ARMS-PCR Based SNP Analysis of MTHFR C677T Allele Using Syber Green in Pancreatic Tumour

Abstract

In human, carcinoma of pancreas, a rare disease and mortality rate is quite high in Indian population. Epidemiological studies support the hypothesis that folate metabolism regulate DNA stability and prevent cancer. Because folate have been linked to dietary supplement  and defect in folate metabolism have been increase risk of developing cancer shows adverse effect on health conditions. In the present study we have assess methylene tetrahydrofolate reductase gene polymorphism (MTHFR) 677 C → T using ARMS PCR based SNP analysis using Syber green in the cases of pancreatic tumour to determine the “risk factor”. Interestingly, our findings reveals that 33.0% frequency (one case) showing mutation of MTHFR 677 TT genotype (rare type) in homozygous condition with Tm value 82.50°C  for mutant 677T allele shifted to 677C allele (83.50°C).Two cases (66%) showing CC (wild type) allele and Tm value 82.83°C for 677C allele. In MTHFR 677TT is a rare mutation and individuals show very low enzymatic activity due to the substitution of alanine to valine. The study further continue to confirm the mutation by   visualization on agarose gel electrophoresis of the same PCR product, again showing (Lane- 2) the band of 50 kb of mutant 677TT allele in the same case, suggesting an relevant role of folate metabolism and subsequent impairment of aberrant DNA methylation during carcinogenesis with increasing “high risk” for the development of carcinoma due to allele TT mutation of MTHFR. However, large samples size is required to further confirm the association.