Primer Design

While most PCR reagents are commercially available for purchase, many laboratories may choose to develop their own. Since multiplex PCR is the simultaneous detection of more than one target sequence in a single reaction tube using more than one primer pair, the co-amplification of two or more targets in a single reaction depends on the compatibility of the PCR primers used in the reaction. When designing and choosing primers for multiplex assays, it is essential to consider the following:

Primers determine which region of the DNA will be amplified by PCR. The primer should be specific to the target. Sequence selection follows a detailed analysis of the DNA for each target, most commonly assisted by various public-domain computer programs. An example is the Basic Local Alignment Search Tool (BLAST^®), an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences. BLAST^® is available on the National Institutes of Health website at http://blast.ncbi.nlm.nih.gov/Blast.cgi.