Reduction of heteroduplex formation in PCR amplification

Abstract

Heteroduplex formation is known to occur during mixed-template polymerase chain reaction (PCR) using universal primers, and may represent a serious problem in several PCR-based analyses. A common way to eliminate heteroduplex formation is to use reconditioning PCR. Because we detected that reconditioning PCR was not always sufficient to prevent heteroduplex formation, we focused on developing methods for the elimination of heteroduplexes during PCR. We detected that the heteroduplex to homoduplex ratio can be decreased by the addition of Taq polymerase and by a decrease in the number of PCR cycles. An appropriate combination of both of these approaches can be a method generally applicable to decrease the formation of heteroduplexes.