Multiplex Polymerase Chain Reaction

Often, studies require the polymerase chain reaction (PCR) amplification of several nucleic acid targets. Examples include the amplification of several exons of the same gene for mutation detection (e.g., p53 or dystrophin) (1 –4 ), the amplification of several different genomes for infectious pathogen identification (5 –9 ), identification of the simultaneous expression of transcripts of interest (10 ), quantification of message or gene copy number (11 ,12 ), and the amplification of a target and control gene to test for the nucleic acid integrity (10 ). When these assays must be performed repetitively on multiple samples, the multiplex PCR, which allows for simultaneous amplification of 2–50 different targets (13 –15 ), is potentially very useful from a perspective of time, and money. However, because multiplex PCR calls for the simultaneous amplification of several different targets, PCR primers optimized for different targets can potentially interact both with other primers (forming the dreaded “primer-dimers”), or with PCR products (“amplicons”) produced by the successful and intended amplification of another target. This can potentially decrease the amplification efficiency of some, or all, targets. Thus, multiplex PCR often necessitates the investment of considerable time and effort. It probably should only be used in situations where the advantage of streamlining multiple assays clearly outweighs the investment of time and resources.