Multiplex polymerase chain reaction

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Multiplex polymerase chain reaction (Multiplex PCR) refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously (as if performing many separate PCR reactions all together in one reaction). This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR.

Multiplex-PCR was first described in 1988 as a method to detect deletions in the dystrophin gene.[1] It has also been used with the steroid sulfatase gene.[2] In 2008, multiplex-PCR was used for analysis of microsatellites and SNPs.[3] In 2020, RT-PCR multiplex assays were designed that combined multiple gene targets from the Center for Diseases and Control in a single reaction to increase molecular testing accessibility and throughput for SARS-CoV-2 diagnostics. [4]

Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple sequences at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis. Alternatively, if amplicon sizes overlap, the different amplicons may be differentiated and visualised using primers that have been dyed with different colour fluorescent dyes. Commercial multiplexing kits for PCR are available and used by many forensic laboratories to amplify degraded DNA samples.


Some of the applications of multiplex PCR include:

  1. Pathogen Identification[5]
  2. High Throughput SNP Genotyping[6]
  3. Mutation Analysis[7]
  4. Gene Deletion Analysis[8]
  5. Template Quantitation[9]
  6. Linkage Analysis[10]
  7. RNA Detection[11]
  8. Forensic Studies[12]
  9. Diet Analysis[13]


  1. ^ Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT (1988). "Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification". Nucleic Acids Research. 16 (23): 11141–11156. doi:10.1093/nar/16.23.11141. PMC 339001. PMID 3205741.
  2. ^ Ballabio A, Ranier JE, Chamberlain JS, Zollo M, Caskey CT (1990). "Screening for steroid sulfatase (STS) gene deletions by multiplex DNA amplification" (PDF). Human Genetics. 84 (6): 571–573. doi:10.1007/BF00210812. hdl:2027.42/47626. PMID 2338343. S2CID 18579745.
  3. ^ Hayden MJ, Nguyen TM, Waterman A, Chalmers KJ (2008). "Multiplex-ready PCR: a new method for multiplexed SSR and SNP genotyping". BMC Genomics. 9: 80. doi:10.1186/1471-2164-9-80. PMC 2275739. PMID 18282271.
  4. ^ Perchetti, GA; Nalla, AK; Huang, ML; Jerome, KR; Greninger, AL (2020). "Multiplexing primer/probe sets for detection of SARS-CoV-2 by qRT-PCR". Journal of Clinical Virology. 129: 104499. doi:10.1016/j.jcv.2020.104499. PMC 7278635. PMID 32535397.
  5. ^ Järvinen, Anna-Kaarina; Laakso, Sanna; Piiparinen, Pasi; Aittakorpi, Anne; Lindfors, Merja; Huopaniemi, Laura; Piiparinen, Heli; Mäki, Minna (2009). "Rapid identification of bacterial pathogens using a PCR- and microarray-based assay". BMC Microbiology. 9: 161. doi:10.1186/1471-2180-9-161. PMC 2741468. PMID 19664269.
  6. ^ Myakishev, M. V. (2001). "High-Throughput SNP Genotyping by Allele-Specific PCR with Universal Energy-Transfer-Labeled Primers". Genome Research. 11 (1): 163–169. doi:10.1101/gr.157901. PMC 311033. PMID 11156625.
  7. ^ Morlan, John; Baker, Joffre; Sinicropi, Dominick (2009). "Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method". PLOS ONE. 4 (2): e4584. Bibcode:2009PLoSO...4.4584M. doi:10.1371/journal.pone.0004584. PMC 2642996. PMID 19240792.
  8. ^ Abbs, S; Bobrow, M (1992). "Analysis of quantitative PCR for the diagnosis of deletion and duplication carriers in the dystrophin gene". Journal of Medical Genetics. 29 (3): 191–196. doi:10.1136/jmg.29.3.191. PMC 1015896. PMID 1552558.
  9. ^ "Welcome | Forensic DNA Profiling Facility" (PDF).
  10. ^ Reis, Andre (1991). "PCR in Linkage Analysis of Genetic Diseases". PCR Topics. pp. 75–79. doi:10.1007/978-3-642-75924-6_15. ISBN 978-3-540-52934-7.
  11. ^ Miyakawa, Y.; Yoshizawa, H.; Mishiro, S.; Machida, A.; Akahane, Y.; Sugai, Y.; Tanaka, T.; Sugiyama, Y.; Okada, S.; Okamoto, H. (August 1990). "Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5'-noncoding region". The Japanese Journal of Experimental Medicine. 60 (4): 215–222. PMID 1963453.
  12. ^ "DNA Evidence: Basics of Analyzing".
  13. ^ Dunshea, Glenn (2009). "DNA-Based Diet Analysis for Any Predator". PLOS ONE. 4 (4): e5252. Bibcode:2009PLoSO...4.5252D. doi:10.1371/journal.pone.0005252. PMC 2668750. PMID 19390570.