遺伝子変異解析における電気泳動法
PCR (polymerase chain reaction) 法, ミスマッチPCR法
1999 年 43 巻 1 号 p. 1-7
詳細
1999 年 43 巻 1 号 p. 1-7
Rapid progress in the molecular technology has stimulated attempts to establish genetic diagnosis of human disease. Practical use of DNA amplification method proved to be very useful in genetical analysis. Polymerase chain reaction (PCR) is one of the most popular method for DNA amplification technology. There are some point mutations which can be detected by the restriction endonuclease digestion of amplified DNA. Such technology is generally called as PCR-RFLP (restriction fragment length polymorphism). However, some mutations do not effect on recognition sites of any restriction endonucleases. In those cases, a new primer can be designed to create any recognition sites. The primer and PCR using the primer are called as mismatched primer and mismatched PCR, respectively. Before and after restriction endonuclease digestion of amplified DNA, they are electrophoresed on agarose gel or polyacrylamide gel to distinguish the cleaved bands. It is important to confirm that the amplified DNAs do only contain the target DNA, without non-specific DNAs. Electrophoretic methods are useful for this purpose. In this experimental protocol we describe the procedures for PCR techniques, restriction endonuclease digestion, and mismatched PCR. Also we describe some examples using these procedures to detect genetic mutations in lactate dehydrogenase and serum cholinesterase genes.
