Selection for 3′-End Triplets for Polymerase Chain Reaction Primers

Abstract

Primer extension by thermostable DNA polymerase in PCR starts from the 3'-end of a primer. If the PCR starting process fails, the entire PCR fails. Primer sequences at the 3'-end often interfere with success in PCR experiments. Over 2000 primer sequences from successful PCR experiments used with varieties of templates and conditions were analyzed for finding frequencies of the 3'-end triplets. This chapter discusses a trend in 3'-end triplet frequencies in primers used in successful PCR experiments and proposes requirements for the 3'-end of a primer. Finally, a method to select primers with the best 3'-end triplets is introduced based on the 3'-end analysis result.