Multiplex PCR

Multiplex PCR (M-PCR) is a demanding technique used for amplification of several discrete genetic loci with multiple PCR primer pairs in a single reaction. Multiplex PCR simultaneously answers several related questions about a specimen without the need for multiple individual PCR reactions. Multiplex PCR is commonly used for verification that amplifiable nucleic acid is present in the sample, for example, amplification of a housekeeping gene in addition to the gene sequence of interest, and to check for the presence of PCR inhibitors that can prevent amplification of target nucleic acid, for example, coamplification of an exogenously added internal control. Multiplex PCR often requires painstaking optimization of PCR conditions and careful design of the multiple primer pairs to prevent the generation of primer-dimers and other nonspecific PCR products that may interfere with the amplification of specific products. Touchdown PCR can be used with multiplex PCR if the primer pairs have different annealing temperatures. Concentrations of individual primer pairs may need to be optimized to account for different amplification efficiencies and competition between the primer pairs.

Examples of Applications of Multiplex PCR

1. Detection of enterovirus and herpes simplex virus (HSV) nucleic acids in cerebrospinal fluid (CSF)

2. Detection of pathogenic enteric bacteria in stool

3. Analysis of multiple BRCA1 loci in a breast cancer patient19

4. Identification of different bacteria in a respiratory infection20

5. Amplification of multide microsatellite loci for bone marrow engraftment analysis

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