A simple PCR-based method to follow and genotype alleles with single nucleotide changes

摘要

PCR-based detection of homozygous wild type and mutant alleles for . ( ) primers design allowing the introduction of an Acc65I site in the PCR product from the mutant allele. Blue nucleotides: sequence of the primers used; Red nucleotides: DNA alterations in the allele; Green nucleotide: PCR-introduced change. Small lettering, intronic sequence; capitals, exons ( ) Representative analysis on a 2,5% agarose gel. Lane L: ladder, the 500pb band is indicated; lanes 1: WT allele; lanes 2: mutant allele; lane 3: heterozygote animal. The fragment amplified from the WT allele is left undigested by Acc65I with a size of 358pb (white star), while the PCR fragment length obtained from mutant allele is 279pb after Acc65I digestion (red star, the remaining 79pb fragment is not visible here).