Km genotyping by polymerase chain reaction (PCR) using allele-specific amplification primers

We developed a method for identifying genetic polymorphisms of the human immunoglobulin kappa light chain, namely the Km allotypes, by allele-specific amplification by means of the polymerase chain reaction (PCR). This procedure consisted of first and second PCR. In the first PCR, the 353 bp fragment of the human immunoglobulin kappa light chain constant gene was amplified without differentiating individual alleles. In the second PCR, the specific sequence in each of the three alleles Km*1, Km*1,2 and Km*3, in the first PCR product was specifically amplified using allele-specific primers. The product of the second PCR was separated by electrophoresis on a polyacrylamide gel and the band was observed by means of UV trans-illumination after staining with ethidium bromide. From DNA extracted from lymphocytes, the specific sequence of each Km allele was selectively amplified, and the Km genotype was identified. Moreover, the Km genotype could be ascertained from whole blood, saliva and hair roots without DNA extraction. No genetic contradiction was found in the results of Km genotyping among parents and their children. The estimated gene frequency of 115 Japanese individuals living in Okayama Prefecture, Japan, was: Km* 3 = 0.739 and Km*1,2 = 0.261.