Mutation Detection Using RT-PCR-RFLP

Genetic analysis by restriction fragment length polymorphism (RFLP) is one of the most common methods used to examine nucleic acids for the presence of known sequence variants. A segment that is to be searched for a mutation is amplified from genomic DNA or cDNA, digested by the appropriate restriction enzyme, and then separated by agarose gel electrophoresis. Although RFLP analysis is a highly sensitive method that is easy to apply for the screening of known sequence variants, many common polymorphisms are the result of single-base substitutions that fail to create or remove any restriction site and, therefore, these cannot be readily typed by simple polymerase chain reaction (PCR) and RFLP analysis. However, the use of a mismatch PCR primer to artificially create a restriction site in the amplified product make it possible to overcome this disadvantage (1 ,2 ). The mismatch primer contains a one-or two-base mismatch near its 3′ end such that the amplified product incorporates or removes a restriction site for the appropriate endonuclease in the presence of a base substitution (3 –5 ). The protocol for this method is the same as standard PCR.