Restriction Site Generating PCR

Some mutations create or abolish RE recognition sites and can easily be detected by PCR-RFLP. Unfortunately, most polymorphisms or mutations do not alter an RE recognition site. In restriction-site generating PCR (RG-PCR) (and a related research technique called PCR-mediated site-directed mutagenesis [PSDM]), an artificial RE recognition site is generated during PCR using a specially designed PCR primer.17,18 The primer contains a base mismatch to the template DNA adjacent to the variable base of the mutation that creates an RE recognition site in the PCR product. The mismatched base in the primer is located near or at the 3' end of the primer, which is near or adjacent to the variable base of the mutation, and together they create a novel restriction site within either the mutant or wild-type amplicon. The presence or absence of the RE recognition site is determined from the pattern of digested fragments by gel electrophoresis. Not all sequences are amenable to the generation of a restriction site, and the amplification efficiency is often decreased due to destabilization of the primer with the mismatch.

Examples of Applications of RG-PCR

1. Identification of KRAS codon 12 mutations in colon cancer19

2. Identification of mutations in the CTFR gene in cystic fibrosis

3. Identification of mutations in the ATM gene in ataxia-telangectasis

Was this article helpful?

0 0

Post a comment