Clinico-pathologic Features and Predictive Factors for Colorectal Cancer Outcome
in the Kingdom of Saudi Arabia
Supplementary Material
Assay to Detect Mutant KRAS
KRAS mutations were assessed in paraffin embedded tissuefrom biopsy tissue as well asnormal colorectal mucosa samples. Formalin-fixed, paraffin-embedded tumor sections were deparaffinizedand air dried, and DNA was isolated using proteinase K and a DNeasy minispin column (Qiagen, Valencia, CA). Mutant. KRAS assay identified seven somatic mutations located in codons 12 and 13 (Gly12Asp Gly12Ala, Gly12Val, Gly12Ser, Gly12Arg, Gly12Cys, and Gly13Asp) usingallele-specific real-time polymerase chain reaction. 19,20 To prevent any cross contamination, disposable scalpel blades were used to cut 10µm thickness sections from each patient sample. The scrapped tissue was de-paraffinized by washing with xylol, re-hydrated with absolute ethanol and dried to get rid of ethanol. DNA was extracted using DNA Extraction kit (High Pure template preparation kit, Roche. Pleaston, New Jersey, USA) according to the manufacturer’s instructions. DNA quality and concentration was determined prior to setting up PCR.
KRAS mutational analysis:
DNA isolated from the sample was quantified and amplified by polymerase chain reaction (PCR) using primers to exon 2 of the KRAS gene. PCR products are subjected to single nucleotide primer extension to detect mutations at codons 12 and 13as previously reported 17 with minor modifications. Briefly, PCR conditions were 95°C for 10 min, followed by 35 cycles of 95°C for 30s, 60°C for 45s, and 72 °C for 45 followed by a final extension for 7 min at 72°C. To remove excess primers, PCR products were purified with Exonuclease I (EXOSAPit, Amersham? (Exol, Amersham BiosciencesUK) and shrimp alkaline phosphatase (Amersham Biosciences, UK). Mutational analysis of the purified PCR products was performed by direct sequencing with Big Dye Terminator kit v1.1 (Applied Biosystems, Boston, MA, USA). Primer extension products are analyzed using capillary gel electrophoresis and fluorescence detection. False positive or negative results may occur for reasons that include genetic variants or somatic heterogeneity of the tissue samples. DNA extracted from three cell lines K562, A549 and HCT116 were used as a negative and positive controls.
II. BRAF Mutation analyses
BRAF mutations were detected utilizing direct sequencing. One hundred nanograms of DNA was amplified in a 20 µL reaction volume containing 2 µL of 10× buffer (Roche), 1.7-2.5 µM of MgCl2, 0.3 µM of each primer pair, 250 µM of dNTPs, and 2.5 unit of DNA polymerase (Roche). The PCR primers were 5'-TGCTTGCTCTGATAGGAAAATG-3' (forward) and 5'-AGCATCTCAGGGCCAAA AAT-3' (reverse). The PCR cycling conditions were as follows: an initial denaturation at 95℃ for 30 seconds, an annealing at 61℃ for 90 seconds decreasing by 0.5℃ every cycle until it reached 57℃, and an elongation step at 72℃ for 90 seconds. This procedure was followed by an additional 30 cycles with an annealing temperature at 57℃, and a final elongation step at 72℃ for 10 minutes. The PCR products were processed for the DNA sequencing reaction using the ABI-PRISM BigDye Terminator version 3.1 (Applied Biosystems) with both forward and reverse sequence-specific primers. Sequence data were generated using the ABI-PRISM 3100 DNA Analyzer (Applied Biosystems). In the analyses on the serially diluted DNA from the BRAF mutant cell line SNU-790, the analytical limit of detection was 12.5%.
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