Chemically Modified Primers for Improved Multiplexed PCR

Abstract

Multiplex PCR is an advantageous technique used in PCR applications to amplify multiple targets in a single reaction. As useful as it is, this technique presents a new set of challenges that further complicates PCR setup. For example, reactions are more prone to off‐target amplifications such as mis‐priming and primer dimer due to the increased number of primer pairs. Furthermore, preferential amplification of certain targets leads to an unequal distribution of amplicon products, making quantification and detection of problematic targets extremely difficult. To improve upon the problems specific to multiplex PCR, we evaluated Hot Start modified primers which contain either one or two thermolabile 4‐oxo‐tetradecyl (OXT) modifications to prevent DNA polymerase extension at low‐stringent temperatures, and that are released after a Hot Start activation step. Herein, we find that the singly‐modified primers provide greater amplification efficiency, specificity, and yield in the multiplex amplification of DNA targets. In reverse transcriptase PCR (RT‐PCR), the doubly‐modified primers have been proven to be the optimal choice. The presence of two thermolabile protecting groups allows for an efficient one‐step RT‐PCR reaction that provides high specificity for multiple targets. TriLink's innovative technology represents a convenient tool for multiplex PCR amplification of DNA and RNA samples.