Abstract 1518: A new multiplex genotyping assay using PNA clamping-assisted fluorescence melting curve analysis

Recent studies on molecular mechanisms of anti-EGFR therapies revealed importance of mutation screening and genotyping of multiple target oncogenes. For example, KRAS mutations have been associated with poor responsiveness to anti-EGFR therapies. Determining the KRAS status of a newly diagnosed cancer patient prior to initiating anti-EGFR therapy would be required in selecting effective therapeutics among tyrosine kinase inhibitor and monoclonal antibody therapy. Peptide nucleic acid (PNA) is an artificially synthesized polymer. The negatively charged deoxyribose phosphate backbone of DNA is replaced by an electroneutral peptide-like backbone consisting of N-(2-aminoethyl) glycine units directly linked to the nucleotide bases. Complementary PNA/DNA complex has stronger binding affinity than complementary DNA/DNA complex. A PNA probe has higher specificity and mismatch discrimination towards its target DNA than a DNA probe. Furthermore, it shows a remarkable biological stability to enzymatic degradation. PNA is not recognized by DNA polymerase so it cannot be used as a primer. PNAClamp™ technique is the PNA-based PCR clamping that selectively amplifies only the target (mutated) DNA sequence as a minor portion in the mixture with the major non-target (wild type) DNA sequences. Therefore, PNAClamp technique provides better limit of detection than conventional PCR method. PANA S-Melting™ (PNA probe-based fluorescence melting curve analysis) based on change in fluorescent signal as a sample thermally denatured (melting temperature shift). This is a powerful tool for mutation detection (screening and genotyping) by a single probe. It can distinguish multiple sequence variants adjacent to each other on a small target region. We developed a new technique, which combines two techniques; PNAClamp and PNA probe-based fluorescence melting curve analysis. This new PNAClamp-assisted melting curve method can discriminate a sequence variant from a wild-type sequence with a relatively large Tm difference. Therefore, it could easily detect presence of sequence variation without an additional data analysis process. This technique can be performed in any real-time PCR machine, which has melting curve analysis function. We tested a new multiplex genotyping assay of KRAS gene mutation using this combined method in lung cancer cell line samples. This new assay could detect a total of seven sequence variants (6 genotypes of codon 12 and 1 genotype of codon 13) in a single closed