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Results for “DNA Copy Number Variations”

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At least 433 records · Page 24Linked to original sources

PubMed1993

PCR-based restriction fragment length polymorphism analysis of DNA sequence diversity of flagellin genes of Campylobacter jejuni and allied species.

Genotypes were analysed within 29 geographically diverse strains of Campylobacter jejuni mainly from human enteritis cases and four strains representing C. coli, C. lari and C. helveticus. A 1723 bp DNA fragment, amplified by the polymerase chain reaction (PCR) from the flagellin (fla) genes, was used to probe genomic Southern blots for restriction site variation at homologous loci. Internal Hin fl restriction fragment length polymorphisms (RFLP) of the fla gene PCR amplification product were also used to characterize isolates. Copy number and polymorphisms in and around the fla genes were detected, and strains were grouped into 10 genotypes on the basis of fla restriction site similarities. Campylobacter jejuni had a fla gene copy number of two and the majority (64%) of strains in the species had common internal fla gene sequences as indicated by Hin fl restriction analysis. A greater degree of diversity was detected around the fla loci in Hae III and Bgl II genomic Southern blots. The results showed that analysis of fla genes provided a novel and fundamental approach to grouping strains and offered a basis for defining a predominant clonal evolutionary line within C. jejuni.

Base Sequence↗

PubMed2006

Retrotransposon-based S-SAP as a platform for the analysis of genetic variation and linkage in globe artichoke.

A high copy number of retrotransposon sequences are present and widely dispersed in plant genomes. Their activity generates a considerable degree of sequence polymorphism. Here, we report the cloning of CYRE-5, a long-terminal repeat carrying retrotransposon-like sequence in Cynara cardunculus L., and its exploitation to develop a DNA fingerprinting assay across 22 accessions, including both cultivated (globe artichoke and cultivated cardoon) and wild (wild cardoon) types. The effectiveness of the sequence-specific amplified polymorphism (S-SAP) platform is compared with that of amplified fragment length polymorphism (AFLP). A genetic linkage analysis, based on a hybrid population between 2 globe artichoke varietal types, resulted in the inclusion of 29 S-SAP loci in the core genetic map, confirming their dispersed distribution across the globe artichoke genome.

Cynara scolymus↗

PubMed1995

Variations of two repetitive DNA sequences in several Triticeae genomes revealed by polymerase chain reaction and sequencing.

Genomes of Triticeae were analyzed using PCR with synthesized primers that were based on two published repetitive DNA sequences, pLeUCD2 (pLe2) and 1-E6hcII-1 (L02368),which were originally isolated from Thinopyrum elongatum. The various genomes produced a 2240 bp PCR product having high homology with the repetitive DNA pLe2. The PCR fragments produced from different genomes differed mainly in amplification quantity and in base composition at 89 variable sites. On the other hand, amplification products from the primer set for L02368 were of different sizes and nucleotide sequences. These results show that the two repetitive DNA sequences have different evolutionary significance. ple2 is present in all genomes tested, although differences in copy number and nucleotide sequence are notable. L02368 is more genome specific, i.e., fewer genomes possess this family of repetitive sequences. It was concluded that the repetitive sequence pLe2 family is an ancient one that existed in progenitor genome prior to divergence of annual and perennial genomes. In contrast, sequences similar to L02368 have only evolved following genome divergence.

Base Sequence↗

PubMed1983

Glucocorticoid regulation of transcription at an amplified, episomal promoter.

The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been introduced into cultured murine cells, using the 69% transforming fragment of bovine papilloma virus type 1 (BPV). Transformed cells contain up to 200 copies of the chimeric molecules per diploid genome. The restriction endonuclease map of the acquired recombinants, as well as the physical structure of the DNA, indicates that the LTR-BPV molecules present in these cells occur exclusively as unintegrated, extrachromosomal episome. When a 72-base pair direct repeat "enhancer" element (derived from the Harvey sarcoma retrovirus) was included in the MMTV LTR-BPV chimeric plasmids, DNA acquired through transfection, with a single exception, was integrated or rearranged or both. The transcriptional potential of the episomal MMTV promoter present in these cells was tested in two ways. First, steady-state levels of MMTV-initiated RNA were measured by quantitative S1 mapping. Second, the relative number of transcription complexes initiated in vivo was determined by using a subnuclear fraction highly enriched for MMTV-BPV minichromosomes in an in vitro transcription extension assay. Both approaches showed that the MMTV LTR present in the episomal state was capable of supporting glucocorticoid hormone-regulated transcription. We have therefore demonstrated the hormone response for the first time in a totally defined primary sequence environment. Significant differences both in the basal level of MMTV-initiated transcription and in the extent of glucocorticoid induction were observed in individual cell lines with similar episomal copy numbers. These phenotypic variations suggest that epigenetic structure is an important component of the mechanism of regulation.

Animals↗

PubMed1981

Analysis of a dispersed repetitive DNA sequence in isogenic lines of Drosophila.

The location of sequences homologous to a cloned D. melanogaster DNA segment, Dm 25, has been examined in polytene chromosomes by hybridization in situ. Dm 25 localizes to multiple sites and shows variation in patterns between different strains and among individuals within wild-type laboratory strains. Analysis of numerous geographically distinct isogenic lines suggests that Dm 25 patterns are determined by germ-line factors and are not the product of strictly somatic events. In general there is wide variation in Dm 25 patterns among different lines, but a significant number of sites are common to two or more distinct lines. Hybridization to restriction digests of genomic DNA suggests that Dm 25 is a moderately repetitive, conserved sequence whose copies are dispersed throughout the genome. Analysis of species other than melanogaster indicates a significant divergence in structure of sequences homologous to Dm 25 as well as a drastic reduction in amount of homology to the melanogaster sequence.

Animals↗

PubMed1997

Characterization of pigs transgenic for human decay-accelerating factor.

BACKGROUND: To prevent the central role played by complement activation in the hyperacute rejection of pig organs transplanted into primates, pigs transgenic for human decay-accelerating factor (HDAF) have recently been produced. The data presented here extend previous immunohistochemical findings by documenting the immunological characterization and the levels of expression of HDAF in these transgenic pigs. METHODS: Animals from 30 independently derived lines were included in this study. HDAF expression was characterized by immunoprecipitation and epitope mapping. Quantitative analysis was performed by radiometric assays followed by Scatchard analysis and by double-determinant radioimmunoassay. Deposition of iC3b on porcine aortic endothelial cells was determined by radioimmunoassay. DNA slot-blot analysis and densitometric scanning were used to evaluate HDAF transgene copy number. RESULTS: The integrity of HDAF expressed by these transgenic pigs could be demonstrated. HDAF was present in 72% of the organs analyzed, although considerable variation in expression occurred, both between animals and within the same pig. High levels of HDAF on porcine aortic endothelial cells resulted in iC3b deposition at levels as low as that detected on human endothelial cells. Twenty-six organs expressed levels of HDAF greater than those observed in the equivalent human tissue. HDAF expression did not correlate with the number of copies of the transgene incorporated into the porcine genome. CONCLUSIONS: Transgenic pigs, which express levels of functional HDAF even greater than those observed in humans, have successfully been produced. Pigs transgenic for human complement inhibiting molecules could represent a source of organs for future clinical xenotransplantation.

Animals↗

PubMed1998

Expression of tetanus toxin fragment C.

1. The yield of fragment C was only modestly affected by Mut phenotype, and the site and type of integration event. 2. Fragment C accumulation was closely correlated with gene dosage and maximal expression levels required high gene copy number. 3. Yields were greatly increased in controlled fermenters, compared to shake-flasks, owing to the high cell density achieved and to an increased efficiency of induction (2.5- to 10-fold). 4. In fermenter inductions of a 14-copy strain, fragment C accumulated to 27% of total protein, giving an estimated yield of 12 g/L. 5. Considerable clonal variation in the level of expression occurred with transplacement transformants, and this was owing to a diversity of different integration events and to differences in gene copy number. 6. These multicopy transplacement events occur by in vivo circularization of transforming DNA fragments followed by repeated single-crossover integration. This is presumably a general phenomenon, such that it should be possible to obtain multicopy integrants from all P. pastoris transplacement transformations.

Alcohol Oxidoreductases↗

PubMed1999

RAPD polymorphisms detected among the flax genotrophs.

The occurrence of environmentally induced heritable changes in certain flax varieties has been shown to be accompanied by changes in the genomic DNA. A large difference in nuclear DNA contents has been characterized between the extreme types, termed genotrophs. The genomic variation between a series of genotrophs has been studied by the polymerase chain reaction using random arbitrary oligonucleotide primers. A total of 320 primers were used in the reactions and 253 polymorphic bands observed. The polymorphic bands were derived from all parts of the genome, namely the highly repetitive. middle-repetitive and low-copy-number sequences. They were also shown to be distributed throughout the genome. In one group of genotrophs, all of which were induced by temperature treatment, there was a clustering of the polymorphisms with a high degree of shared polymorphisms. These results are in agreement with earlier studies showing that a dispersed fraction of the genome is susceptible to variation when environmentally induced heritable changes occur.

DNA, Plant↗

PubMed2006

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

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

DNA↗

PubMed1994

The DNA rearrangement associated with facioscapulohumeral muscular dystrophy involves a heterochromatin-associated repetitive element: implications for a role of chromatin structure in the pathogenesis of the disease.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant form of muscular dystrophy. The FSHD locus has been linked to the most distal genetic markers on the long arm of chromosome 4. Recently, a probe was identified that detects an EcoRI fragment length polymorphism which segregates with the disease in most FSHD families. Within the EcoRI fragment lies a tandem array of 3.2 kb repeats. In several familial cases and four independent sporadic FSHD mutations, the variation in size of the EcoRI fragment was due to a decrease in copy number of the 3.2 kb repeats. To gain further insight into the relationship between the tandem array and FSHD, a single 3.2 kb repeat unit was characterized. Fluorescence in situ hybridization (FISH) demonstrates that the 3.2 kb repeat cross-hybridizes to several regions of heterochromatin in the human genome. In addition, DNA sequence analysis of the repeat reveals a region which is highly homologous to a previously identified family of heterochromatic repeats, LSau. FISH on interphase chromosomes demonstrates that the tandem array of 3.2 kb repeats lies within 215 kb of the 4q telomere. Together, these results suggest that the tandem array of 3.2 kb repeats, tightly linked to the FSHD locus, is contained in heterochromatin adjacent to the telomere. In addition, they are consistent with the hypothesis that the gene responsible for FSHD may be subjected to position effect variegation because of its proximity to telomeric heterochromatin.

Base Sequence↗

PubMed1998

Tandemly repeated sequences in the mitochondrial DNA control region and phylogeography of the Pike-Perches Stizostedion.

DNA sequences from the mitochondrial DNA control region are used to test the phylogeographic relationships among the pike-perches, Stizostedion (Teleostei: Percidae) and to examine patterns of variation. Sequences reveal two types of variability: single nucleotide polymorphisms and 6 to 14 copies of 10- to 11-base-pair tandemly repeated sequences. Numbers of copies of the tandem repeats are found to evolve too rapidly to detect phylogenetic signal at any taxonomic level, even among populations. Sequence similarities of the tandem repeats among Stizostedion and other percids suggest concerted evolutionary processes. Predicted folding of the tandem repeats and their proximity to termination-associated sequences indicate that secondary structure mediates slipped-strand mispairing among the d-loop, heavy, and light strands. Neighbor-joining and maximum parsimony analyses of sequences indicate that the genus is divided into clades on the continents of North America and Eurasia. Calibrating genetic distances with divergence times supports the hypothesis that Stizostedion dispersed from Eurasia to North America across a North Pacific Beringial land bridge approximately 4 million years before present, near the beginning of the Pliocene Epoch. The North American S. vitreum and S. canadense appear separated by about 2.75 million years, and the Eurasian S. lucioperca and S. volgensis are diverged by about 1.8 million years, suggesting that speciation occurred during the late Pliocene Epoch.

Animals↗

PubMed2007

Statistical methods for determining the accuracy of quantitative polymerase chain reaction-based tests.

Quantitative polymerase chain reaction (PCR)-based tests are used in several different scientific fields to determine levels of a target DNA sequence of interest (the target molecule). The accuracy of quantitative PCR-based tests can be assessed by using the assay to determine the number of copies of the target molecule in a sample with a known concentration of the target molecule. For example, a sample with a known concentration of a target DNA sequence is serially diluted into replicate aliquots and these are tested to determine if the observed quantity of the target is close to the expected quantity (given the concentration in the original sample and the dilution). Statistical methods that are conventionally used to assess the accuracy of these assays do not take into account the variability in the number of target molecules in each aliquot from the original sample. We develop methods that take into account this extra variation and which determine the accuracy of quantitative PCR-based tests in estimating the number of target molecules based on a set of assays of serial dilutions from an original sample with a known concentration of target molecules. These methods are applied to data from an experiment to test the accuracy of a real-time PCR assay at low HIV-1 DNA copy levels.

DNA, Viral↗

PubMed2006

Detection of small genomic imbalances using microarray-based multiplex amplifiable probe hybridization.

Array-based genome-wide screening methods were recently introduced to clinical practice in order to detect small genomic imbalances that may cause severe genetic disorders. The continuous advancement of such methods plays an extremely important role in diagnostic genetics and medical genomics. We have modified and adapted the original multiplex amplifiable probe hybridization (MAPH) to a novel microarray format providing an important new diagnostic tool for detection of small size copy-number changes in any locus of human genome. Here, we describe the new array-MAPH diagnostic method and show proof of concept through fabrication, interrogation and validation of a human chromosome X-specific array. We have developed new bioinformatic tools and methodology for designing and producing amplifiable hybridization probes (200-600 bp) for array-MAPH. We designed 558 chromosome X-specific probes with median spacing 238 kb and 107 autosomal probes, which were spotted onto microarrays. DNA samples from normal individuals and patients with known and unknown chromosome X aberrations were analyzed for validation. Array-MAPH detected exactly the same deletions and duplications in blind studies, as well as other unknown small size deletions showing its accuracy and sensitivity. All results were confirmed by fluorescence in situ hybridization and probe-specific PCR. Array-MAPH is a new microarray-based diagnostic tool for the detection of small-scale copy-number changes in complex genomes, which may be useful for genotype-phenotype correlations, identification of new genes, studying genetic variation and provision of genetic services.

Chromosome Aberrations↗

PubMed2005

Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes.

A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software. This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA.

Biosensing Techniques↗

PubMed2004

Chromosome 1 trisomy compromises the virulence of Candida albicans.

Although increases in chromosome copy number typically have devastating developmental consequences in mammals, fungal cells such as Saccharomyces cerevisiae seem to tolerate trisomies without obvious impairment of growth. Here, we demonstrate that two commonly used laboratory strains of the yeast Candida albicans, CAI-4 and SGY-243, can carry three copies of chromosome 1. Although the trisomic strains grow well in the laboratory, Ura+ derivatives of CAI-4, carrying three copies of chromosome 1, are avirulent in the intravenously inoculated mouse model, unlike closely related strains carrying two copies of chromosome 1. Furthermore, changes in chromosome copy number occur during growth in an animal host and during growth in the presence of growth-inhibiting drugs. These results suggest that chromosome copy number variation provides a mechanism for genetic variation in this asexual organism.

Base Sequence↗

PubMed2004

Distribution of Activator (Ac) throughout the maize genome for use in regional mutagenesis.

A collection of Activator (Ac)-containing, near-isogenic W22 inbred lines has been generated for use in regional mutagenesis experiments. Each line is homozygous for a single, precisely positioned Ac element and the Ds reporter, r1-sc:m3. Through classical and molecular genetic techniques, 158 transposed Ac elements (tr-Acs) were distributed throughout the maize genome and 41 were precisely placed on the linkage map utilizing multiple recombinant inbred populations. Several PCR techniques were utilized to amplify DNA fragments flanking tr-Ac insertions up to 8 kb in length. Sequencing and database searches of flanking DNA revealed that the majority of insertions are in hypomethylated, low- or single-copy sequences, indicating an insertion site preference for genic sequences in the genome. However, a number of Ac transposition events were to highly repetitive sequences in the genome. We present evidence that suggests Ac expression is regulated by genomic context resulting in subtle variations in Ac-mediated excision patterns. These tr-Ac lines can be utilized to isolate genes with unknown function, to conduct fine-scale genetic mapping experiments, and to generate novel allelic diversity in applied breeding programs.

Chromosome Mapping↗

PubMed1987

OriC plasmids do not affect timing of chromosome replication in Escherichia coli K12.

The variability of the time interval between successive rounds of chromosome replication was estimated by density-shift experiments, by measuring the conversion of heavy DNA to hybrid density and light DNAs upon transfer of a steady-state culture growing in medium with [13C]glucose and 15NH4Cl to medium with light isotopes. The coefficient of variation (CV%) for the interreplication time of the Escherichia coli K12 chromosome was found to be 17%, i.e. similar to that for interdivision time. The presence of additional copies of oriC in the cell on a high copy number plasmid did not increase the CV of interreplication time. It is concluded that a single rate-limiting event is unlikely to time the initiation of chromosome replication. The regulation of initiation at oriC and the coordination with cell division is discussed.

Chromosomes, Bacterial↗

PubMed1988

Strain variation of R5 direct repeats in the right-hand portion of the long unique segment of varicella-zoster virus DNA.

We located a region of interstrain size variability in a short segment in an area at the right-hand end of the long unique sequence of the varicella-zoster viral genome. Varicella-zoster virus strains isolated in a district of Japan were classified into three groups on the basis of the size of this segment. Sequence comparison of the variable segment among strains from different groups revealed that the tandem direct repeat, R5, in the segment was variable among strains. R5, which was first discovered in a European strain (Dumas), contained a direct duplication of 88-base-pair (bp) elements separated by a 24-bp element (A.J. Davison and J.E. Scott, J. Gen. Virol. 67:1759-1816, 1986). We found that one 88-bp element and one 24-bp element constitute a repeating unit whose copy number varied from one to three among strains. The simplest R5 we detected was similar to that of Dumas, but there were a few base mismatches between these two R5 structures.

Base Sequence↗