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PCR Primer Pairs (pcr + primer_pair)
Selected AbstractsSimultaneous PCR Detection of the Two Major Bacterial Pathogens of GeraniumJOURNAL OF PHYTOPATHOLOGY, Issue 2 2002D. L. GLICK Xanthomonas campestris pv. pelargonii (Xcp) and Ralstonia solanacearum (Rs) are the two most important bacterial pathogens of commercially cultivated geraniums (Pelargonium spp.), both causing bacterial wilt and leaf spot. Asymptomatic infections are important reservoirs of infections in commercial growing facilities. Our objective was to design a multiplex PCR (Polymerase Chain Reaction) assay to detect infection by either or both of these pathogens. We used a previously characterized PCR primer pair for Xcp that amplifies a region of 200 bp. In addition, we designed a new primer pair specific for Rs that amplifies a region of 822 bp. With these two primer pairs, we could detect either or both pathogens. As geranium tissue extracts frequently contain inhibitors of the PCR process, a negative PCR could result from either an accurate indication that the plant was pathogen-free or from a false negative assay. We therefore designed `amplification competence' primers, targeting a portion of the geranium 18 s rRNA gene, and generating a 494-bp amplification product that confirms amplification competence and validates a negative assay result. Thus, the triple primer pair multiplex PCR screens for the two most important bacterial pathogens of geraniums simultaneously confirms amplification competence for each geranium sample. [source] Novel polymerase chain reaction primers for the specific detection of bacterial copper P-type ATPases gene sequences in environmental isolates and metagenomic DNALETTERS IN APPLIED MICROBIOLOGY, Issue 6 2010R. De la Iglesia Abstract Aims:, In the last decades, the worldwide increase in copper wastes release by industrial activities like mining has driven environmental metal contents to toxic levels. For this reason, the study of the biological copper-resistance mechanisms in natural environments is important. Therefore, an appropriate molecular tool for the detection and tracking of copper-resistance genes was developed. Methods and Results:, In this work, we designed a PCR primer pair to specifically detect copper P-type ATPases gene sequences. These PCR primers were tested in bacterial isolates and metagenomic DNA from intertidal marine environments impacted by copper pollution. As well, T-RFLP fingerprinting of these gene sequences was used to compare the genetic composition of such genes in microbial communities, in normal and copper-polluted coastal environments. New copper P-type ATPases gene sequences were found, and a high degree of change in the genetic composition because of copper exposure was also determined. Conclusions:, This PCR based method is useful to track bacterial copper-resistance gene sequences in the environment. Significance and Impact of the Study:, This study is the first to report the design and use of a PCR primer pair as a molecular marker to track bacterial copper-resistance determinants, providing an excellent tool for long-term analysis of environmental communities exposed to metal pollution. [source] DNA 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] Molecular characterization of swine leucocyte antigen class I genes in outbred pig populationsANIMAL GENETICS, Issue 4 2009C.-S. Ho Summary The highly polymorphic swine leucocyte antigen (SLA) genes are one of the most important determinants in swine immune responses to infectious diseases, vaccines, and in transplantation success. Study of SLA influence requires accurate and effective typing methods. We developed a simple and rapid method to type alleles at the three classical SLA class I loci (SLA-1, SLA-3 and SLA-2) using the PCR-sequence-specific primer (PCR-SSP) strategy. This typing system relies on 47 discriminatory PCR primer pairs designed to amplify the SLA class I alleles by groups that have similar sequence motifs. We applied this low-resolution group-specific typing method to characterize the SLA class I alleles present in three outbred pig populations (n = 202). Alleles from 24 class I allele groups corresponding to 56 class I genotypes were detected. We also identified 23 low-resolution SLA class I haplotypes in these pigs and found haplotypes Lr-1.0 (SLA-1*01XX- SLA-3*01XX- SLA-2*01XX) and Lr-4.0 (SLA-1*04XX- SLA-3*04XX- SLA-2*04XX) in all three pig populations with a high prevalence. Over 80% of the pigs examined (n = 162) were found to bear at least one of these haplotypes, resulting in a combined haplotype frequency of nearly 50%. This PCR-SSP-based typing system demonstrates a reliable and unambiguous detection of SLA class I alleles, and can be used to effectively investigate the SLA diversity in outbred pig populations. It will help to identify the role of SLA antigens in disease-resistant pigs and may facilitate the development of effective vaccines. [source] Interspecies and intergenus transferability of barley and wheat D-genome microsatellite markersANNALS OF APPLIED BIOLOGY, Issue 3 2010A. Castillo A selection of 147 wheat D-genome and 130 barley genomic simple sequence repeat (gSSR) markers were screened for their utility in Hordeum chilense, as an alien donor genome for cereal breeding. Fifty-eight wheat D-genome and 71 barley PCR primer pairs consistently amplified products from H. chilense. Nineteen wheat D-genome and 20 barley gSSR markers were polymorphic and allowed wide genome coverage of the H. chilense genome. Twenty-three of the wheat D-genome and 11 barley PCR primer pairs were suitable for studying the introgressions of H. chilense into wheat, amplifying H. chilense products of distinct size. In 88% of the markers tested, H. chilense products were maintained in the expected homeologous linkage group, as revealed by the analysis of wheat/H. chilense addition lines. Twenty-nine microsatellite markers (eight gSSRs and 21 expressed sequence tags-SSRs) uniformly distributed across the genome were tested for their utility in genetic diversity analysis within the species. Three genetic clusters are reported, in accordance with previous morphological and amplified fragment length polymorphism data. These results show that it is possible to discriminate the three previously established germplasm groups with microsatellite markers. The reported markers represent a valuable resource for the genetic characterisation of H. chilense, for the analysis of its genetic variability, and as a tool for wheat introgression. This is the first intraspecific study in a collection of H. chilense germplasm using microsatellite markers. [source] | ||||||||||