Detection of single-base mutations by reaction of DNA heteroduplexes with a water-soluble carbodiimide followed by primer extension: application to products from the polymerase chain reaction

Abstract

A new method was developed for the detection of single-base mutations in DNA. The polymerase chain reaction was used to prepare DNA fragments of up to 1 kb. Fragments that differed by a single-base were combined, denatured and renatured to generate heteroduplexes. The heteroduplexes were reacted with a water-soluble carbodiimide under conditions in which the carbodiimide modified Gs and Ts that were not base paired. The DNA was then used as a template for primer extension with Taq DNA polymerase under conditions in which extension terminated at the site of the carbodiimide-modified base and generated a 32P-labeled fragment that was identified by polyacrylamide gel electrophoresis as a fragment smaller than the full length product. The procedure detected all four general classes of single-base mutations in several different sequence contexts. The site of the mutation was located to within about 15 bp. Extension with both a 5'- and a 3'-primer made it possible to confirm the site of the mutation in most DNA samples or detect a mutation in heteroduplexes even if a G or T in one strand was unreactive because of its sequence context. The procedure appears to have several advantages over previously published techniques.