LIFE09200604007 Tabish - Homi Bhabha National Institute

Results
Objective 3
To genotype selected candidate genes involved in DNA repair, carcinogen
metabolism, apoptosis and cell cycle regulation.
The association of various gene polymorphisms with cancer has been frequently
reported. The candidate genes influencing susceptibility to cancer includes genes falling
mainly in important carcinogenesis pathways of carcinogen metabolism, DNA repair,
cell cycle regulation and cell death control. Variation in any of these mechanisms could
result in accumulation of cell with genetic alteration in critical genes leading to
tumourigenesis. In the present study 22 single nucleotide polymorphisms (SNPs) in
candidate genes involved in DNA repair, apoptosis, cell cycle regulation and
carcinogen metabolism were genotyped and a G Score was calculated from the number
of variant alleles.
4.3.1 Genomic DNA extraction
High molecular weight genomic DNA was isolated from whole blood or the pellet
obtained after isolating PBLs from the whole blood for cell line preparation. The
quality of DNA was assessed on 0.8% agarose gels stained with ethidium bromide.
Approximately 5-20μg of good quality DNA was obtained from each sample. Extreme
care was taken to avoid cross contamination of samples. DNA was diluted to a
concentration of 50ng/μl for performing PCR amplification while the stock DNA was
stored at 0 °C till further use.
4.3.2 Genotyping of genes by PCR-RFLP
Genotyping of 15 SNPs was performed by PCR and PCR-RFLP in 30 samples. Each
sample was subjected to PCR for amplification of the genes of interest and to confirm
the successful amplification PCR products were run on agarose gel. Multiplex PCR was
done to assess the null genotype of GSTT1 and GSTM1 genes. In the PCR, to
investigate null genotype, IFN was used as internal control to rule out amplification
failure. For RFLP, PCR products were digested with respective restriction enzymes and
the digested products were analyzed on 2% agarose gel (Fig. 36a, Fig. 36b, Fig. 36c).
Depending upon the obtained digested product the genotype was recorded as
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