This study was approved by the Jamia Millia Islamia and Sher-e-kashmir Institute of Medical Sciences Bioethics Committee. Informed consent was obtained from all participants of the study. The total sample consisted of 450 colorectal cases (CRC group) and 450 apparently healthy (Healthy group) subjects. The information of age, smoking status, gender, tissue grade, tumor node involvement etc. is summarized in Table 3.
Sample collection, DNA isolation and Genotyping of SNP:
Blood samples were collected in EDTA coated vials. Human Genomic DNA was extracted as per laboratory protocol from control and case subjects using Phenol-Chloroform method. DNA was quantified using NanoDrop ND-1000 spectrophotometer. Extracted DNA was amplified by PCR for rs2253310 and rs4946936 using the oligonucleotide primers given in the Table1. 100ng of isolated DNA, 0.5µl of forward and reverse primers, 200µl of 200 μM of each deoxynucleotide triphosphates (dNTPs) (deoxyadenosine triphosphate [dATP], deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP) along with 1 unit of DNA polymerase was used. PCR cycling procedures were as follows: 95°C for 5minutes, 34 cycles of 94°C for 30 seconds, 58°C (rs2253310) and 59°C (rs4946936) for 30 seconds and elongation at 72°C for 30 seconds. The final amplicon elongation was performed at 72°C for 5 minutes. PCR products were visualized by electrophoresis on 1.5% agarose gel containing 2 µl of ethidium bromide.
Analysis of PCR products using RFLP and SSCP
rs2253310C>G was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) FIG. 1(a) and (b). The PCR product (321bp) was digested by DpnI restriction enzyme (THERMO SCIENTIFIC) at 37°C overnight. The C- allele carrying PCR product is cleaved once by the enzyme giving rise to two fragments (127bp and 194bp), where as the G-allele gave only one band (321bp). The digested products were separated by electrophoresis on 2.5% ethidium bromide-stained agarose gels and visualized under UV transilluminator.
rs4946936C>T was analyzed using PCR-RFLP. The PCR products of (224bp) were then digested with BfmI (THERMO SCIENTIFIC) overnight at 37°C for 1-16 hours as per the manufacturers protocol. The sizes of the restriction fragments of the PCR products were 152bp and 72bp for the wild type homozygote (CC), 224bp for the TT genotype and 224bp, 152bp and 72bp for the heterozygote (CT). Since sequence variation often goes undetected, because each enzyme used scans only a small number of potentially variable nucleotide positions, therefore, single-strand conformation polymorphism (SSCP), further provided useful alternative for the direct analysis of sequence variation.
SSCP assay was performed in 16µl reaction mixture containing 4µl of PCR product and 12µl of denaturing loading dye (95% formamide, 20 mM ethylenediaminetetraacetic acid (EDTA), 0.05% bromophenol blue, and 0.05% xylene cyanol) and heat- denatured at 95°C for 10 min and afterward immediately snap-chilled on ice prior to polyacrylamide gel electrophoresis (PAGE). Single-stranded amplicons were loaded onto 8 % polyacrylamide gel(29:1 acrylamide to bisacrylamide) containing 50 mM of Tris– borate (pH 7.5) and 2.5 mM of EDTA prepared with 1× Tris–borate–EDTA (TBE) buffer and electrophoresed for 12-14 h at constant temperature. The gel was subjected to silver nitrate staining to visualize the SSCP bands (Fig. 1(c) and 2(c). Samples demonstrating differences in band-shifts with respect to the wild-type bands were categorized as mutants.
To analyze the genotype of rs2253310 and rs4946936 in a more accurate and fast manner, we used direct sequencing method to confirm the results of RFLP and PCR coupled SSCP. The genotypes of all the 10% samples, determined by direct sequencing Fig. 1(d) and 2(d) showed the exact pattern as shown by RFLP and SSCP. Discrepancies were not observed.
Statistical package for social sciences (SPSS) software version 17.0 was used for data analysis. 3x2chi-square test was performed to compare the overall distribution of genotype and allelic frequencies of cases and controls. The odd ratios (OR) were calculated from genotypic frequency and allelic frequency with 95% confidence interval (95%CI) with adjustment for age. All P-values were corrected for multiple comparisons according to Bonferroni method. LD pattern and population haplotype frequencies for the SNPs were estimated using SHEsis (freely available software). Fisher’s exact test was performed for determining the association of haplotypes with diseased condition.