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Total Genomic DNA (total + genomic_dna)
Selected AbstractsDNA sequence analysis of interlocus recombination between the human T-cell receptor gamma variable (GV) and beta diversity-joining (BD/BJ) sequences on chromosome 7 (inversion 7)ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2002Scott W. Ballinger Abstract V(D)J recombinase-mediated recombination between the T-cell receptor (TCR) gamma variable (GV) genes at chromosome 7p15 and the TCR beta joining (BJ) genes at 7q35 leads to the formation of a hybrid TCR gene. These TCR gamma/beta interlocus rearrangements occur at classic V(D)J recombination signal sequences (RSS) and, because the loci are in an inverted orientation, result in inversion events that are detectable in the chromosome structure as inv(7)(p15;q35). Similar rearrangements involving oncogenes and either TCR or immunoglobulin genes mediated by the V(D)J recombinase are found in lymphoid malignancies. Oligonucleotide primers that allow polymerase chain reaction (PCR) amplification across the inv(7) genomic recombination junction sequence have been described. Southern blot analysis has been primarily used to confirm the GV/BJ hybrid nature of the product, with limited information on the DNA sequence of these recombinations. We have modified this PCR method using total genomic DNA from the mononuclear cells in peripheral blood samples to increase specificity and to allow direct sequencing of the translocation junction that results from the recombination between the GV1 and BJ1 families of TCR genes in 25 examples from 11 individuals (three adults, one child, six newborns, and one ataxia telangiectasia (AT) patient). We focused on samples from newborns based on previous studies indicating that the predominant hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutations in newborns are V(D)J recombinase-mediated deletion events and that the frequency of these mutations decreases with increasing age. Although the dilution series-based PCR assay utilized does not yield sharply defined quantitative endpoints, results of this study strongly suggest that inv(7) recombinations in newborns occur at equal or lower frequencies than those seen in adults. Consistent with the PCR primer pairs, all sequenced products contain a GV1 and a BJ1 segment and most also contain a BD1 segment. GV1s2 and 1s4 were the most frequently found GV1 genes (8 and 9 examples, respectively) and BJ1s5 and 1s6 were the most frequently found BJ1 genes (9 and 10 examples, respectively). These results demonstrate the effectiveness of this methodology for assessing GV/BJ interlocus rearrangements mediated by V(D)J recombinase. Environ. Mol. Mutagen. 40:85,92, 2002. © 2002 Wiley-Liss, Inc. [source] Identification of genetic aberrations on chromosome 22 outside the NF2 locus in schwannomatosis and neurofibromatosis type 2,HUMAN MUTATION, Issue 6 2005Patrick G. Buckley Abstract Schwannomatosis is characterized by multiple peripheral and cranial nerve schwannomas that occur in the absence of bilateral 8th cranial nerve schwannomas. The latter is the main diagnostic criterion of neurofibromatosis type 2 (NF2), which is a related but distinct disorder. The genetic factors underlying the differences between schwannomatosis and NF2 are poorly understood, although available evidence implicates chromosome 22 as the primary location of the gene(s) of interest. To investigate this, we comprehensively profiled the DNA copy number in samples from sporadic and familial schwannomatosis, NF2, and a large cohort of normal controls. Using a tiling-path chromosome 22 genomic array, we identified two candidate regions of copy number variation, which were further characterized by a PCR-based array with higher resolution. The latter approach allows the detection of minute alterations in total genomic DNA, with as little as 1.5,kb per measurement point of nonredundant sequence on the array. In DNA derived from peripheral blood from a schwannomatosis patient and a sporadic schwannoma sample, we detected rearrangements of the immunoglobulin lambda (IGL) locus, which is unlikely to be due to a B-cell specific somatic recombination of IGL. Analysis of normal controls indicated that these IGL rearrangements were restricted to schwannomatosis/schwannoma samples. In the second candidate region spanning GSTT1 and CABIN1 genes, we observed a frequent copy number polymorphism at the GSTT1 locus. We further describe missense mutations in the CABIN1 gene that are specific to samples from schwannomatosis and NF2 and make this gene a plausible candidate for contributing to the pathogenesis of these disorders. Hum Mutat 26(6), 540,549, 2005. © 2005 Wiley-Liss, Inc. [source] Quantitative analysis of total mitochondrial DNA: Competitive polymerase chain reaction versus real-time polymerase chain reactionJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2004Hari K. Bhat Abstract An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 ± 1.01 × 104 molecules/ng total genomic DNA using competitive PCR vs 4.90 ± 0.84 × 104 molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:180,186, 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20024 [source] Gerbera jamesonii, a New Host of Fusarium oxysporum f.sp. tracheiphilumJOURNAL OF PHYTOPATHOLOGY, Issue 1 2010Marco Troisi Abstract The random amplified polymorphic DNA (RAPD) technique was used to analyze the total genomic DNA of pathogenic isolates of Fusarium oxysporum on Gerbera jamesonii by comparing them to representatives of the formae speciales chrysanthemi and tracheiphilum. A close genetic relationship was observed among most of the new isolates from G. jamesonii. They shared RAPD markers with the tested representatives of the forma specialis chrysanthemi. Some isolates of those tested from diseased G. jamesonii were placed in a different cluster, which included representative isolates of forma specialis tracheiphilum. This is the first report of F. oxysporum f.sp. tracheiphilum on G. jamesonii. A rapid protocol for DNA extraction directly from fungal colonies grown on potato dextrose agar allowed complete analysis in less than 4 h. [source] A short-cut DNA extraction from cod caviarJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 3 2006Futoshi Aranishi Abstract Caviars represent the most consumed form of fish roe products. Due to high demand, ingredient roes of fish are often susceptible to illegal substitution with those of related fish. This study developed a simple and inexpensive protocol enabling the rapid extraction of DNA of acceptable quality and amount to PCR amplification from both cod caviars and their ingredient pollack roes. The protocol was based on extracting total genomic DNA from eggs using urea and a Chelex 100 chelating resin, and could be completed in less than 15 min. Approximately 8 µg of DNA were reproducibly obtained from single eggs of cod caviars and pollack roes in eight individual experiments, and the quality and amount of DNA were sufficient to serve as template for hundreds of PCR reactions of polymorphic DNA markers for phylogenetic analysis. Being applicable to various caviars, this protocol can be useful to detect illegal substitution among ingredient roes of related fishes in PCR-based food inspection. Copyright © 2005 Society of Chemical Industry [source] Five hundred and fifty microsatellite markers for the study of the honeybee (Apis mellifera L.) genomeMOLECULAR ECOLOGY RESOURCES, Issue 2 2003Michel Solignac Summary Microsatellites are currently considered the most useful genetic markers with wide applications in genomics, quantitative and population genetics. We present here the structure of the core sequence of 552 microsatellites, together with the sequences of the primers and the length of the sequenced allele. These microsatellites were isolated from several libraries constructed from either fractions of total genomic DNA or from clones of a bacterial artificial chromosome (BAC) library. All 552 loci are polymorphic in the honeybee. Many of them were also successfully amplified in three other species of Apis: A. cerana (58%), A. dorsata (59%) and A. florea (38%). A summary of the variability of 36 loci in the three main evolutionary lineages of A. mellifera is given. [source] Molecular cytogenetic analysis of a durum wheat ×Thinopyrum distichum hybrid used as a new source of resistance to Fusarium head blight in the greenhousePLANT BREEDING, Issue 5 2001Q. Chen Abstract Fusarium head blight (FHB, scab), caused by Fusarium graminearum Schwabe, is a serious and damaging disease of wheat. Although some hexaploid wheat lines express a good level of resistance to FHB, the resistance available in hexaploid wheat has not yet been transferred to durum wheat. A germplasm collection of Triticum durum× alien hybrid lines was tested as a potential source of resistance to FHB under controlled conditions. Their FHB reaction was evaluated in three tests against conidial suspensions of three strains of F. graminearum at the flowering stage. Two T. durum×Thinopyrum distichum hybrid lines, ,AFR4' and ,AFR5,, expressed a significantly higher level of resistance to the spread of FHB than other durum-alien hybrid lines and a resistant common wheat line ,Nyu-Bay'. Genomic in situ hybridization using total genomic DNA from alien grass species demonstrated that ,AFR5' had 13 or 14 alien genome chromosomes plus 27 or 28 wheat chromosomes, while ,AFR4' had 22 alien genome and 28 wheat chromosomes. All of the alien chromosomes present in these two lines belonged to the J genome. ,AFR5' is likely to be more useful as a source of FHB resistance than ,AFR4' because of its relatively normal meiotic behaviour, high fertility and fewer number of alien chromosomes. ,AFR5' shows good potential as a source for transferring FHB resistance gene into wheat. The development of T. durum addition lines carrying resistance genes from ,AFR5' is underway. [source] | |||||||||||||