A modified simple RFLP-PCR method for single nucleotide polymorphism (SNP) typing

We describe a modified single nucleotide polymorphism (SNP) typing method based on the restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR). This is a simple, economical method without the need for special equipment. For most SNP loci, a common restriction endonuclease (Hind III, EcoR I or BamH I) recognizing site (RER) can be introduced into one allelic form, but not the other by two rounds of mismatched PCR. The flanking regions can be changed by as many as five bases after PCR amplification with specially designed mismatching primers so the genotypes can be distinguished after digestion of the PCR products with corresponding endonucleases.