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The page below is a sample from the LabCE course PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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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 is dependent on the compatibility of the PCR primers used in the reaction. When designing and choosing primers for multiplex assays, it is important to consider the following:
  • Primer design
  • Primer annealing temperature
  • Primer concentration
  • Concentration of dNTPs, magnesium chloride (MgCl2), polymerase, and salts
Primers are the determining factor of what region of the DNA will be amplified by PCR. The primer should be specific to the target. The selection of the sequence follows a detailed analysis of the DNA for each target, most commonly assisted by a variety of public domain computer programs. An example is the Basic Local Alignment Search Tool (BLAST®), which is 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. Accessed November 26, 2018.