( fGTPases1. Mutant activated forms...)Mutation detection for the,,,K- rasand P16 genes

f GTPases1. Mutant, activated forms of Ras proteins, which are frequently observed in cancer, have an impaired GTPase activity rendering the protein resistant to inactivation by regulatory GAP proteins2. The ability to detect changes in the region of this gene which codes for activation is essential. For our purposes, this test was used to ultimately determine a cancer patients eligibility into a clinical trial for a peptide vaccine. The normal form of codon 12 codes for glycine. Known mutations observed at codon 12 are: aspartic acid, valine, serine, cysteine, alanine, arginine, and asparagine. The PCR primers chosen amplified an initial product of 157 base pairs.
The forward primer had a mismatch incorporated into it in order to create a BstNI site. This fragment was cut with BstNI, amplified (with an internal reverse primer), and cut again with BstNI3. The restriction enzymes purpose was to trim away excess normal DNA sequences and enrich for any mutant sequences. In mutated samples, a BstNI site was not created, and therefore not recognized. Tumors will inherently contain normal tissue infiltrated throughout. Unless laser capture microdissection is incorporated, normal tissue cannot be removed by simple microtomy. Hence, we used BstNI to assist in normal DNA sequence removal. The second nested PCR amplification produced a 135-base pair band. If the sample was wild-type, BstNI recognized its site and trimmed the product to a 106 bp size. If the sample was mutated, the digested product remained at 135 bp. For P16, a 198 bp fragment was generated. For normal samples, a single band appeared on the gel image. For mutated samples, a doublet band and sometimes a triplet band were observed. This corresponded 100 % with DNA sequence data. In instances of degraded or low amounts of initial DNA template, the quality of the PCR product was
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