Optimisation of multiple PCR using a combination of Full Factorial Design and three-dimensional Simplex optimisation method

Abstract

An alternative approach for optimisation of multiple polymerase chain reaction (PCR) by means of experimental design techniques is described. In initial experiments, the effects of independent variables: deoxyribonucleotides (dNTPs), Mg²⁺ and primers on multiple PCR were determined by Full Factorial Design. According to the results, each factor (stand-alone or composed ones) influences the amplification of individual genes in multiple PCR in a different way. A three-dimensional Simplex optimisation strategy was used for studying and consequently for improving multiple PCR yields. Using optimised concentrations of dNTPs, Mg²⁺ and primers in PCR, an improved simultaneous amplification of pkaC and pepC was achieved. Moreover, also in duplex RT-PCR the optimised PCR significantly increased the yield of both transcripts. The outlined optimisation strategy could be adopted to any multiple PCR or RT–PCR.