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

also viewed on the chip before wasting the reagents and time to sequence it. Often formalin-fixed tissues or lymph node metastases do not present good quality DNA template for PCR reactions. The chip images were a qualitative as well as a quantitative tool that indicated if a sample needed to be repeated to increase its yield. The resolution was also better on the Agilent 2100 bioanalyzer platform than on ethidium-stained agarose gels. Chips were safer to work with and preparation time was reduced dramatically.
Materials and methods
Tissues
Sample blocks were obtained through the National Cancer Institutes Naval Medical Oncology Branch. Ten 5-m sections were deparaffinized and extracted using the Series III kit from Xtrana. For P16, DNA was received already extracted in barcoded 96- well plates. We quantitated the DNA via spectrophotometry and it was diluted to 10 ng for starting material.
PCR
Nested PCR was performed for K-ras with final reaction concentrations of 0.25 M each primer3 (Invitrogen), 0.2 mM dNTPs (Roche), 1X PCR buffer (Roche), 1.25 U Taq polymerase (Roche), HPLC-grade water (Fisher), and Amp Enhancer (Xtrana). 50 l were added to the bound DNA on the Xtrana tubes. For samples not extracted using the Xtrana kit, the Amp Enhance solution was eliminated and solubilized template was incorporated. This reaction was cycled 18 times using ABIs 9700 thermal cycler under the following conditions: 94 C for 45 seconds, 55 C for 1 minute, and 72 C for 90 seconds. This was purified using the Wizard kit (Promega). The eluant was used as template in a BstNI reaction with BSA (New England BioLabs). This reaction was purified using the MinElute kit (Qiagen). Then an aliquot was used in the second round of PCR; the same conditions
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