( placed in theAgilent 2100 bioanalyze...)Mutation detection for the,,,K- rasand P16 genes

placed in the Agilent 2100 bioanalyzer and the double-stranded DNA 1000 assay software was run. Twelve samples could be run in a 30-40 minute time frame. Data was saved to our server and analyzed using the Agilent analysis software package provided.
Results and discussion
Our established PCR assays enable the analysis of two gene regions relevant to cancer. The significance of these diagnostics relies on accurate interpretation of visual data. First, we compared mutant versus wild type samples for K-ras codon 12. The sizing differences, as well as the intensity of heterozygous bands would be difficult to discern on a standard agarose gel, not to mention timeconsuming. Applying the microfluidic technology of Agilents equipment, we were able to easily confirm our data with that of DNA sequencing. The sizing and quantitation offered by the software were invaluable to eliminating any guesswork previously involved in this assay. Figure 1 displays wildtype (lanes 10 and 11) versus mutated samples (lanes 4-7), as well as a heterozygous mutation (lanes 2 and 3). PCR products are in even-numbered lanes, and PCR products subjected to BstNI digests are in odd-numbered lanes. The ladder is in lane 1 and water negative controls are in lanes 8 and 9.

Figure 2 displays the corresponding DNA sequence chromatograms for these samples. The analysis of the exon 3 region of the P16 gene was researched. At first, the discovery of multiple bands on the chip image was thought to be primer dimer or an annealing stringency problem. After comparing normal and mutated samples from DNA sequencing data to the Agilent data, the correspondence was perfect. The PCR product at size 198 bp was the main band. Only mutated samples showed these extra bands. The mutated samples were found to be
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