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

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.     

Allelic Variation in the Porcine MYF5 Gene Detected by PCR–SSCP

The MYF5 gene has been reported to be integral to muscle growth and development, and hence it has been considered as a candidate gene for meat selection programs in pig. To ascertain whether there was variation in the porcine MYF5 gene, we have developed a method of PCR–single-strand conformational polymorphism (PCR–SSCP) analysis. In this study, two coding regions of the MYF5 gene were investigated. Four unique SSCP patterns were detected in exon 1 and three patterns were identified in exon 3. Two SNPs detected in exon 1 led to a non-synonymous alanine/proline substitution. A nucleotide change in exon 3 did not affect the amino acid sequence. Five extended haplotypes were observed across the two regions. The variation detected in this study might underpin the development of gene markers for improved muscle growth in pig breeding.