Multiplex polymerase chain reaction

Multiplex polymerase chain reaction (Multiplex PCR) refers to the use of polymerase chain reaction to amplify several different DNA targets (genes) simultaneously (as if performing many separate PCR reactions all together in one reaction). This process amplifies genomic DNA 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]

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. 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.

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
9. Diet Analysis

Software

Visual OMP - Software for Multiplex PCR Primer design

PrimerPlex - Software for Multiplex PCR Primer design

uMelt Batch 1.5 - Software for Predicting Melting Curves for Multiplex PCR Products (sum of curves)

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.
  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.