IMPROVED SEPARATION OF PCR AMPLIFIED VNTR ALLELES BY A VERTICAL POLYACRYLAMIDE-GEL ELECTROPHORESIS

The effect of a stacking gel, the pH and crosslinking agent concentration on the resolution and sharpness of PCR amplified VNTR alleles in a vertical discontinuous polyacrylamide gel electrophoresis system was investigated. The experiments show that the use of a low crosslinking agent concentration, a stacking gel and a wide pH difference between the gel buffer and the electrophoresis buffer at the beginning of the electrophoresis resulted in reduced band width and increasing resolution in silver-stained polyacrylamide gels. The importance of sharp DNA fragments is especially emphasized when analyzing multi-allelic DNA loci, that exhibit alleles differing from only few bp to few dozen bp in length, such as variable number of tandem repeat (VNTR) or short tandem repeat (STR) loci.