Multiplex polymerase chain reaction

About: Multiplex polymerase chain reaction is a research topic. Over the lifetime, 6409 publications have been published within this topic receiving 221244 citations.

Abundance of Multiple High-Risk Human Papillomavirus (HPV) Infections Found in Cervical Cells Analyzed by Use of an Ultrasensitive HPV Genotyping Assay

Abstract: PCR methods enable the detection of a large variety of human papillomavirus (HPV) genotypes that infect the anogenital tract. However, PCR with consensus primers, general primers, and, to a lesser extent, broad-spectrum primers may underrepresent the true prevalence of HPV, especially the true prevalence of multiple infections. We compared the rate of HPV positivity determined by a broad-spectrum PCR with primers BSGP5+ and BSGP6+ (BS-PCR) coupled to an established bead-based multiplex HPV genotyping (MPG) assay with the rate of HPV positivity determined by a multiplex PCR with type-specific primers (TS-PCR) coupled to a newly developed MPG assay for 735 selected cervical scraping samples. While the primers used for the BS-PCR are located within the L1 region of the HPV genome, the primers used for the TS-PCR target the E7 gene. The overall rates of positivity for the 19 HPV types included in both assays were 60.9% and 72.2% by the BS-PCR and the TS-PCR, respectively, and the two assays found multiple infections in 34.8% and 58.0% of the specimens, respectively. Both HPV detection assays allowed the semiquantitative detection of HPV types and identified the same dominant HPV type in 66.6% of the multiple infections. In conclusion, the TS-PCR-MPG assay significantly increased the rate of detection of HPV DNA and the number of infections with multiple HPV types detected and demonstrated that the prevalence of low-copy-number HPV infections in the anogenital tract may be strongly underestimated by conventional HPV amplification methods, especially in cases of multiple infections. As a consequence, PCR-TS-MPG appears to be highly suited for analysis of the significance of multiple infections in the development of cervical cancer and for the study the natural history and the latency of HPV.

Application of Multiplex PCR and Flourescence-Based, Semi-Automated Allele Sizing Technology for Genotyping Plant Genetic Resources

Abstract: For future progress, effective conservation of plant genetic resources will require the integration of technologies and protocols that provide for the acquisition of large quantities of genetic information for improved gene and genotype identification. As a contribution to this integration, simple sequence repeat (SSR) marker analysis in Brassica spp. was evaluated as a prototype system for establishing the potential of multiplex polymerase chain reactions (PCRs) coupled with fluorescence-based DNA detection and semi-automated sizing technology for mass genotyping of plant genetic resources. Consistent results were achieved for multiplex PCRs (simultaneous amplification of several genetic markers in a single reaction) with 11 fluorescently labeled primer pairs. Comparison of computer-derived estimates of fragment sizes between gels demonstrated that automated sizing was accurate enough to achieve reliable and repeatable plant genotypes. To establish a genetic basis for putative SSR loci previously identified in Brassica spp., segregation data were analyzed. Three polymorphic SSRs in Brassica napus L. (canola and rapeseed) exhibited simple Mendelian inheritance and were not linked. One of these polymorphic loci was duplicated, i.e., a single copy marked each of the aa and cc genomes originating from progenitor species B. rapa and B. oleracea, respectively. Technologies that provide for high genetic resolution and throughput at reasonable costs will find ready application in plant genetic resources conservation. In addition, these same tools and marker systems can be applied to questions of pedigree analysis and intellectual property rights.

High‐throughput multiplex SNP genotyping with MALDI‐TOF mass spectrometry: Practice, problems and promise

Abstract: Single nucleotide polymorphisms (SNPs) are currently being identified and mapped at a remarkable pace, providing a rich genetic resource with vast potential for disease gene discovery, pharmacogenetics, and understanding the origins of modern humans. High-throughput, cost effective genotyping methods are essential in order to make the most advantageous and immediate use of these SNP data. We have incorporated the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) in our laboratory as a tool for differentiating genotypes based on the mass of the variant DNA sequence, and have utilized this method for production scale SNP genotyping. We have combined a 4 μl PCR amplification reaction using 3 ng of genomic DNA with a secondary enzymatic reaction (mini-sequencing) containing oligonucleotide primers that anneal immediately upstream of the polymorphic site, dideoxynucleotides, and a thermostable polymerase used to extend the PCR product by a single base pair. Mass spectrometry (MS) analysis of mini-sequencing reactions was performed using a MALDI-TOF instrument (Voyager-DE, Perseptive Biosystems, Framingham, MA). We performed both single and multiplex PCR and mini-sequencing reactions, and genotyped seven different variant sites in a random sample of 989 individuals. Genotypes generated with MS methods were compared with genotypes produced using a 5′ exonuclease fluorescence-based assay (Taqman, Applied Biosystems, Foster City, CA) and a gel-based genotyping protocol. Because multiple polymorphisms can be detected in a single reaction, the MS technique provides a cost-effective and efficient method for high-throughput genotyping. Hum Mutat 17:296–304, 2001. © 2001 Wiley-Liss, Inc.