Genotyping the Mouse Severe Combined Immunodeficiency Mutation Using the Polymerase Chain Reaction with Confronting Two-Pair Primers (PCR-CTPP)

Abstract

An allele specific polymerase chain reaction with confronting two-pair primers (PCR-CTPP) was developed as an assay for genotyping the mouse Prkdc^scid gene mutation (former name scid). The reverse primer (WR) was designed to include the antisense nucleotide (A) specific for the wild type allele at the 3’ end with the counterpart forward primer (F) upstream. The other forward primer (MF) was designed to include the sense nucleotide (A) specific for the Prkdc^scid mutation at the 3’ end with the other counterpart reverse primer (R) downstream. PCR was performed in a single tube with these two pairs of primers. The products specific for each allele extended by F/WR (101 bp) or MF/R (180 bp) were visualized with common PCR products (257 bp) extended by F/R, and three genotypes of mice (Prkdc^scid/Prkdc^scid, Prkdc^scid/+, and +/+) were clearly distinguished.