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Mutation detection for the,,,K- rasand P16 genes

Mutations in the K-ras gene codon 12 region can lead to cancer, for example of the colon, pancreas, liver, spleen, stomach or lungs. The CDKN2A/P16 gene is a familial melanoma gene. Routine PCR and DNA sequencing methods can identify exactly which point mutation is present in patient tissue samples. Freshly frozen tumor sections direct from surgeries can be utilized, as well as archived paraffin-embedded specimens. Prior to DNA sequencing of K-ras, the nested PCR products are digested with a restriction enzyme and electrophoresed for quality and sizing purposes. A sample can be determined to be either wild-type or mutated simply by comparing the size of the PCR band to the size of the digested PCR band on a DNA chip. This analysis demonstrated the separation of PCR fragments from 135 bp to 106 bp. DNA sequencing is then utilized to verify the chip results. If a sample is shown to be mutated, sequencing can pinpoint the exact mutation. For P16 exon 3, the PCRs are electrophoresed on the Agilent 2100 bioanalyzer, purified, and sequenced. Heterozygous mutations can be resolved accurately within 1015 % of base pair length using the Agilent 2100 bioanalyzer. The Lab-on-a-chip technology is a novel and important as well as rapid step in these diagnostic and quality control assays. In this Application Note we demonstrate how extra bands on Agilents chip image correlate to mutated DNA sequences.
Technology that is currently available for SNP detection include the SpectruMedix platform, dHPLC, real-time PCR, DNA sequencing, the Nanogen cartridge platform, and the Affymetrix chip platform. We found that using the Agilent platform we could also detect single nucleotide polymorphisms, and verified this with sequencing analysis. The human K-ras gene is a member of the Ras family o
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