Multiplex polymerase chain reaction (Multiplex PCR) refers to the use of
polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed st ...
to amplify several different DNA sequences simultaneously (as if performing many separate PCR reactions all together in one reaction). This process
amplifies DNA
Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
in samples using multiple
primers and a temperature-mediated
DNA polymerase
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create t ...
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
Dystrophin is a rod-shaped cytoplasmic protein, and a vital part of a protein complex that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix through the cell membrane. This complex is variously known as the costa ...
gene. It has also been used with the
steroid sulfatase gene. In 2008, multiplex-PCR was used for analysis of
microsatellite
A microsatellite is a tract of repetitive DNA in which certain Sequence motif, DNA motifs (ranging in length from one to six or more base pairs) are repeated, typically 5–50 times. Microsatellites occur at thousands of locations within an organ ...
s and
SNPs. 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
Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) is a strain of coronavirus that causes COVID-19, the respiratory illness responsible for the COVID-19 pandemic. The virus previously had the Novel coronavirus, provisional nam ...
diagnostics.
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.
Applications
Some of the
applications of multiplex PCR include:
# Pathogen Identification
# High Throughput SNP Genotyping
# Mutation Analysis
# Gene Deletion Analysis
# Template Quantitation
# Linkage Analysis
# RNA Detection
# Forensic Studies
# Diet Analysis
References
{{Portal bar, Biology
Molecular biology
Laboratory techniques
Amplifiers
Polymerase chain reaction