Methods (molecular, IHC, frozen)
Multiplex PCR

Topic Completed: 1 July 2010

Minor changes: 15 February 2019

Copyright: 2008-2018,, Inc.

PubMed Search: Multiplex PCR[TI] pathology free full text[sb]

Rodney E. Shackelford, D.O., Ph.D.
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Table of Contents
Definition / general
Cite this page: Shackelford R. Multiplex PCR. website. Accessed January 18th, 2021.
Definition / general
  • In multiplex PCR, several discrete sequences are amplified simultaneously in the same reaction using multiple primer pairs
  • The technique is used to verify that an amplifiable sequence is present in a specific sample or to amplify multiple sequences within one reaction
  • Usually the sequence being searched for is coamplified with a known housekeeping gene, which is always present and easily amplified
  • If only the housekeeping gene is amplified, then the other sequence is absent from the original sample
  • If neither sequence is amplified, then a PCR inhibitor is likely present
  • Up to 20 different reactions can be run simultaneously, thus lowering the amount of sample used, reducing the reagents consumed and collecting far more information per reaction, while simplifying data analyses
  • This technique is widely used in forensics, tissue identification, detection of different species of viral nucleic acids within cerebrospinal fluid and transplantation engraftment studies, where it can use single nucleotide polymorphisms and other variation in DNA sequences to identify tissue sample origins
  • The main disadvantages are the difficulty, sometimes extreme, in optimizing the PCR reaction so that each sequence amplification is roughly as efficient as the other reaction within the same reaction chamber and designing primers that will not interact with each other (i.e. primer dimmers), resulting in nonspecific amplifications
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