MUTATION DETECTION AND TYPING OF POLYMORPHIC LOCI THROUGH DOUBLE-STRAND CONFORMATION ANALYSIS

Variations, such as nucleotide substitutions, deletions and insertions , within genes can affect the function of the gene product and in some cases be deleterious. Screening for known allelic variation is import ant for determining disease and gene associations'. Techniques which t arget specific mutations such as restriction enzyme polymorphism and o ligonucleotide probe or PCR primer reactivity are useful for the detec tion of specific mutations, but these techniques are not generally eff ective for the identification of new mutations. Approaches for measuri ng changes in DNA conformation have been developed, based on the princ iple that DNA fragments which differ in nucleotide composition exhibit different mobilities after separation by polyacrylamide gel electroph oresis (PAGE; refs 2,3). Here we describe a conformation-based mutatio n detection system, double-strand conformation analysis (DSCA), which provides a simple means to detect genetic variants and to type complex polymorphic loci, We demonstrate the application of DSCA to detect ge netic polymorphisms such as a single-nucleotide difference within DNA fragments of up to 979 base pairs in length. We present the applicatio n of DSCA in detecting four different mutations in the cystic fibrosis gene (CFTR) and 131 different alleles encoded by HLA class I genes.