Improved Polymerase Chain Reaction Method for Genotyping Lep∨ob Mouse Using Mismatched Allele-Specific Primers
2009
Rhee, S.D., Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea | Kim, H.Y., Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea | Jung, W.H., Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea | Sohn, J.Y., SolGent Co. Ltd., Daejeon, Republic of Korea | Kho, M.S., SolGent Co. Ltd., Daejeon, Republic of Korea | Cheon, H.G., Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea
The previously reported methods for genotyping Lep∨ob mice have shortcoming of either inconveniency or poor reproducibility. Therefore we improved up these protocols by adding the mismatched base pairs in the allele-specific primers for stable discrimination of single base pair mismatch and amplifying the polymerase chain reaction (PCR) product in one tube. This new simplified genotyping method results in higher reproducibility even with crudely prepared DNA. When the method was used to select heterozygotic breeders to produce obese mice, 256 pairs of the all 287 mating pairs produced obese offspring, providing 90% or greater genotyping efficiency. This method using PCR with mismatched allele specific primers is appropriate and efficient for genotyping a large number of Lep∨ob mutants.
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