Simultaneous genotyping of single nucleotide polymorphisms in the IL-6, IL-10, TNFalpha and TNFbeta genes.
Single nucleotide polymorphisms (SNP) in the human IL-6, IL-10, TNFalpha and TNFbeta genes have been associated with gene function and susceptibility to disease. In this study, primers containing mismatches at 1-3 nucleotide positions were designed to incorporate a new restriction site recognized by endonucleases AlwNI, BcgI, BglI, BsaBI, BslI, BstXI, EcoNI or XcmI for genotyping SNPs in the IL-6 gene (position - 174), IL-10 gene (positions -592 and -1082), TNFalpha gene (positions -238, - 308 and -863) and TNFbeta gene (position + 249) by mismatched polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP). Our results show that appropriately designed BslI-based mismatched PCR/RFLP assays can be successfully used to determine the genotypes for approximately 40% of SNPs. The mismatched PCR strategy can be coupled with multiplex-amplification to enable simple and rapid determination of several SNP genotypes in a single reaction.
Tseng
L-H
LH
Department of Medical Genetics, Pathology and Oncology, National Taiwan University Hospital and Graduate Institutes of Clinical Medicine and Cancer Research Center, National Taiwan University, Taipei, Taiwan.
Chen
P-J
PJ
Lin
M-T
MT
Singleton
K
K
Martin
E G
EG
Yen
A-H
AH
Chuang
S-M
SM
Martin
P J
PJ
Hansen
J A
JA
D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers; D005838:Genotype; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics; D008233:Lymphotoxin-alpha / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D016207:Cytokines; D017931:DNA Primers; D015850:Interleukin-6; D008233:Lymphotoxin-alpha; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10