Polymerase chain reaction haplotyping using 3′ mismatches in the forward and reverse primers: application to the biallelic polymorphisms of tumor necrosis factor and lymphotoxin

A polymerase chain reaction with sequence-specific primers (PCR-SSP) system that operates under identical conditions to HLA phototyping was devised for characterizing polymorphisms in tumor necrosis factor (TNF) and lymphotoxin α (LT-α). Mismatches at the 3′ end were incorporated into the forward and reverse primers of each PCR so as to unequivocally establish the cis / trans status between the biallelic sites. Three previously described biallelic polymorphisms in TNF and three in LT-α were characterized in a 24-reaction PCR-SSP system. The method was used to genotype 20 cell lines and 201 HLA class I and II typed controls from the United Kingdom at the TNF and LT-α loci. Population frequencies of TNF haplotypes were determined as was linkage disequilibrium with HLA-A, B, Cw, DRB1 and DQB1 loci. In each gene there were 8 theoretical polymorphic combinations; 4 were observed in TNF and 4 in LT-α. A total of 11 TNF-LT-α haplotypes were determined from apparent homozygous controls and statistical analysis