Polymerase chain reaction haplotyping using 3' mismatches in the forward and reverse primers: application to the biallelic polymorphisms of tumor necrosis factor and lymphotoxin alpha.
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 alpha (LT-alpha). 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-alpha 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-alpha 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-alpha. A total of 11 TNF-LT-alpha haplotypes were determined from apparent homozygous controls and statistical analysis.
Fanning
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GC
NOS, Oxford Transplant Centre, United Kingdom. gfanning@bioch.ox.ac.uk
Bunce
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M
Black
C M
CM
Welsh
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KI
D000483:Alleles; D001483:Base Sequence; D017931:DNA Primers; D005787:Gene Frequency; D005838:Genotype; D006239:Haplotypes; D006801:Humans; D015810:Linkage Disequilibrium; D008233:Lymphotoxin-alpha / Q000235:genetics*; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D009626:Terminology as Topic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D017931:DNA Primers; D008233:Lymphotoxin-alpha; D009692:Nucleic Acid Heteroduplexes; D014409:Tumor Necrosis Factor-alpha