Sequencing of double-stranded polymerase chain reaction products for mutation analysis

This report describes a reproducible, straightforward approach to sequencing double-stranded DNA products from the polymerase chain reaction (PCR) for analysis of mutations. The sequencing protocol is a modification of that published by Kretz (Kretz et al., 1989) and has been successful in the hands of a number of investigators working on diverse projects. Following this procedure, PCR DNA products generated from bacterial sources (including pBR322 and F' derivatives), as well as cDNA and genomic DNA from both hamster and human cell lines, have been sequenced with equal success. Close attention to the molar ratio of nucleotides to double-stranded DNA template present during the labeling reaction ensures best results.