s-RT-MELT: a novel technology for mutation screening.

The fast growing understanding of genetic pathways that mediate cancer etiology, biology, and personalized medicine leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables expanded-throughput enzymatic mutation scanning in clinical cancer samples for germline or low-level somatic mutations, or for SNP genotyping. GC-clamp-containing PCR products from tumor and normal cells are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in parallel. Mismatches are converted to double strand breaks using a DNA endonuclease (Surveyor), and poly A oligonucleotide tails are enzymatically attached at the position of the mutations. A novel application of PCR that operates at low denaturation temperatures enables the selective amplification of mutation-containing DNA fragments and high-throughput, closed-tube mutation scanning via melting curve analysis on conventional or nanotechnology real-time PCR platforms. We have applied s-RT-MELT in the screening of TP53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer.