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.

Multiplex PCR: Critical Parameters and Step-by-Step Protocol

Abstract: By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.

Single-step species identification of bivalve larvae using multiplex polymerase chain reaction

Abstract: One of the biggest obstacles to studying recruitment variation in marine bivalves is the need to collect and process large numbers of plankton samples. Larval bivalves are notoriously difficult, if not impossible, to identify to species using morphological criteria alone. Remote time-series collections could satisfy the sampling challenge, but efficient identification techniques must be developed to obtain species-specific data. Thus, we have developed a multiplex polymerase chain reaction (PCR) identification assay in which a single reaction is capable of accurate and efficient discrimination of five target bivalve species based on the size of cytochrome oxidase I products. The assay was tested with cultured and field-sampled larvae as well as adult genomic DNAs. Using a single whole larva as template, multiplex PCR reactions were capable of discriminating among the commercially important bivalves: Mercenaria mercenaria, Argopecten irradians, Mulinia lateralis, Spisula solidissima and Mya arenaria. Overall accuracy was 92%, including very few false positives. The efficiency of this assay stems from its ability to discriminate multiple target species with a single molecular step that ultimately can be automated to process large numbers of larvae.