Multiplex PCR is a widespread molecular biology technique widely used in Short Tandem Repeat and Single Nucleotide Polymorphism Analysis. Multiplex PCR is a polymerase chain reaction in which multiple DNA templates can be amplified by using multiple primer pairs in a single reaction. The method was first described in 1988 to detect deletions in the dystrophin gene.
Optimization of reagents is an important step in Multiplex PCR so that an equal amount of amplicons are produced in a PCR reaction. Amount and concentration of primers, Deoxy nucleotide triphosphate, adjuvants (Bovine serum albumin, Dimethyl sulfoxide, glycerol), Buffer, Magnesium Chloride, DNA template, and polymerase should be optimized in 25 microliters. Adjuvants aids in easy template denaturation in multiplex PCR.
Primers should be 18-30 bases in length and shorter primer lengths are not specific to the template DNA. The primers must posses similar annealing temperature (55 to72C) to ensure specific priming. Melting temperature should be equal to or less than 5C differences. Primers should not interact among themselves forming primer dimers and Guanine-Cytosine content should be 40-60 %.
United States uses 13 Combined DNA Index System (CODIS) STR loci for doing multiplex PCR simultaneously and input the data onto the CODIS database for identifying criminals or missing people. In multiplex PCR, extension time should be increased to fully copy all target DNA. The procedure saves money, time and expedites forensic casework in labs.