Multiplex polymerase chain reaction

Multiplex polymerase chain reaction (Multiplex PCR) is a modification of polymerase chain reaction in order to rapidly detect deletions or duplications in a large gene. This process amplifies genomic DNA samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. 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]

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 genes 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.

Commercial multiplexing kits for PCR are available and used by many forensic laboratories to amplify degraded DNA samples. Commercial kits have a number of advantages over in-house multiplexing methods. Quality control measures are undertaken by the manufacturer of the kit and ensure that reactions are uniform across all kits. This avoids the preparation of PCR master mixes which require pipetting and use of multiple assay tubes, increasing the risk of operator error and contamination. This increased reliability allows profiles obtained from commercial kits to be admitted into court which is pivotal in large criminal trials. The use of specific kits over a number of laboratories also allows for profile results to be compared as long as the same STR markers have been used in each kit.

Applications

Some of the applications of multiplex PCR include:
1. Pathogen Identification
2. High Throughput SNP Genotyping
3. Mutation Analysis
4. Gene Deletion Analysis
5. Template Quantitation
6. Linkage Analysis
7. RNA Detection
8. Forensic Studies

Software

PrimerPlex - Software for Multiplex PCR Primer design

uMelt Batch 1.5 - Software for Predicting Melting Curves for Multiplex PCR Products

References

  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. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=339001. 
  2. ^ Ballabio A, Ranier JE, Chamberlain JS, Zollo M, Caskey CT (1990). "Screening for steroid sulfatase (STS) gene deletions by multiplex DNA amplification". Human Genetics 84 (6): 571–573. doi:10.1007/BF00210812. PMID 2338343. 
  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. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2275739.