Detection of sequence variation and genotyping of polymorphic genes using polymerase chain reaction stem-loop conformational polymorphism analysis

Abstract

Here we describe a technique we have named polymerase chain reaction stem-loop conformational polymorphism (PCR-SLCP) analysis for screening DNA sequence variation. This technique uses PCR primers with adapters at the 5' ends to generate amplicons containing inverted terminal repeat sequences. These enable the formation of specific conformers for single-stranded molecules on denaturation, and sequence variation in the loop region may affect the structure of these and their mobilities during electrophoresis. The technique has the ability to resolve sequence variation in amplicons that cannot be resolved using PCR single-strand conformational polymorphism (PCR-SSCP). PCR-SLCP is simple and sensitive, and the results are highly repeatable.