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.

https://www.future-science.com/doi/pdf/10.2144/97233rr01

Multiplex PCR: critical parameters and step-by-step protocol.

PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance.

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Multiplex PCR: Optimization and Application in Diagnostic Virology

The ability to select short DNA oligonucleotide sequences capable of binding solely to their intended target is of great importance in developing nucleic acid based detection technologies. Applicat...

https://www.future-science.com/doi/pdf/10.2144/04372ST03

AutoDimer: a screening tool for primer-dimer and hairpin structures.

Preimplantation genetic diagnosis

Fundamentals of Forensic DNA Typing , Fundamentals of Forensic DNA Typing , کتابخانه دیجیتال جندی شاپور اهواز

/pdf/fundamentals-of-forensic-dna-typing-2rl7l3h9ow.pdf

Fundamentals of Forensic DNA Typing

An Introduction to PCR Primer Design and Optimization of Amplification Reactions

Dystrophin gene deletions account for up to 68% of all Duchenne (DMD) and Becker (BMD) muscular dystrophy mutations. In affected males, these deletions can be detected easily using multiplex PCR tests which monitor for exon presence. In addition, quantitative dosage screening can discriminate female carriers. We previously analyzed multiplex PCR products by gel electrophoresis and quantitation of fluorescently labeled primers with the Gene Scanner™ in order to test carrier status. These multiplex PCR protocols detect DMD gene deletions adequately, but require up to 18 pairs of fluorochrome-labeled primers. We previously described two alternative fluorescent labeling strategies, each with approximately 1,000-fold greater sensitivity than ethidium bromide staining, which can be used to quantify the products of multiplex PCR. The first method uses the DNA intercalating thiazole orange dye TOTO-1 to stain PCR products after 20 cycles. In the second method, fluorescein-12,2′-dUTP is incorporated into products during PCR as a fluorescent tag for subsequent quantitative dosage studies. Both methods label all multiplexed exons including the 506 bp exon 48 fragment that is difficult to detect and quantify by standard ethidium bromide staining. Using this approach, we determined DMD/BMD carrier status in 24 unrelated families using a fluorescent fragment analyzer. Analysis of fluorochrome-labeled PCR products facilitates quantitative multiplex PCR for gene-dosage analysis. © 1993 Wiley-Liss, Inc.

Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies.

A system of four short tandem repeat loci (HUMVWF31A, HUMTH01, HUMF13A1, and HUMFES/FPS) has been tested in co-amplification with forensic (post-mortem and post-coital) DNA samples. Semiautomated DNA typing was employed to analyze polymerase chain reaction (PCR) products formed by extension of primers labeled with a fluorescent dye at the 5'-terminus. Most DNA extracts could be typed, although a few required the addition of bovine serum albumin or a pretreatment by ultrafiltration in order to obtain sufficient signal for typing. Balanced signals for the alleles were obtained frequently across the loci, although preferential amplifications of HUMTH01 was observed often with the forensic samples. Band splitting due to nontemplate nucleotide addition to the blunt ends of the amplimers was frequently detected for the DNA extracted from the forensic samples. A data-base was constructed for the African-American population and compared with a Caucasian database. Few differences were observed across the two populations, except at the locus HUMTH01. The fluorescence-based system facilitates large-scale databasing, because the PCR products run off the gel, allowing more than one set of samples to be analyzed per run. Polyacrylamide gel reuse did not diminish genotyping accuracy.

Forensic applications of a rapid, sensitive, and precise multiplex analysis of the four short tandem repeat loci HUMVWF31/A, HUMTH01, HUMF13A1, and HUMFES/FPS

Fluorescence-based, multiplex allele-specific PCR (MASPCR) detection of the ΔF508 deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene

James M. Robertson

Papers

An Introduction to PCR Primer Design and Optimization of Amplification Reactions

Duchenne/Becker muscular dystrophy carrier detection using quantitative PCR and fluorescence-based strategies.

Forensic applications of a rapid, sensitive, and precise multiplex analysis of the four short tandem repeat loci HUMVWF31/A, HUMTH01, HUMF13A1, and HUMFES/FPS

Fluorescence-based, multiplex allele-specific PCR (MASPCR) detection of the ΔF508 deletion in the cystic fibrosis transmembrane conductance regulator (CFTR) gene