Improving sequencing quality from PCR products containing long mononucleotide repeats

Abstract

Stutter products are a common artifact in the PCR amplification of frequently used genetic markers that contain mononucleotide simple sequence repeats. Despite the importance of accurate determination of nucleotide sequence and allele size, there has been little progress toward decreasing the formation of stutter products during PCR. In this study, we tested the effects of lowered extension temperatures, inclusion of co-solutes in PCR, PCR cycle number, and the use of different polymerases on sequence quality for a set of sequences containing mononucleotide A/T repeats of 10–17 bp. Our analyses showed that sequence quality of mononucleotide repeats ≤15 bp is greatly improved with the use of proofreading DNA polymerases fused to nonspecific dsDNA binding domains. Our findings also suggest that the number of nucleotides with which the DNA polymerase interacts may be the most important factor in the reduction of slipped-strand mispairings in vitro.