A Novel PCR-RFLP Detection Method Using an Optimized Set of Restriction Enzymes

A number of new and useful mutation detection methods have evolved in recent years enabled by the advent of polymerase chain reaction (PCR) (Grompe, 1993). These methods can be divided into two groups: (1 ) the PCR-based techniques aimed at scanning DNA sequences for unknown mutations and (2 ) the techniques for identification of known polymorphisms. The essence of the first group is sensitivity, and this group is represented by single-strand conformation polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HA), RNase A cleavage, chemical mismatch cleavage, Escherichia coli mismatch repair enzyme-based analysis (Grompe, 1993), and restriction endonuclease fingerprinting (Liu and Sommer, 1995). Once a polymorphism is detected by the techniques described above, there is a clear necessity to switch to a simpler technique. The methods designed for simplicity and speed of genotyping large numbers of individuals are represented by oligodeoxynucleotide hybridization assay, the oligonucleotide ligation assay, allele-specific PCR amplification or, if the DNA variation is detected by a restriction enzyme (RE), PCR-restriction fragment length polymorphism (RFLP) (Grompe, 1993).