu, Chin-San; Chow, Wong-Ho; Shuch, Brian; Cheng, Wen-Ling; Lin, Ta-Tsung; Moore, Lee E; Lan, Qing; Rothman, Nathaniel; Purdue, Mark P

    2014-05-01

    Mitochondrial DNA (mtDNA) is vulnerable to mutations, and the number of copies of mtDNA per cell may increase to compensate for DNA damage. Case-control studies have reported associations between altered mtDNA copy number and risk of renal cell carcinoma (RCC); however, this association has not been investigated prospectively. We conducted a nested case-control study (252 cases and 504 controls) of RCC risk in relation to pre-diagnostic leukocyte mtDNA copy number in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. mtDNA copy number was measured in triplicate using a fluorescence-based quantitative PCR assay; samples from 22 cases and 36 controls could not be assayed, leaving 230 cases and 468 controls for analysis. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression. High mtDNA copy number was associated with an increased risk of RCC, both overall (highest quartile versus lowest: OR = 2.0, 95% CI = 1.2-3.2; P trend = 0.002) and among cases diagnosed ≥6 years after blood collection (OR = 2.6, 95% CI = 1.4-5.0; P trend = 0.003). These findings did not differ significantly by sex, body mass index, history of hypertension or smoking status (P interaction ≥ 0.3). Results of this study suggest that high pre-diagnostic leukocyte mtDNA copy number, a suspected marker of oxidative DNA damage and mitochondrial dysfunction, is associated with increased future RCC risk. PMID:24398668

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

    PubMed

    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

  18. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. 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

  1. 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

  2. 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

  3. De-methylation of displacement loop of mitochondrial DNA is associated with increased mitochondrial copy number and nicotinamide adenine dinucleotide subunit 2 expression in colorectal cancer.

    PubMed

    Gao, Jinhang; Wen, Shilei; Zhou, Hongying; Feng, Shi

    2015-11-01

    DNA methylation occurs in the displacement loop (D-loop) region of mammals; however, D-loop regions of certain tumor tissue types were found to be de‑methylated. Whether hypomethylation of the D‑loop region is involved in the regulation of the mitochondrial DNA (mtDNA) copy number and nicotinamide adenine dinucleotide subunit 2 (ND‑2) expressions in colorectal cancer has remained elusive. In the present study, the association between methylation status of the D‑loop region, mtDNA copy number and ND‑2 expression was investigated in 65 colorectal cancer specimens and their corresponding non‑cancerous tissues. In addition, a de‑methylation experiment was performed on the Caco‑2 colorectal cancer cell line by using 5‑aza-2'-deoxycytidine (5‑Aza). The methylation rate of the D‑loop region in all 65 colorectal cancer tissues was markedly reduced when compared with that of their corresponding non‑cancerous tissues (13.8 vs. 81.5%; P<0.05). Furthermore, the methylation rate of the D‑loop region in colorectal cancer tissues was markedly decreased in clinicopathological stages III and IV compared with that in clinicopathological stages I and II (7.1 and 0% vs. 25 and 16%; P<0.05). In addition, the mean relative mtDNA copy number and ND‑2 expression in colorectal cancer tissues were increased compared with those in the corresponding non‑cancerous tissues. De‑methylation of the D‑loop region was associated with an elevated mtDNA copy number and an increased ND‑2 expression. Furthermore, the mtDNA copy number and ND‑2 expression in Caco‑2 cells were significantly increased after 5‑Aza treatment. In conclusion, de‑methylation of the D‑loop region is likely to be involved in the regulation of the mtDNA copy number and ND-2 expression. PMID:26323487

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26500031

  5. 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. PMID:26688582

  6. Impacts of low temperature preservation on mitochondrial DNA copy number in oocytes of the hard coral Echinopora sp.

    PubMed

    Tsai, Sujune; Thongpooe, Preeyanuch; Kuo, Fu-Wen; Lin, Chiahsin

    2016-07-01

    Given the current threats to coral reefs worldwide, there is an urgent need to develop protocols for the cryopreservation of reef-building corals. However, chilling may alter coral mitochondrial distribution and membrane potential, resulting in reduced ATP production. The aim of this study was to investigate the impacts of chilling on mitochondrial DNA copy number (CN) in oocytes of the hard coral Echinopora sp. Oocytes were exposed to 0.5 M, 1 M or 2 M methanol at 5, 0 or -5 °C for 2, 4, 8 and 16 h. When oocytes were chilled with no cryoprotectant (CPT) or 1 M methanol at 5 or 0 °C, the mtDNA CNs initially increased at hour 2 of incubation, although it decreased significantly over the 16 h of incubation in chilled oocytes at -5 °C. The mtDNA CN increased and picked in 0.5 M methanol at 5 °C and 0 °C at hour 8 of incubation in chilled oocytes indicating that the high mtDNA CN of these oocytes is probably responsible for withstanding high chilling sensitivity. We currently propose that 0.5 M methanol is the optimal CPT for oocytes of Echinopora sp., and potentially other reef corals. PMID:25901989

  7. 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

  8. 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. PMID:26068926

  9. The role of mitochondrial DNA copy number, variants, and haplotypes in farm animal developmental outcome.

    PubMed

    Tsai, Tesha; St John, Justin C

    2016-07-01

    The vast majority of cellular energy is generated through the process of oxidative phosphorylation, which takes place in the electron transport chain in the mitochondria. The electron transport chain is encoded by 2 genomes, the chromosomal and the mitochondrial genomes. Mitochondrial DNA is associated with a number of traits, which include tolerance to heat, growth and physical performance, meat and milk quality, and fertility. Mitochondrial genomes can be clustered into groups known as mtDNA haplotypes. Mitochondrial DNA haplotypes are a potential genetic source for manipulating phenotypes in farm animals. The use of assisted reproductive technologies, such as nuclear transfer, allows favorable chromosomal genetic traits to be mixed and matched with sought after mtDNA haplotype traits. As a result super breeds can be generated. PMID:27345311

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

    PubMed

    Kervio, Eric; Sosson, Marilyne; Richert, Clemens

    2016-07-01

    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

  12. 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

  13. 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. PMID:23133665

  14. Phylogeny of the Genus Chrysanthemum L.: Evidence from Single-Copy Nuclear Gene and Chloroplast DNA Sequences

    PubMed Central

    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. PMID:23133665

  15. 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). PMID:25768890

  16. Human subtelomeric copy number gains suggest a DNA replication mechanism for formation: beyond breakage – fusion - bridge for telomere stabilization

    PubMed Central

    Yatsenko, Svetlana A.; Hixson, Patricia; Roney, Erin K.; Scott, Daryl A.; Schaaf, Christian P.; Ng, Yu-tze; Palmer, Robbin; Fisher, Richard B.; Patel, Ankita; Cheung, Sau Wai; Lupski, James R.

    2012-01-01

    Constitutional deletions of distal 9q34 encompassing the EHMT1 (euchromatic histone methyltransferase 1) gene, or loss-of-function point mutations in EHMT1, are associated with the 9q34.3 microdeletion, also known as Kleefstra syndrome [MIM#610253]. We now report further evidence for genomic instability of the subtelomeric 9q34.3 region as evidenced by copy number gains of this genomic interval that include duplications, triplications, derivative chromosomes and complex rearrangements. Comparisons between the observed shared clinical features and molecular analyses in 20 subjects suggest that increased dosage of EHMT1 may be responsible for the neurodevelopmental impairment, speech delay, and autism spectrum disorders revealing the dosage sensitivity of yet another chromatin remodeling protein in human disease. Five patients had 9q34 genomic abnormalities resulting in complex deletion-duplication or duplication-triplication rearrangements; such complex triplications were also observed in six other subtelomeric intervals. Based on the specific structure of these complex genomic rearrangements (CGR) a DNA replication mechanism is proposed confirming recent findings in C elegans telomere healing. The end-replication challenges of subtelomeric genomic intervals may make them particularly prone to rearrangements generated by errors in DNA replication. PMID:22890305

  17. Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae.

    PubMed Central

    Zelenaya-Troitskaya, O; Newman, S M; Okamoto, K; Perlman, P S; Butow, R A

    1998-01-01

    Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf2p in mtDNA transactions. We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium. The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control. Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein.These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination. PMID:9581629

  18. 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

  19. 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

  20. 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

  1. Kinetic Analysis of Human PrimPol DNA Polymerase Activity Reveals a Generally Error-Prone Enzyme Capable of Accurately Bypassing 7,8-Dihydro-8-oxo-2′-deoxyguanosine

    PubMed Central

    2015-01-01

    Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg2+ as a cofactor, PrimPol binds primer-template DNA with low affinity Kd,DNA values (∼200–1200 nM). DNA binding is enhanced 34-fold by Mn2+ (Kd,DNA = 27 nM). The pol activity of PrimPol is increased 400–1000-fold by Mn2+ compared to Mg2+ based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ∼100–2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ∼1.7-fold more accurate when Mg2+ is the cofactor compared to Mn2+. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn2+) to 6-fold (Mg2+) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ∼25% efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ∼2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ∼0.04%. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn2+, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription. PMID:25255211

  2. Implementing Prenatal Diagnosis Based on Cell-Free Fetal DNA: Accurate Identification of Factors Affecting Fetal DNA Yield

    PubMed Central

    Barrett, Angela N.; Zimmermann, Bernhard G.; Wang, Darrell; Holloway, Andrew; Chitty, Lyn S.

    2011-01-01

    Objective Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used. Methods Microfluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples. Results Total cell-free DNA quantity increased significantly with time in samples stored in K3EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4°C did not prevent these changes. Conclusion When samples can be processed within eight hours of blood draw, K3EDTA tubes can be used. Prolonged transfer times in K3EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests. PMID:21998643

  3. Postoperative hepatitis B virus reactivation in hepatitis B virus-related hepatocellular carcinoma patients with hepatitis B virus DNA levels <500 copies/mL

    PubMed Central

    Xie, Zhi-Bo; Wang, Xiao-Bo; Fu, De-Liang; Zhong, Jian-Hong; Yang, Xia-Wei; Li, Le-Qun

    2016-01-01

    Background Patients with hepatocellular carcinoma have the risk of postoperative hepatitis B virus (HBV) reactivation (PHR). Antiviral therapy was given to patients with detectable HBV DNA levels but not to patients with undetectable HBV DNA levels. Methods In this retrospective study, 258 patients were enrolled (HBV DNA levels <500 copies/mL group, n=159, and HBV DNA levels >500 copies/mL group, n=99). Results A total of 50 patients (19.4%) had PHR. The following significant factors related to PHR were found: without antiviral therapy (hazard ratio [HR] =0.17, 95% confidence interval [CI] 0.031–0.911), hepatitis B e antigen positivity (HR =5.20, 95% CI 1.931–14.007), hepatitis B core antigen S1 positivity (HR =2.54, 95% CI 1.116–5.762), preoperative HBV DNA levels ≥500 copies/mL (HR =1.28, 95% CI 1.085–2.884), hepatic inflow occlusion (HR =3.60, 95% CI 1.402–9.277), moderate liver cirrhosis or more (HR =2.26, 95% CI 1.001–5.121), and blood transfusion (HR =2.89, 95% CI 0.836–10.041). Recurrence-free survival time was significantly shorter in patients with PHR (23.06±2.46 months) than in patients without PHR (29.30±1.27 months). Conclusion Antiviral therapy could efficiently decrease the incidence of PHR. Patients with HBV DNA levels <500 copies/mL still have the risk of PHR. PHR remained as a prognostic risk factor for hepatocellular carcinoma recurrence and recurrence-free survival. PMID:27524913

  4. QuantiSNP: an Objective Bayes Hidden-Markov Model to detect and accurately map copy number variation using SNP genotyping data.

    PubMed

    Colella, Stefano; Yau, Christopher; Taylor, Jennifer M; Mirza, Ghazala; Butler, Helen; Clouston, Penny; Bassett, Anne S; Seller, Anneke; Holmes, Christopher C; Ragoussis, Jiannis

    2007-01-01

    Array-based technologies have been used to detect chromosomal copy number changes (aneuploidies) in the human genome. Recent studies identified numerous copy number variants (CNV) and some are common polymorphisms that may contribute to disease susceptibility. We developed, and experimentally validated, a novel computational framework (QuantiSNP) for detecting regions of copy number variation from BeadArray SNP genotyping data using an Objective Bayes Hidden-Markov Model (OB-HMM). Objective Bayes measures are used to set certain hyperparameters in the priors using a novel re-sampling framework to calibrate the model to a fixed Type I (false positive) error rate. Other parameters are set via maximum marginal likelihood to prior training data of known structure. QuantiSNP provides probabilistic quantification of state classifications and significantly improves the accuracy of segmental aneuploidy identification and mapping, relative to existing analytical tools (Beadstudio, Illumina), as demonstrated by validation of breakpoint boundaries. QuantiSNP identified both novel and validated CNVs. QuantiSNP was developed using BeadArray SNP data but it can be adapted to other platforms and we believe that the OB-HMM framework has widespread applicability in genomic research. In conclusion, QuantiSNP is a novel algorithm for high-resolution CNV/aneuploidy detection with application to clinical genetics, cancer and disease association studies. PMID:17341461

  5. Methods for accurate quantification of LTR-retrotransposon copy number using short-read sequence data: a case study in Sorghum.

    PubMed

    Ramachandran, Dhanushya; Hawkins, Jennifer S

    2016-10-01

    Transposable elements (TEs) are ubiquitous in eukaryotic genomes and their mobility impacts genome structure and function in myriad ways. Because of their abundance, activity, and repetitive nature, the characterization and analysis of TEs remain challenging, particularly from short-read sequencing projects. To overcome this difficulty, we have developed a method that estimates TE copy number from short-read sequences. To test the accuracy of our method, we first performed an in silico analysis of the reference Sorghum bicolor genome, using both reference-based and de novo approaches. The resulting TE copy number estimates were strikingly similar to the annotated numbers. We then tested our method on real short-read data by estimating TE copy numbers in several accessions of S. bicolor and its close relative S. propinquum. Both methods effectively identify and rank similar TE families from highest to lowest abundance. We found that de novo characterization was effective at capturing qualitative variation, but underestimated the abundance of some TE families, specifically families of more ancient origin. Also, interspecific reference-based mapping of S. propinquum reads to the S. bicolor database failed to fully describe TE content in S. propinquum, indicative of recent TE activity leading to changes in the respective repetitive landscapes over very short evolutionary timescales. We conclude that reference-based analyses are best suited for within-species comparisons, while de novo approaches are more reliable for evolutionarily distant comparisons. PMID:27295958

  6. 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. PMID:27299603

  7. DISPLAR: an accurate method for predicting DNA-binding sites on protein surfaces

    PubMed Central

    Tjong, Harianto; Zhou, Huan-Xiang

    2007-01-01

    Structural and physical properties of DNA provide important constraints on the binding sites formed on surfaces of DNA-targeting proteins. Characteristics of such binding sites may form the basis for predicting DNA-binding sites from the structures of proteins alone. Such an approach has been successfully developed for predicting protein–protein interface. Here this approach is adapted for predicting DNA-binding sites. We used a representative set of 264 protein–DNA complexes from the Protein Data Bank to analyze characteristics and to train and test a neural network predictor of DNA-binding sites. The input to the predictor consisted of PSI-blast sequence profiles and solvent accessibilities of each surface residue and 14 of its closest neighboring residues. Predicted DNA-contacting residues cover 60% of actual DNA-contacting residues and have an accuracy of 76%. This method significantly outperforms previous attempts of DNA-binding site predictions. Its application to the prion protein yielded a DNA-binding site that is consistent with recent NMR chemical shift perturbation data, suggesting that it can complement experimental techniques in characterizing protein–DNA interfaces. PMID:17284455

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

    PubMed Central

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

    2013-01-01

    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. PMID:23468533

  9. Generation of Backbone-Free, Low Transgene Copy Plants by Launching T-DNA from the Agrobacterium Chromosome1[W][OA

    PubMed Central

    Oltmanns, Heiko; Frame, Bronwyn; Lee, Lan-Ying; Johnson, Susan; Li, Bo; Wang, Kan; Gelvin, Stanton B.

    2010-01-01

    In both applied and basic research, Agrobacterium-mediated transformation is commonly used to introduce genes into plants. We investigated the effect of three Agrobacterium tumefaciens strains and five transferred (T)-DNA origins of replication on transformation frequency, transgene copy number, and the frequency of integration of non-T-DNA portions of the T-DNA-containing vector (backbone) into the genome of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). Launching T-DNA from the picA locus of the Agrobacterium chromosome increases the frequency of single transgene integration events and almost eliminates the presence of vector backbone sequences in transgenic plants. Along with novel Agrobacterium strains we have developed, our findings are useful for improving the quality of T-DNA integration events. PMID:20023148

  10. Asexual Populations of the Human Malaria Parasite, Plasmodium falciparum, Use a Two-Step Genomic Strategy to Acquire Accurate, Beneficial DNA Amplifications

    PubMed Central

    Ahyong, Vida; Patrapuvich, Rapatbhorn; White, John; Gujjar, Ramesh; Phillips, Margaret A.; DeRisi, Joseph; Rathod, Pradipsinh K.

    2013-01-01

    Malaria drug resistance contributes to up to a million annual deaths. Judicious deployment of new antimalarials and vaccines could benefit from an understanding of early molecular events that promote the evolution of parasites. Continuous in vitro challenge of Plasmodium falciparum parasites with a novel dihydroorotate dehydrogenase (DHODH) inhibitor reproducibly selected for resistant parasites. Genome-wide analysis of independently-derived resistant clones revealed a two-step strategy to evolutionary success. Some haploid blood-stage parasites first survive antimalarial pressure through fortuitous DNA duplications that always included the DHODH gene. Independently-selected parasites had different sized amplification units but they were always flanked by distant A/T tracks. Higher level amplification and resistance was attained using a second, more efficient and more accurate, mechanism for head-to-tail expansion of the founder unit. This second homology-based process could faithfully tune DNA copy numbers in either direction, always retaining the unique DNA amplification sequence from the original A/T-mediated duplication for that parasite line. Pseudo-polyploidy at relevant genomic loci sets the stage for gaining additional mutations at the locus of interest. Overall, we reveal a population-based genomic strategy for mutagenesis that operates in human stages of P. falciparum to efficiently yield resistance-causing genetic changes at the correct locus in a successful parasite. Importantly, these founding events arise with precision; no other new amplifications are seen in the resistant haploid blood stage parasite. This minimizes the need for meiotic genetic cleansing that can only occur in sexual stage development of the parasite in mosquitoes. PMID:23717205

  11. 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.

  12. 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

  13. Regionalized pathology correlates with augmentation of mtDNA copy numbers in a patient with myoclonic epilepsy with ragged-red fibers (MERRF-syndrome).

    PubMed

    Brinckmann, Anja; Weiss, Claudia; Wilbert, Friederike; von Moers, Arpad; Zwirner, Angelika; Stoltenburg-Didinger, Gisela; Wilichowski, Ekkehard; Schuelke, Markus

    2010-01-01

    Human patients with myoclonic epilepsy with ragged-red fibers (MERRF) suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G). In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV), transcription factors (NRF1 and TFAM), and VDAC1 (Porin) as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89-100%. However, mtDNA copy numbers were increased 3-7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs). Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that "futile" stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome. PMID:20976001

  14. Array CGH identifies distinct DNA copy number profiles of oncogenes and tumor suppressor genes in chromosomal- and microsatellite-unstable sporadic colorectal carcinomas.

    PubMed

    Lassmann, Silke; Weis, Roland; Makowiec, Frank; Roth, Jasmine; Danciu, Mihai; Hopt, Ulrich; Werner, Martin

    2007-03-01

    DNA copy number changes represent molecular fingerprints of solid tumors and are as such relevant for better understanding of tumor development and progression. In this study, we applied genome-wide array comparative genomic hybridization (aCGH) to identify gene-specific DNA copy number changes in chromosomal (CIN)- and microsatellite (MIN)-unstable sporadic colorectal cancers (sCRC). Genomic DNA was extracted from microdissected, matching normal colorectal epithelium and invasive tumor cells of formalin-fixed and paraffin-embedded tissues of 22 cases with colorectal cancer (CIN = 11, MIN = 11). DNA copy number changes were determined by aCGH for 287 target sequences in tumor cell DNAs, using pooled normal DNAs as reference. aCGH data of tumor cell DNAs was confirmed by fluorescence in situ hybridization (FISH) for three genes on serial tissues as those used for aCGH. aCGH revealed DNA copy number changes previously described by metaphase CGH (gains 7, 8q, 13q, and 20q; losses 8p, 15q, 18q, and 17p). However, chromosomal regions 20q, 13q, 7, and 17p were preferentially altered in CIN-type tumors and included DNA amplifications of eight genes on chromosome 20q (TOP1, AIB1, MYBL2, CAS, PTPN1, STK15, ZNF217, and CYP24), two genes on chromosome 13q (BRCA2 and D13S25), and three genes on chromosome 7 (IL6, CYLN2, and MET) as well as DNA deletions of two genes on chromosome 17p (HIC1 and LLGL1). Finally, additional CIN-tumor-associated DNA amplifications were identified for EXT1 (8q24.11) and MYC (8q24.12) as well as DNA deletions for MAP2K5 (15q23) and LAMA3 (18q11.2). In contrast, distinct MIN-tumor-associated DNA amplifications were detected for E2F5 (8p22-q21.3), GARP (11q13.5-q14), ATM (11q22.3), KAL (Xp22.3), and XIST (Xq13.2) as well as DNA deletions for RAF1 (3p25), DCC (18q21.3), and KEN (21q tel). aCGH revealed distinct DNA copy number changes of oncogenes and tumor suppressor genes in CIN- and MIN-type sporadic colorectal carcinomas. The identified candidate

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

    PubMed Central

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

    2011-01-01

    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. PMID:21808029

  16. A MATLAB-based tool for accurate detection of perfect overlapping and nested inverted repeats in DNA sequences

    PubMed Central

    Sreeskandarajan, Sutharzan; Flowers, Michelle M.; Karro, John E.; Liang, Chun

    2014-01-01

    Summary: Palindromic sequences, or inverted repeats (IRs), in DNA sequences involve important biological processes such as DNA–protein binding, DNA replication and DNA transposition. Development of bioinformatics tools that are capable of accurately detecting perfect IRs can enable genome-wide studies of IR patterns in both prokaryotes and eukaryotes. Different from conventional string-comparison approaches, we propose a novel algorithm that uses a cumulative score system based on a prime number representation of nucleotide bases. We then implemented this algorithm as a MATLAB-based program for perfect IR detection. In comparison with other existing tools, our program demonstrates a high accuracy in detecting nested and overlapping IRs. Availability and implementation: The source code is freely available on (http://bioinfolab.miamioh.edu/bioinfolab/palindrome.php) Contact: liangc@miamioh.edu or karroje@miamioh.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24215021

  17. 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

  18. Mitochondrial DNA copy number in peripheral blood leukocytes and the risk of clear cell renal cell carcinoma.

    PubMed

    Melkonian, Stephanie C; Wang, Xin; Gu, Jian; Matin, Surena F; Tannir, Nizar M; Wood, Christopher G; Wu, Xifeng

    2015-02-01

    Variation of mitochondrial DNA copy number (mtDNAcn) in peripheral blood leukocytes has been associated with the risk of various cancers, including renal cell carcinoma (RCC). We assessed the association between mtDNAcn and clear cell RCC (ccRCC) risk in 608 cases and 629 controls frequency-matched on age and gender. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for age, gender, body mass index, smoking status, history of hypertension, total energy intake and physical activity. Our results suggest an association between low mtDNAcn and ccRCC risk (OR = 1.28, 95% CI: 0.97-1.68, P = 0.09). Lower mtDNAcn was associated with increased ccRCC risk in younger individuals (age <60, OR = 1.68, 95% CI: 1.13-2.49, P = 0.01), women (OR = 1.66, 95% CI: 1.03-2.73, P = 0.04), individuals without history of hypertension (OR = 1.62, 95% CI: 1.09-2.41, P = 0.02) and individuals with low physical activity levels (OR = 1.55, 95% CI: 1.02-2.37, P = 0.05). We observed significant and marginally significant interactions between both age and history of hypertension and mtDNAcn in elevating ccRCC risk (P for interaction = 0.04 and 0.07, respectively). Additionally, low mtDNAcn was associated with ccRCC risk in younger individuals with low levels of physical activity [ORs and 95% CI for medium and low physical activity levels, respectively, 2.31 (1.18-4.52) and 2.09 (1.17-3.75), P interaction = 0.04]. To our knowledge, this is the first report to investigate the role of mtDNAcn in the ccRCC subtype and the first to suggest that this association may be modified by risk factors including age, gender, history of hypertension and physical activity. PMID:25524925

  19. Mitochondrial DNA copy number in peripheral blood leukocytes and the risk of clear cell renal cell carcinoma

    PubMed Central

    Melkonian, Stephanie C.; Wang, Xin; Gu, Jian; Matin, Surena F.; Tannir, Nizar M.; Wood, Christopher G.; Wu, Xifeng

    2015-01-01

    Variation of mitochondrial DNA copy number (mtDNAcn) in peripheral blood leukocytes has been associated with the risk of various cancers, including renal cell carcinoma (RCC). We assessed the association between mtDNAcn and clear cell RCC (ccRCC) risk in 608 cases and 629 controls frequency-matched on age and gender. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for age, gender, body mass index, smoking status, history of hypertension, total energy intake and physical activity. Our results suggest an association between low mtDNAcn and ccRCC risk (OR = 1.28, 95% CI: 0.97–1.68, P = 0.09). Lower mtDNAcn was associated with increased ccRCC risk in younger individuals (age <60, OR = 1.68, 95% CI: 1.13–2.49, P = 0.01), women (OR = 1.66, 95% CI: 1.03–2.73, P = 0.04), individuals without history of hypertension (OR = 1.62, 95% CI: 1.09–2.41, P = 0.02) and individuals with low physical activity levels (OR = 1.55, 95% CI: 1.02–2.37, P = 0.05). We observed significant and marginally significant interactions between both age and history of hypertension and mtDNAcn in elevating ccRCC risk (P for interaction = 0.04 and 0.07, respectively). Additionally, low mtDNAcn was associated with ccRCC risk in younger individuals with low levels of physical activity [ORs and 95% CI for medium and low physical activity levels, respectively, 2.31 (1.18–4.52) and 2.09 (1.17–3.75), P interaction = 0.04]. To our knowledge, this is the first report to investigate the role of mtDNAcn in the ccRCC subtype and the first to suggest that this association may be modified by risk factors including age, gender, history of hypertension and physical activity. PMID:25524925

  20. Inhalable particulate matter and mitochondrial DNA copy number in highly exposed individuals in Beijing, China: a repeated-measure study

    PubMed Central

    2013-01-01

    Background Mitochondria are both a sensitive target and a primary source of oxidative stress, a key pathway of air particulate matter (PM)-associated diseases. Mitochondrial DNA copy number (MtDNAcn) is a marker of mitochondrial damage and malfunctioning. We evaluated whether ambient PM exposure affects MtDNAcn in a highly-exposed population in Beijing, China. Methods The Beijing Truck Driver Air Pollution Study was conducted shortly before the 2008 Beijing Olympic Games (June 15-July 27, 2008) and included 60 truck drivers and 60 office workers. Personal PM2.5 and elemental carbon (EC, a tracer of traffic particles) were measured during work hours using portable monitors. Post-work blood samples were obtained on two different days. Ambient PM10 was averaged from 27 monitoring stations in Beijing. Blood MtDNAcn was determined by real-time PCR and examined in association with particle levels using mixed-effect models. Results In all participants combined, MtDNAcn was negatively associated with personal EC level measured during work hours (β=−0.059, 95% CI: -0.011; -0.0006, p=0.03); and 5-day (β=−0.017, 95% CI: -0.029;-0.005, p=0.01) and 8-day average ambient PM10 (β=−0.008, 95% CI: -0.043; -0.008, p=0.004) after adjusting for possible confounding factors, including study groups. MtDNAcn was also negatively associated among office workers with EC (β=−0.012, 95% CI: -0.022;-0.002, p=0.02) and 8-day average ambient PM10 (β=−0.030, 95% CI: -0.051;-0.008, p=0.007). Conclusions We observed decreased blood MtDNAcn in association with increased exposure to EC during work hours and recent ambient PM10 exposure. Our results suggest that MtDNAcn may be influenced by particle exposures. Further studies are required to determine the roles of MtDNAcn in the etiology of particle-related diseases. PMID:23628000

  1. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. Plant DNA Barcodes Can Accurately Estimate Species Richness in Poorly Known Floras

    PubMed Central

    Costion, Craig; Ford, Andrew; Cross, Hugh; Crayn, Darren; Harrington, Mark; Lowe, Andrew

    2011-01-01

    Background Widespread uptake of DNA barcoding technology for vascular plants has been slow due to the relatively poor resolution of species discrimination (∼70%) and low sequencing and amplification success of one of the two official barcoding loci, matK. Studies to date have mostly focused on finding a solution to these intrinsic limitations of the markers, rather than posing questions that can maximize the utility of DNA barcodes for plants with the current technology. Methodology/Principal Findings Here we test the ability of plant DNA barcodes using the two official barcoding loci, rbcLa and matK, plus an alternative barcoding locus, trnH-psbA, to estimate the species diversity of trees in a tropical rainforest plot. Species discrimination accuracy was similar to findings from previous studies but species richness estimation accuracy proved higher, up to 89%. All combinations which included the trnH-psbA locus performed better at both species discrimination and richness estimation than matK, which showed little enhanced species discriminatory power when concatenated with rbcLa. The utility of the trnH-psbA locus is limited however, by the occurrence of intraspecific variation observed in some angiosperm families to occur as an inversion that obscures the monophyly of species. Conclusions/Significance We demonstrate for the first time, using a case study, the potential of plant DNA barcodes for the rapid estimation of species richness in taxonomically poorly known areas or cryptic populations revealing a powerful new tool for rapid biodiversity assessment. The combination of the rbcLa and trnH-psbA loci performed better for this purpose than any two-locus combination that included matK. We show that although DNA barcodes fail to discriminate all species of plants, new perspectives and methods on biodiversity value and quantification may overshadow some of these shortcomings by applying barcode data in new ways. PMID:22096501

  4. Estimation of copy number using SYBR Green: confounding by AT-rich DNA and by variation in amplicon length.

    PubMed

    Colborn, James M; Byrd, Brian D; Koita, Ousmane A; Krogstad, Donald J

    2008-12-01

    Although SYBR Green is used to estimate copy number, its fluorescence varies with amplicon length and adenine/thymine (AT) content. As a result, threshold cycle (Ct) values obtained using real-time polymerase chain reaction (PCR) are lower for longer amplicons (P<0.001) and amplicons with greater AT content (P<0.001). In contrast, neither amplicon length nor AT content affects the Ct with TaqMan probes or LUX-labeled primers. Because SYBR Green yields lower Cts with AT-rich templates and longer templates, it overestimates copy number for those templates. Therefore, sequence-specific methods such as TaqMan probes or LUX-labeled primers should be considered when using real-time PCR to estimate copy number if the amplicons generated are AT-rich or vary in length. PMID:19052298

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

    PubMed

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

    2016-01-01

    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. PMID:26966900

  6. Single mammalian cells compensate for differences in cellular volume and DNA copy number through independent global transcriptional mechanisms

    PubMed Central

    Padovan-Merhar, Olivia; Nair, Gautham P.; Biaesch, Andrew; Mayer, Andreas; Scarfone, Steven; Foley, Shawn W.; Wu, Angela R.; Churchman, L. Stirling; Singh, Abhyudai; Raj, Arjun

    2015-01-01

    Summary Individual mammalian cells exhibit large variability in cellular volume even with the same absolute DNA content and so must compensate for differences in DNA concentration in order to maintain constant concentration of gene expression products. Using single molecule counting and computational image analysis, we show that transcript abundance correlates with cellular volume at the single cell level due to increased global transcription in larger cells. Cell fusion experiments establish that increased cellular content itself can directly increase transcription. Quantitative analysis shows that this mechanism measures the ratio of cellular volume to DNA content, mostly likely through sequestration of a transcriptional factor to DNA. Analysis of transcriptional bursts reveals a separate mechanism for gene dosage compensation after DNA replication that enables proper transcriptional output during early and late S-phase. Our results provide a framework for quantitatively understanding the relationships between DNA content, cell size and gene expression variability in single cells. PMID:25866248

  7. Four DNA methylation biomarkers in biliary brush samples accurately identify the presence of cholangiocarcinoma

    PubMed Central

    Andresen, Kim; Boberg, Kirsten Muri; Vedeld, Hege Marie; Honne, Hilde; Jebsen, Peter; Hektoen, Merete; Wadsworth, Christopher A.; Clausen, Ole Petter; Lundin, Knut E.A.; Paulsen, Vemund; Foss, Aksel; Mathisen, Øystein; Aabakken, Lars; Schrumpf, Erik; Lothe, Ragnhild A.

    2015-01-01

    Early detection of the highly aggressive malignancy cholangiocarcinoma (CCA) remains a challenge but has the potential to render the tumor curable by surgical removal. This study evaluates a biomarker panel for the diagnosis of CCA by DNA methylation analyses of biliary brush samples. The methylation status of 13 candidate genes (CDO1, CNRIP1, DCLK1, FBN1, INA, MAL, SEPT9, SFRP1, SNCA, SPG20, TMEFF2, VIM, and ZSCAN18) was investigated in 93 tissue samples (39 CCAs and 54 nonmalignant controls) using quantitative methylation‐specific polymerase chain reaction. The 13 genes were further analyzed in a test series of biliary brush samples (15 CCAs and 20 nonmalignant primary sclerosing cholangitis controls), and the methylation status of the four best performing markers was validated (34 CCAs and 34 primary sclerosing cholangitis controls). Receiver operating characteristic curve analyses were used to evaluate the performance of individual biomarkers and the combination of biomarkers. The 13 candidate genes displayed a methylation frequency of 26%‐82% in tissue samples. The four best‐performing genes (CDO1, CNRIP1, SEPT9, and VIM) displayed individual methylation frequencies of 45%‐77% in biliary brushes from CCA patients. Across the test and validation biliary brush series, this four‐gene biomarker panel achieved a sensitivity of 85% and a specificity of 98%, with an area under the receiver operating characteristic curve of 0.944. Conclusion: We report a straightforward biomarker assay with high sensitivity and specificity for CCA, outperforming standard brush cytology, and suggest that the biomarker panel, potentially in combination with cytological evaluation, may improve CCA detection, particularly among primary sclerosing cholangitis patients. (Hepatology 2015;61:1651–1659) PMID:25644509

  8. 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)

  9. Subspecies of DNA polymerase alpha from calf thymus with different fidelity in copying synthetic template-primers.

    PubMed Central

    Brosius, S; Grosse, F; Krauss, G

    1983-01-01

    Three different subspecies of DNA polymerase alpha from calf thymus sedimenting at 9 S, 7 S and 5.7 S have been investigated with respect to their accuracy of in vitro DNA synthesis on poly (dA) (dT)16 and poly d(AT) as template-primers. Our results indicate that the structure of DNA polymerase alpha has a strong influence on the accuracy of DNA synthesis. The 9 S enzyme shows a misincorporation frequency of about 1:100 000. An error rate of 1:15 000 is measured for the 7 S species. The 5.7 S enzyme for which an error rate of 1:3 000 is determined, has to be considered as error prone. Lowering the rate of DNA synthesis leads to a decrease in fidelity. The single stranded DNA binding protein from E.coli increases the accuracy of the 5.7 S and the 7 S enzyme by a factor of two. Mn2+ decreases the fidelity of all three subspecies in a concentration dependent manner. PMID:6866763

  10. Detection of low copy human papilloma virus DNA and mRNA in routine paraffin sections of cervix by non-isotopic in situ hybridisation.

    PubMed Central

    Burns, J; Graham, A K; Frank, C; Fleming, K A; Evans, M F; McGee, J O

    1987-01-01

    In analysing human papilloma virus (HPV) infection of the cervix in formalin fixed paraffin sections by non-isotopic in situ hybridisation two main problems were found: detachment of sections from the glass during hybridisation and probe detection; inadequate sensitivity and inability to assess sensitivity of the in situ procedure. The first problem was investigated by assessing the efficiency of various tissue adhesives individually and in combination. The second problem was addressed by optimising conditions for DNA unmasking, hybridisation, and biotinylated probe detection. Sensitivity of the final in situ procedure developed was assessed by using the detection of pHY2.1 repeats as a built-in control. Extrapolation of data showed that less than 10 copies of HPV DNA can be visualised by these procedures. HPV nucleic acid, mainly in the form of DNA, was detected not only in koilocytic nuclei but also in suprabasal cells in condylomas and CIN lesions. HPV mRNA was also visualised in the cytoplasm (and probably also nuclei) of the same cell types. These non-isotopic in situ procedures give results comparable to those obtained with radiolabelled probes, but they are less time consuming and provide better morphological resolution. Images Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 Fig 8 PMID:2821078

  11. 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. PMID:27617230

  12. 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

  13. DNA copy number aberrations and disruption of the p16Ink4a/Rb pathway in radiation-induced and spontaneous rat mammary carcinomas.

    PubMed

    Iizuka, Daisuke; Imaoka, Tatsuhiko; Takabatake, Takashi; Nishimura, Mayumi; Kakinuma, Shizuko; Nishimura, Yukiko; Shimada, Yoshiya

    2010-08-01

    Chromosomal amplifications and deletions are thought to be important events in spontaneous and radiation-induced carcinogenesis. To clarify how ionizing radiation induces mammary carcinogenesis, we characterized genomic copy number aberrations for gamma-ray-induced rat mammary carcinomas using microarray-based comparative genomic hybridization. We examined 14 carcinomas induced by gamma radiation (2 Gy) and found 26 aberrations, including trisomies of chromosomes 4 and 10 for three and one carcinomas, respectively, an amplification of the chromosomal region 1q12 in two carcinomas, and deletions of the chromosomal regions 3q35q36, 5q32 and 7q11 in two, two and four carcinomas, respectively. These aberrations were not observed in seven spontaneous mammary carcinomas. The expression of p16Ink4a and p19Arf, which are located in the chromosomal region 5q32, was always up-regulated except for a carcinoma with a homozygous deletion of region 5q32. The up-regulation was not accounted for by gene mutations or promoter hypomethylation. However, the amounts of Rb and its mRNA were down-regulated in these carcinomas, indicating a disruption of the p16Ink4a/Rb pathway. This is the first report of array CGH analysis for radiation-induced mammary tumors, which reveals that they show distinct DNA copy number aberration patterns that are different from those of spontaneous tumors and those reported previously for chemically induced tumors. PMID:20681787

  14. Identification of Multiple DNA Copy Number Alterations Including Frequent 8p11.22 Amplification in Conjunctival Squamous Cell Carcinoma

    PubMed Central

    Asnaghi, Laura; Alkatan, Hind; Mahale, Alka; Othman, Maha; Alwadani, Saeed; Al-Hussain, Hailah; Jastaneiah, Sabah; Yu, Wayne; Maktabi, Azza; Edward, Deepak P.; Eberhart, Charles G.

    2014-01-01

    Purpose. Little is known about the molecular alterations that drive formation and growth of conjunctival squamous cell carcinoma (cSCC). We therefore sought to identify genetic changes that could be used as diagnostic markers or therapeutic targets. Methods. The DNA extracted from 10 snap-frozen cSCC tumor specimens and 2 in situ carcinomas was analyzed using array-based comparative genomic hybridization (aCGH), and further examined with NanoString and quantitative PCR. Results. The number of regions of DNA loss ranged from 1 to 23 per tumor, whereas gains and amplifications ranged from 1 to 15 per tumor. Most large regions of chromosomal gain and loss were confirmed by NanoString karyotype analysis. The commonest alteration was amplification of 8p11.22 in 9 tumors (75%), and quantitative PCR analysis revealed 100-fold or greater overexpression of ADAM3A mRNA from 8p11.22 locus. In addition, recurring losses were observed at 14q13.2 and 22q11.23, both lost in 5 (42%) of the 12 tumors, and at 12p13.31, lost in 4 (33%) of the 12 samples. Of the eight loci associated with the DNA damage repair syndrome xeroderma pigmentosum, three showed loss of at least one allele in our aCGH analysis, including XPA (9q22.33, one tumor), XPE/DDB2 (11p11.2, one tumor) and XPG/ERCC5 (13q33.1, three tumors). Conclusions. Conjunctival SCC contains a range of chromosomal alterations potentially important in tumor formation and growth. Amplification of 8p11.22 and overexpression of ADAM3A suggests a potential role for this protease. Our findings also suggest that defects in DNA repair loci are important in sporadic cSCC. PMID:25491297

  15. A DNA target-enrichment approach to detect mutations, copy number changes and immunoglobulin translocations in multiple myeloma.

    PubMed

    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

  16. 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 Central

    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. PMID:26247464

  17. Accurate quantification of episomal HIV-1 two-long terminal repeat circles by use of optimized DNA isolation and droplet digital PCR.

    PubMed

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2015-02-01

    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

  18. 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

  19. Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA.

    PubMed

    Stenger, D C; Revington, G N; Stevenson, M C; Bisaro, D M

    1991-09-15

    Agrobacterium-mediated inoculation of Nicotiana benthamiana plants with Ti plasmids containing tandem genome repeats derived from different strains of the gemini-virus beet curly top virus (BCTV) resulted in the production of unit-length recombinant progeny genomes in systemically infected plants. When two putative plus-strand origins of replication were present in constructs used as inocula, a replicational escape mechanism was favored that resulted in progeny genomes of a single predominant genotype. The genotype was dependent upon the arrangement of repeated parental genomes in the inocula. Sequencing across the junction between parental BCTV strains in the recombinant progeny allowed mapping of the plus-strand origin of replication to a 20-base-pair sequence within the conserved hairpin found in all geminivirus genomes. In contrast, when inocula contained tandemly repeated BCTV genome sequences but only a single conserved hairpin, a number of different progeny genotypes were simultaneously replicated in infected plants, a result expected if unit-length viral genomes were generated by random intramolecular recombination events. These results and other considerations indicate that geminivirus DNA replication occurs by a rolling-circle mechanism. PMID:1896448

  20. Analysis of copy number variation using quantitative interspecies competitive PCR.

    PubMed

    Williams, Nigel M; Williams, Hywel; Majounie, Elisa; Norton, Nadine; Glaser, Beate; Morris, Huw R; Owen, Michael J; O'Donovan, Michael C

    2008-10-01

    Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls. PMID:18697816

  1. 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

  2. Tension on dsDNA bound to ssDNA-RecA filaments may play an important role in driving efficient and accurate homology recognition and strand exchange

    PubMed Central

    Vlassakis, Julea; Feinstein, Efraim; Yang, Darren; Tilloy, Antoine; Weiller, Dominic; Kates-Harbeck, Julian; Coljee, Vincent; Prentiss, Mara

    2013-01-01

    It is well known that during homology recognition and strand exchange the double stranded DNA (dsDNA) in DNA/RecA filaments is highly extended, but the functional role of the extension has been unclear. We present an analytical model that calculates the distribution of tension in the extended dsDNA during strand exchange. The model suggests that the binding of additional dsDNA base pairs to the DNA/RecA filament alters the tension in dsDNA that was already bound to the filament, resulting in a non-linear increase in the mechanical energy as a function of the number of bound base pairs. This collective mechanical response may promote homology stringency and underlie unexplained experimental results.

  3. 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

  4. Development of a Novel Reference Plasmid for Accurate Quantification of Genetically Modified Kefeng6 Rice DNA in Food and Feed Samples

    PubMed Central

    Zhang, Xiujie; Wan, Yusong

    2013-01-01

    Reference plasmids are an essential tool for the quantification of genetically modified (GM) events. Quantitative real-time PCR (qPCR) is the most commonly used method to characterize and quantify reference plasmids. However, the precision of this method is often limited by calibration curves, and qPCR data can be affected by matrix differences between the standards and samples. Here, we describe a digital PCR (dPCR) approach that can be used to accurately measure the novel reference plasmid pKefeng6 and quantify the unauthorized variety of GM rice Kefeng6, eliminating the issues associated with matrix effects in calibration curves. The pKefeng6 plasmid was used as a calibrant for the quantification of Kefeng6 rice by determining the copy numbers of event- (77 bp) and taxon-specific (68 bp) fragments, their ratios, and their concentrations. The plasmid was diluted to five different concentrations. The third sample (S3) was optimized for the quantification range of dPCR according to previous reports. The ratio between the two fragments was 1.005, which closely approximated the value certified by sequencing, and the concentration was found to be 792 copies/μL. This method was precise, with an RSD of ~3%. These findings demonstrate the advantages of using the dPCR method to characterize reference materials. PMID:24324952

  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. Building accurate sequence-to-affinity models from high-throughput in vitro protein-DNA binding data using FeatureREDUCE.

    PubMed

    Riley, Todd R; Lazarovici, Allan; Mann, Richard S; Bussemaker, Harmen J

    2015-01-01

    Transcription factors are crucial regulators of gene expression. Accurate quantitative definition of their intrinsic DNA binding preferences is critical to understanding their biological function. High-throughput in vitro technology has recently been used to deeply probe the DNA binding specificity of hundreds of eukaryotic transcription factors, yet algorithms for analyzing such data have not yet fully matured. Here, we present a general framework (FeatureREDUCE) for building sequence-to-affinity models based on a biophysically interpretable and extensible model of protein-DNA interaction that can account for dependencies between nucleotides within the binding interface or multiple modes of binding. When training on protein binding microarray (PBM) data, we use robust regression and modeling of technology-specific biases to infer specificity models of unprecedented accuracy and precision. We provide quantitative validation of our results by comparing to gold-standard data when available. PMID:26701911

  7. 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. PMID:26243938

  8. MBRidge: an accurate and cost-effective method for profiling DNA methylome at single-base resolution

    PubMed Central

    Cai, Wanshi; Mao, Fengbiao; Teng, Huajing; Cai, Tao; Zhao, Fangqing; Wu, Jinyu; Sun, Zhong Sheng

    2015-01-01

    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. PMID:26078362

  9. 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. PMID:26078362

  10. Single copy insertion of transgenes in C. elegans

    PubMed Central

    Frøkjær-Jensen, Christian; Davis, M. Wayne; Hopkins, Christopher E.; Newman, Blake; Thummel, Jason M.; Olesen, Søren-Peter; Grunnet, Morten; Jorgensen, Erik M.

    2009-01-01

    Currently transgenes in C. elegans are generated by injecting DNA into the germline. The DNA assembles into a semi-stable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method called MosSCI (Mos1-mediated Single Copy Insertion) that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double strand break in non-coding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single copy insertions can be obtained in less than two weeks by injecting approximately twenty animals. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines. PMID:18953339

  11. Accurate and easy-to-use assessment of contiguous DNA methylation sites based on proportion competitive quantitative-PCR and lateral flow nucleic acid biosensor.

    PubMed

    Xu, Wentao; Cheng, Nan; Huang, Kunlun; Lin, Yuehe; Wang, Chenguang; Xu, Yuancong; Zhu, Longjiao; Du, Dan; Luo, Yunbo

    2016-06-15

    Many types of diagnostic technologies have been reported for DNA methylation, but they require a standard curve for quantification or only show moderate accuracy. Moreover, most technologies have difficulty providing information on the level of methylation at specific contiguous multi-sites, not to mention easy-to-use detection to eliminate labor-intensive procedures. We have addressed these limitations and report here a cascade strategy that combines proportion competitive quantitative PCR (PCQ-PCR) and lateral flow nucleic acid biosensor (LFNAB), resulting in accurate and easy-to-use assessment. The P16 gene with specific multi-methylated sites, a well-studied tumor suppressor gene, was used as the target DNA sequence model. First, PCQ-PCR provided amplification products with an accurate proportion of multi-methylated sites following the principle of proportionality, and double-labeled duplex DNA was synthesized. Then, a LFNAB strategy was further employed for amplified signal detection via immune affinity recognition, and the exact level of site-specific methylation could be determined by the relative intensity of the test line and internal reference line. This combination resulted in all recoveries being greater than 94%, which are pretty satisfactory recoveries in DNA methylation assessment. Moreover, the developed cascades show significantly high usability as a simple, sensitive, and low-cost tool. Therefore, as a universal platform for sensing systems for the detection of contiguous multi-sites of DNA methylation without external standards and expensive instrumentation, this PCQ-PCR-LFNAB cascade method shows great promise for the point-of-care diagnosis of cancer risk and therapeutics. PMID:26914373

  12. 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. PMID:25515665

  13. BASIC: A New Biopart Assembly Standard for Idempotent Cloning Provides Accurate, Single-Tier DNA Assembly for Synthetic Biology.

    PubMed

    Storch, Marko; Casini, Arturo; Mackrow, Ben; Fleming, Toni; Trewhitt, Harry; Ellis, Tom; Baldwin, Geoff S

    2015-07-17

    The ability to quickly and reliably assemble DNA constructs is one of the key enabling technologies for synthetic biology. Here we define a new Biopart Assembly Standard for Idempotent Cloning (BASIC), which exploits the principle of orthogonal linker based DNA assembly to define a new physical standard for DNA parts. Further, we demonstrate a new robust method for assembly, based on type IIs restriction enzyme cleavage and ligation of oligonucleotides with single stranded overhangs that determine the assembly order. It allows for efficient, parallel assembly with great accuracy: 4 part assemblies achieved 93% accuracy with single antibiotic selection and 99.7% accuracy with double antibiotic selection, while 7 part assemblies achieved 90% accuracy with double antibiotic selection. The linkers themselves may also be used as composable parts for RBS tuning or the creation of fusion proteins. The standard has one forbidden restriction site and provides for an idempotent, single tier organization, allowing all parts and composite constructs to be maintained in the same format. This makes the BASIC standard conceptually simple at both the design and experimental levels. PMID:25746445

  14. Copy number variation and mutation

    NASA Astrophysics Data System (ADS)

    Clark, Brian; Weidner, Jacob; Wabick, Kevin

    2009-11-01

    Until very recently, the standard model of DNA included two genes for each trait. This dated model has given way to a model that includes copies of some genes well in excess of the canonical two. Copy number variations in the human genome play critical roles in causing or aggravating a number of syndromes and diseases while providing increased resistance to others. We explore the role of mutation, crossover, inversion, and reproduction in determining copy number variations in a numerical simulation of a population. The numerical model consists of a population of individuals, where each individual is represented by a single strand of DNA with the same number of genes. Each gene is initially assigned to one of two traits. Fitness of the individual is determined by the two most fit genes for trait one, and trait two genetic material is treated as a reservoir of junk DNA. After a sufficient number of generations, during which the genetic distribution is allowed to reach a steady-state, the mean numberof genes per trait and the copy number variation are recorded. Here, we focus on the role of mutation and compare simulation results to theory.

  15. 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

    2014-01-01

    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

  16. Computational method for estimating DNA copy numbers in normal samples, cancer cell lines, and solid tumors using array comparative genomic hybridization.

    PubMed

    Abkevich, Victor; Iliev, Diana; Timms, Kirsten M; Tran, Thanh; Skolnick, Mark; Lanchbury, Jerry S; Gutin, Alexander

    2010-01-01

    Genomic copy number variations are a typical feature of cancer. These variations may influence cancer outcomes as well as effectiveness of treatment. There are many computational methods developed to detect regions with deletions and amplifications without estimating actual copy numbers (CN) in these regions. We have developed a computational method capable of detecting regions with deletions and amplifications as well as estimating actual copy numbers in these regions. The method is based on determining how signal intensity from different probes is related to CN, taking into account changes in the total genome size, and incorporating into analysis contamination of the solid tumors with benign tissue. Hidden Markov Model is used to obtain the most likely CN solution. The method has been implemented for Affymetrix 500K GeneChip arrays and Agilent 244K oligonucleotide arrays. The results of CN analysis for normal cell lines, cancer cell lines, and tumor samples are presented. The method is capable of detecting copy number alterations in tumor samples with up to 80% contamination with benign tissue. Analysis of 178 cancer cell lines reveals multiple regions of common homozygous deletions and strong amplifications encompassing known tumor suppressor genes and oncogenes as well as novel cancer related genes. PMID:20706610

  17. Loop Mediated Isothermal Amplification (LAMP) Accurately Detects Malaria DNA from Filter Paper Blood Samples of Low Density Parasitaemias

    PubMed Central

    González, Iveth J.; Polley, Spencer D.; Bell, David; Shakely, Delér; Msellem, Mwinyi I.; Björkman, Anders; Mårtensson, Andreas

    2014-01-01

    Background Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots. Methods and Findings Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6–782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94–99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0–4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1–98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2–99.9) and 76.9% (95%CI 46.2–95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1–100) in both study groups. Conclusion Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low

  18. Building accurate sequence-to-affinity models from high-throughput in vitro protein-DNA binding data using FeatureREDUCE

    PubMed Central

    Riley, Todd R; Lazarovici, Allan; Mann, Richard S; Bussemaker, Harmen J

    2015-01-01

    Transcription factors are crucial regulators of gene expression. Accurate quantitative definition of their intrinsic DNA binding preferences is critical to understanding their biological function. High-throughput in vitro technology has recently been used to deeply probe the DNA binding specificity of hundreds of eukaryotic transcription factors, yet algorithms for analyzing such data have not yet fully matured. Here, we present a general framework (FeatureREDUCE) for building sequence-to-affinity models based on a biophysically interpretable and extensible model of protein-DNA interaction that can account for dependencies between nucleotides within the binding interface or multiple modes of binding. When training on protein binding microarray (PBM) data, we use robust regression and modeling of technology-specific biases to infer specificity models of unprecedented accuracy and precision. We provide quantitative validation of our results by comparing to gold-standard data when available. DOI: http://dx.doi.org/10.7554/eLife.06397.001 PMID:26701911

  19. 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 software. (JN)

  20. 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. PMID:25765159

  1. Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[a]pyrene dG adduct

    PubMed Central

    Liu, Yang; Yang, Yeran; Tang, Tie-Shan; Zhang, Hui; Wang, Zhifeng; Friedberg, Errol; Yang, Wei; Guo, Caixia

    2014-01-01

    DNA polymerase κ (Polκ) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a]pyrene diol epoxide (BPDE)-N2-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-N2-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 (mPolκ52–516) leads to reduced polymerization activity, and the mutant PGM252–516 but not PGM152–516 can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPolκ52–516 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. PMID:24449898

  2. Isolation and characterization of a set of disease resistance-gene analogs (RGAs) from wild rice, Zizania latifolia Griseb. I. Introgression, copy number lability, sequence change, and DNA methylation alteration in several rice-Zizania introgression lines.

    PubMed

    Chen, Yu; Long, Likun; Lin, Xiuyun; Guo, Wanli; Liu, Bao

    2006-02-01

    Eight resistance-gene analogs (RGAs) were isolated from wild rice, Zizania latifolia Griseb., by degenerate primers designed according to conserved motifs at or around the nucleotide-binding site (NBS) of known NBS-containing plant resistance genes. The 8 RGAs were classified into 6 distinct groups based on their deduced amino acid sequence similarity of 60% or greater. Gel-blot hybridization of each of the RGAs to 4 rice - Z. latifolia intro gression lines indicated an array of changes at either introgressed Zizania RGAs or, more likely, their rice homologs. The changes included dramatic increase in copy number, modification at the primary DNA sequence, and alteration in DNA methylation patterns. PMID:16498465

  3. Accurate prediction of the binding free energy and analysis of the mechanism of the interaction of replication protein A (RPA) with ssDNA.

    PubMed

    Carra, Claudio; Cucinotta, Francis A

    2012-06-01

    The eukaryotic replication protein A (RPA) has several pivotal functions in the cell metabolism, such as chromosomal replication, prevention of hairpin formation, DNA repair and recombination, and signaling after DNA damage. Moreover, RPA seems to have a crucial role in organizing the sequential assembly of DNA processing proteins along single stranded DNA (ssDNA). The strong RPA affinity for ssDNA, K(A) between 10(-9)-10(-10) M, is characterized by a low cooperativity with minor variation for changes on the nucleotide sequence. Recently, new data on RPA interactions was reported, including the binding free energy of the complex RPA70AB with dC(8) and dC(5), which has been estimated to be -10 ± 0.4 kcal mol(-1) and -7 ± 1 kcal mol(-1), respectively. In view of these results we performed a study based on molecular dynamics aimed to reproduce the absolute binding free energy of RPA70AB with the dC(5) and dC(8) oligonucleotides. We used several tools to analyze the binding free energy, rigidity, and time evolution of the complex. The results obtained by MM-PBSA method, with the use of ligand free geometry as a reference for the receptor in the separate trajectory approach, are in excellent agreement with the experimental data, with ±4 kcal mol(-1) error. This result shows that the MM-PB(GB)SA methods can provide accurate quantitative estimates of the binding free energy for interacting complexes when appropriate geometries are used for the receptor, ligand and complex. The decomposition of the MM-GBSA energy for each residue in the receptor allowed us to correlate the change of the affinity of the mutated protein with the ΔG(gas+sol) contribution of the residue considered in the mutation. The agreement with experiment is optimal and a strong change in the binding free energy can be considered as the dominant factor in the loss for the binding affinity resulting from mutation. PMID:22116609

  4. How to accurately bypass damage

    PubMed Central

    Broyde, Suse; Patel, Dinshaw J.

    2016-01-01

    Ultraviolet radiation can cause cancer through DNA damage — specifically, by linking adjacent thymine bases. Crystal structures show how the enzyme DNA polymerase η accurately bypasses such lesions, offering protection. PMID:20577203

  5. Array comparative genomic hybridization identifies a distinct DNA copy number profile in renal cell cancer associated with hereditary leiomyomatosis and renal cell cancer.

    PubMed

    Koski, Taru A; Lehtonen, Heli J; Jee, Kowan J; Ninomiya, Shinsuke; Joosse, Simon A; Vahteristo, Pia; Kiuru, Maija; Karhu, Auli; Sammalkorpi, Heli; Vanharanta, Sakari; Lehtonen, Rainer; Edgren, Henrik; Nederlof, Petra M; Hietala, Marja; Aittomäki, Kristiina; Herva, Riitta; Knuutila, Sakari; Aaltonen, Lauri A; Launonen, Virpi

    2009-07-01

    Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome with cutaneous and uterine leiomyomatosis as well as renal cell cancer (RCC) as its clinical manifestations. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (fumarase) gene. In this study, we used array comparative genomic hybridization to identify the specific copy number changes characterizing the HLRCC-associated RCCs. The study material comprised formalin-fixed paraffin-embedded renal tumors obtained from Finnish patients with HLRCC. All 11 investigated tumors displayed the papillary type 2 histopathology typical for HLRCC renal tumors. The most frequent copy number changes detected in at least 3/11 (27%) of the tumors were gains in chromosomes 2, 7, and 17, and losses in 13q12.3-q21.1, 14, 18, and X. These findings provide genetic evidence for a distinct copy number profile in HLRCC renal tumors compared with sporadic RCC tumors of the same histopathological subtype, and delineate chromosomal regions that associate with this very aggressive form of RCC. PMID:19373782

  6. Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence.

    PubMed

    Kamath-Loeb, A S; Loeb, L A; Johansson, E; Burgers, P M; Fry, M

    2001-05-11

    Werner syndrome (WS) is an inherited disorder characterized by premature aging and genomic instability. The protein encoded by the WS gene, WRN, possesses intrinsic 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities. WRN helicase resolves alternate DNA structures including tetraplex and triplex DNA, and Holliday junctions. Thus, one function of WRN may be to unwind secondary structures that impede cellular DNA transactions. We report here that hairpin and G'2 bimolecular tetraplex structures of the fragile X expanded sequence, d(CGG)(n), effectively impede synthesis by three eukaryotic replicative DNA polymerases (pol): pol alpha, pol delta, and pol epsilon. The constraints imposed on pol delta-catalyzed synthesis are relieved, however, by WRN; WRN facilitates pol delta to traverse these template secondary structures to synthesize full-length DNA products. The alleviatory effect of WRN is limited to pol delta; neither pol alpha nor pol epsilon can traverse template d(CGG)(n) hairpin and tetraplex structures in the presence of WRN. Alleviation of pausing by pol delta is observed with Escherichia coli RecQ but not with UvrD helicase, suggesting a concerted action of RecQ helicases and pol delta. Our findings suggest a possible role of WRN in rescuing pol delta-mediated replication at forks stalled by unusual DNA secondary structures. PMID:11279038

  7. Impact of the DNA extraction method on 2-LTR DNA circle recovery from HIV-1 infected cells

    PubMed Central

    Badralmaa, Yunden; Natarajan, Ven

    2013-01-01

    Detection of episomal 2-LTR DNA circles is used as a marker for the ongoing virus replication in patients infected with HIV-1, and efficient extraction of episomal DNA is critical for accurate estimation of the 2-LTR circles. The impact of different methods of DNA extraction on the recovery of 2-LTR circles was compared using mitochondrial DNA extracted as an internal control. The bacterial plasmid DNA isolation method extracted less than 10% of cellular DNA, 40% of mitochondrial DNA and 12-20 % of the input 2-LTR DNA. The total DNA isolation method recovered about 70% of mitochondrial DNA and 45% of the input 2-LTR DNA. The total nucleic acid isolation method recovered 90% of mitochondrial DNA and 60% of the input 2-LTR DNA. Similar results were obtained when the DNA was extracted from HIV-1 infected cells. Plasmid DNA isolation could not distinguish between 12 and 25 copies of 2-LTR DNA per million cells, whereas the total nucleic acid isolation showed a consistent and statistically significant difference between 12 and 25 copies. In conclusion, the total nucleic acid isolation method is more efficient than the plasmid DNA isolation method in recovering mitochondrial DNA and 2-LTR DNA circles from HIV-1 infected cells. PMID:23773807

  8. 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

  9. 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

  10. An Algorithm for Inferring Complex Haplotypes in a Region of Copy-Number Variation

    PubMed Central

    Kato, Mamoru; Nakamura, Yusuke; Tsunoda, Tatsuhiko

    2008-01-01

    Recent studies have extensively examined the large-scale genetic variants in the human genome known as copy-number variations (CNVs), and the universality of CNVs in normal individuals, along with their functional importance, has been increasingly recognized. However, the absence of a method to accurately infer alleles or haplotypes within a CNV region from high-throughput experimental data hampers the finer analyses of CNV properties and applications to disease-association studies. Here we developed an algorithm to infer complex haplotypes within a CNV region by using data obtained from high-throughput experimental platforms. We applied this algorithm to experimental data and estimated the population frequencies of haplotypes that can yield information on both sequences and numbers of DNA copies. These results suggested that the analysis of such complex haplotypes is essential for accurately detecting genetic differences within a CNV region between population groups. PMID:18639202

  11. 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.

  12. Specific in situ hepatitis B viral double mutation (HBVDM) detection in urine with 60 copies ml(-1) analytical sensitivity in a background of 250-fold wild type without DNA isolation and amplification.

    PubMed

    Kirimli, Ceyhun E; Shih, Wei-Heng; Shih, Wan Y

    2015-03-01

    We have examined in situ detection of hepatitis B virus 1762T/1764A double mutation (HBVDM) in urine using a (Pb(Mg(1/3)Nb(2/3))O3)(0.65)(PbTiO3)(0.35) (PMN-PT) piezoelectric plate sensor (PEPS) coated with a 16-nucleotide (nt) probe DNA (pDNA) complementary to the HBVDM. The in situ mutation (MT) detection was carried out in a flow with the PEPS vertically situated at the center of the flow in a background of wild type (WT). For validation, this detection was followed by detection in the mixture of MT fluorescent reporter microspheres (FRMs) (MT FRMs) and WT FRMs that emitted different fluorescence colours and were designed to specifically bind to MT and WT, respectively. At 30 °C and 4 ml min(-1), a PEPS was shown to specifically detect HBVDM in situ with 60 copies ml(-1) analytical sensitivity in a background of clinically-relevant 250-fold more WT in 30 min without DNA isolation, amplification, or labelling as validated by the visualization of the captured MT FRMs and WT FRMs following FRM detection where the captured MT FRMs outnumbered the WT FRMs by a factor of 5 to 1. PMID:25599103

  13. A continental-wide perspective: the genepool of nuclear encoded ribosomal DNA and single-copy gene sequences in North American Boechera (Brassicaceae).

    PubMed

    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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. Tensor GSVD of patient- and platform-matched tumor and normal DNA copy-number profiles uncovers chromosome arm-wide patterns of tumor-exclusive platform-consistent alterations encoding for cell transformation and predicting ovarian cancer survival.

    PubMed

    Sankaranarayanan, Preethi; Schomay, Theodore E; Aiello, Katherine A; Alter, Orly

    2015-01-01

    The number of large-scale high-dimensional datasets recording different aspects of a single disease is growing, accompanied by a need for frameworks that can create one coherent model from multiple tensors of matched columns, e.g., patients and platforms, but independent rows, e.g., probes. We define and prove the mathematical properties of a novel tensor generalized singular value decomposition (GSVD), which can simultaneously find the similarities and dissimilarities, i.e., patterns of varying relative significance, between any two such tensors. We demonstrate the tensor GSVD in comparative modeling of patient- and platform-matched but probe-independent ovarian serous cystadenocarcinoma (OV) tumor, mostly high-grade, and normal DNA copy-number profiles, across each chromosome arm, and combination of two arms, separately. The modeling uncovers previously unrecognized patterns of tumor-exclusive platform-consistent co-occurring copy-number alterations (CNAs). We find, first, and validate that each of the patterns across only 7p and Xq, and the combination of 6p+12p, is correlated with a patient's prognosis, is independent of the tumor's stage, the best predictor of OV survival to date, and together with stage makes a better predictor than stage alone. Second, these patterns include most known OV-associated CNAs that map to these chromosome arms, as well as several previously unreported, yet frequent focal CNAs. Third, differential mRNA, microRNA, and protein expression consistently map to the DNA CNAs. A coherent picture emerges for each pattern, suggesting roles for the CNAs in OV pathogenesis and personalized therapy. In 6p+12p, deletion of the p21-encoding CDKN1A and p38-encoding MAPK14 and amplification of RAD51AP1 and KRAS encode for human cell transformation, and are correlated with a cell's immortality, and a patient's shorter survival time. In 7p, RPA3 deletion and POLD2 amplification are correlated with DNA stability, and a longer survival. In Xq, PABPC5

  20. Tensor GSVD of Patient- and Platform-Matched Tumor and Normal DNA Copy-Number Profiles Uncovers Chromosome Arm-Wide Patterns of Tumor-Exclusive Platform-Consistent Alterations Encoding for Cell Transformation and Predicting Ovarian Cancer Survival

    PubMed Central

    Sankaranarayanan, Preethi; Schomay, Theodore E.; Aiello, Katherine A.; Alter, Orly

    2015-01-01

    The number of large-scale high-dimensional datasets recording different aspects of a single disease is growing, accompanied by a need for frameworks that can create one coherent model from multiple tensors of matched columns, e.g., patients and platforms, but independent rows, e.g., probes. We define and prove the mathematical properties of a novel tensor generalized singular value decomposition (GSVD), which can simultaneously find the similarities and dissimilarities, i.e., patterns of varying relative significance, between any two such tensors. We demonstrate the tensor GSVD in comparative modeling of patient- and platform-matched but probe-independent ovarian serous cystadenocarcinoma (OV) tumor, mostly high-grade, and normal DNA copy-number profiles, across each chromosome arm, and combination of two arms, separately. The modeling uncovers previously unrecognized patterns of tumor-exclusive platform-consistent co-occurring copy-number alterations (CNAs). We find, first, and validate that each of the patterns across only 7p and Xq, and the combination of 6p+12p, is correlated with a patient’s prognosis, is independent of the tumor’s stage, the best predictor of OV survival to date, and together with stage makes a better predictor than stage alone. Second, these patterns include most known OV-associated CNAs that map to these chromosome arms, as well as several previously unreported, yet frequent focal CNAs. Third, differential mRNA, microRNA, and protein expression consistently map to the DNA CNAs. A coherent picture emerges for each pattern, suggesting roles for the CNAs in OV pathogenesis and personalized therapy. In 6p+12p, deletion of the p21-encoding CDKN1A and p38-encoding MAPK14 and amplification of RAD51AP1 and KRAS encode for human cell transformation, and are correlated with a cell’s immortality, and a patient’s shorter survival time. In 7p, RPA3 deletion and POLD2 amplification are correlated with DNA stability, and a longer survival. In Xq

  1. 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 Central

    Snowdin, Jacob W.; Hsiung, Chia-Heng; Kesterson, Daniel G.; Kamath, Vasudeva G.

    2015-01-01

    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. PMID:26248377

  2. 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.

  3. Mitochondrial copy number and risk of breast cancer: a pilot study.

    PubMed

    Shen, Jie; Platek, Mary; Mahasneh, Amjad; Ambrosone, Christine B; Zhao, Hua

    2010-01-01

    It has been proposed that the copy number of mitochondria DNA (mtDNA) per cell reflects gene-environment interactions between unknown hereditary factors and exposures affecting levels of oxidative stress. However, whether copy number of mtDNA could be a risk predictor of oxidative stress-related human cancers, such as breast cancer, remains to be determined. To explore the role of mtDNA copy number in breast cancer etiology, we analyzed mtDNA copy number in whole blood from 103 patients with breast cancer and 103 matched control subjects and examined in relation to endogenous antioxidants. Case patients with breast cancer had a statistically significantly higher mtDNA copy number than control subjects (median: 1.29 vs. 0.80, P<0.01). High mtDNA copy number (above the median in controls) was associated with a statistically significantly increased risk of breast cancer, compared with low copy number (Odds ratio (OR)=4.67, 95% CI: 2.45-8.92), with a statistically significant dose-response relationship in trend analysis (P<0.01). Moreover, mtDNA copy number was significantly inversely associated with several important endogenous oxidants and antioxidants in blood in either the cases (total glutathione, CuZn-SOD activity and myeloperoxidase (MPO)) or the controls (catalase (CAT) activity). These results suggest the mtDNA copy number could be associated with risk of breast cancer, perhaps through an oxidative stress mechanism. PMID:19788937

  4. 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.

  5. Magnetic nanoparticles-cooperated fluorescence sensor for sensitive and accurate detection of DNA methyltransferase activity coupled with exonuclease III-assisted target recycling.

    PubMed

    Xue, Qingwang; Zhang, Youna; Xu, Shuling; Li, Haibo; Wang, Lei; Li, Rui; Zhang, Yuanfu; Yue, Qiaoli; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng; Wang, Huaisheng

    2015-11-21

    A fluorescence magnetic biosensor for the DNA methyltransferase activity was developed based on the cooperative amplification by combining the magnetic nanoparticles synergistic exonuclease III (Exo III)-assisted circular exponential amplification and a supramolecular structure ZnPPIX/G-quadruplex. First, a duplex DNA probe, which was constructed by the hybridization of a quadruplex-forming oligomer with a molecular beacon, was assembled on the magnetic nanoparticles (MNPs) as a reporter. A hairpin probe (HP)-containing sequence of GATC was used as the methylation substrate of DNA adenine methyltransferase (DAM). Once HP was methylated by DAM, it could be recognized and cleaved by Dpn I, which allows the release of a single-stranded DNA. The DNA (tDNA1) then hybridizes to the MNP probe, which then triggers the exonuclease III-mediated target exponential recycling reaction. Simultaneously, numerous quadruplex forming oligomers are liberated and folded into the G-quadruplex-ZnPPIX complexes with the help of zinc(ii)-protoporphyrin IX(ZnPPIX) on the MNP surface to give a remarkable fluorescence response. In the developed sensor, a small amount of target DAM can be converted to a large number of stable DNA triggers, leading to remarkable amplification of the target. Moreover, using MNPs as a vector of the sensor may reduce the interference from the real samples, which increases the anti-interference of the sensing system. Based on this unique amplification strategy, a very low detection limit down to 2.0 × 10(-4) U mL(-1) was obtained. Furthermore, the sensor could be used to evaluate the DAM activity in different growth stages of E. coli cells and screen Dam MTase inhibitors. Therefore, the strategy proposed here provides a promising platform for monitoring the activity and inhibition of DNA MTases and has great potential to be applied further in early clinical diagnostics and medical research. PMID:26421322

  6. 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 Central

    Lu, Y.; Rong, C.Z.; Zhao, J.Y.; Lao, X.J.; Xie, L.; Li, S.; Qin, X.

    2016-01-01

    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. PMID:27580005

  7. 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

    2016-01-01

    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. PMID:27580005

  8. Copying could be costly.

    PubMed

    Allen, A

    1992-02-01

    Thanks to modern technology, photocopying is quick, easy, and inexpensive. Or is it? One of the largest awards ever made for copyright infringement was levied in a case involving the use of material copied from publications without permission. This article presents a short discussion about US and international copyright protection, fair use, and work for hire. Suggestions for additional information are offered. PMID:1735873

  9. 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)

  10. 69. Photographic copy of blueprint copy of original elevations drawing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    69. Photographic copy of blueprint copy of original elevations drawing (C. Howard Crane, Kenneth Franzheim, architects, Chicago: sheet $6, 1922). Blueprint copy located at William J. Bachman & Partners (Architects)), 5930 Hohman Avenue, Hammond, Indiana 46230, (219) 932-6006. - Indiana Hotel, 5116 Hohman Avenue, Hammond, Lake County, IN

  11. Copying Machine Improvement

    NASA Technical Reports Server (NTRS)

    1981-01-01

    Manufacturer of the Model 2210 copying machine was looking for a plastic valve bushing material that could be produced by a low-cost injection molding process to replace the unsuitable valve bushing they were using. NERAC conducted a computer search of the NASA database and was able to supply Nashua Corporation with several technical reports in their area of interest. Information aided the company's development of a urethane valve bushing which solved the problem and created a dramatic reduction in unit cost.

  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. PMID:26474073

  14. Diversity of human copy number variation and multicopy genes.

    PubMed

    Sudmant, Peter H; Kitzman, Jacob O; Antonacci, Francesca; Alkan, Can; Malig, Maika; Tsalenko, Anya; Sampas, Nick; Bruhn, Laurakay; Shendure, Jay; Eichler, Evan E

    2010-10-29

    Copy number variants affect both disease and normal phenotypic variation, but those lying within heavily duplicated, highly identical sequence have been difficult to assay. By analyzing short-read mapping depth for 159 human genomes, we demonstrated accurate estimation of absolute copy number for duplications as small as 1.9 kilobase pairs, ranging from 0 to 48 copies. We identified 4.1 million "singly unique nucleotide" positions informative in distinguishing specific copies and used them to genotype the copy and content of specific paralogs within highly duplicated gene families. These data identify human-specific expansions in genes associated with brain development, reveal extensive population genetic diversity, and detect signatures consistent with gene conversion in the human species. Our approach makes ~1000 genes accessible to genetic studies of disease association. PMID:21030649

  15. 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.

  16. 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

    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 (-α(3.7)), 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 (α(codon 59)α/α(CS)α) 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 (α(codon 59)α/α(Paksé)α) after nine years of being misdiagnosis with Hb Adana and HbCS (α(codon 59)α/α(CS)α). 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

  17. 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

  18. Rare Copy Number Variants

    PubMed Central

    Grozeva, Detelina; Kirov, George; Ivanov, Dobril; Jones, Ian R.; Jones, Lisa; Green, Elaine K.; St Clair, David M.; Young, Allan H.; Ferrier, Nicol; Farmer, Anne E.; McGuffin, Peter; Holmans, Peter A.; Owen, Michael J.; O’Donovan, Michael C.; Craddock, Nick

    2015-01-01

    Context Recent studies suggest that copy number variation in the human genome is extensive and may play an important role in susceptibility to disease, including neuropsychiatric disorders such as schizophrenia and autism. The possible involvement of copy number variants (CNVs) in bipolar disorder has received little attention to date. Objectives To determine whether large (>100 000 base pairs) and rare (found in <1% of the population) CNVs are associated with susceptibility to bipolar disorder and to compare with findings in schizophrenia. Design A genome-wide survey of large, rare CNVs in a case-control sample using a high-density microarray. Setting The Wellcome Trust Case Control Consortium. Participants There were 1697 cases of bipolar disorder and 2806 nonpsychiatric controls. All participants were white UK residents. Main Outcome Measures Overall load of CNVs and presence of rare CNVs. Results The burden of CNVs in bipolar disorder was not increased compared with controls and was significantly less than in schizophrenia cases. The CNVs previously implicated in the etiology of schizophrenia were not more common in cases with bipolar disorder. Conclusions Schizophrenia and bipolar disorder differ with respect to CNV burden in general and association with specific CNVs in particular. Our data are consistent with the possibility that possession of large, rare deletions may modify the phenotype in those at risk of psychosis: those possessing such events are more likely to be diagnosed as having schizophrenia, and those without them are more likely to be diagnosed as having bipolar disorder. PMID:20368508

  19. Principles and concepts of DNA replication in bacteria, archaea, and eukarya.

    PubMed

    O'Donnell, Michael; Langston, Lance; Stillman, Bruce

    2013-07-01

    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

  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. 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. PMID:26569395

  2. 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

  3. 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)

  4. 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

  5. 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

  6. Digital genotyping of macrosatellites and multicopy genes reveals novel biological functions associated with copy number variation of large tandem repeats.

    PubMed

    Brahmachary, Manisha; Guilmatre, Audrey; Quilez, Javier; Hasson, Dan; Borel, Christelle; Warburton, Peter; Sharp, Andrew J

    2014-06-01

    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 modulating

  7. 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

  8. 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

  9. 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. PMID:26923213

  10. Copy number variants, aneuploidies, and human disease.

    PubMed

    Martin, Christa Lese; Kirkpatrick, Brianne E; Ledbetter, David H

    2015-06-01

    In the perinatal setting, chromosome imbalances cause a range of clinically significant disorders and increase the risk for other particular phenotypes. As technologies have improved to detect increasingly smaller deletions and duplications, collectively referred to as copy number variants (CNVs), clinicians are learning the significant role that these types of genomic variants play in human disease and their high frequency in ∼ 1% of all pregnancies. This article highlights key aspects of CNV detection and interpretation used during the course of clinical care in the prenatal and neonatal periods. Early diagnosis and accurate interpretation are important for targeted clinical management. PMID:26042902