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 PCR’s
are electrophoresed on the Agilent 2100 bioanalyzer, purified, and
sequenced. Heterozygous mutations can be resolved accurately within
10–15 % 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 Agilent’s chip image correlate to
mutated DNA sequences.
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