P3.11 Novel Mismatch-Suppression Strategy Enabling Allele-Specific Cdna Synthesis and Ultrasensitive Detection of Rna Variants

Abstract

ABSTRACT Although various DNA mutation detection assays can identify the presence of specific mutations, the expression level of the mutant alleles of coding genes may vary [ 1 ] and affect the phenotype of cells or tissues harboring these mutations. As a result, a quantitative assay for analysis of mutant RNA transcripts could more accurately reflect the effect brought upon the cells or tissues by the presence of a specific mutation. We have utilized a moderately thermo-stable reverse transcription enzyme to develop a novel strategy, named ART-PCR (Allele-specific Reverse Transcription - PCR), for ultra-sensitive detection and quantification of mutant RNA in the presence of excessive amounts of the wildtype transcripts. Unlike conventional approaches, the ART-PCR assay achieves allele-specific priming in a hot-start cDNA synthesis utilizing a mutation-specific primer with a 5' tail of unrelated sequence and a competitive wildtype-specific primer without a tail ( Fig. 1A ). The mutation-specific cDNA products are selectively amplified and detected in a PCR reaction containing a common-locus forward primer and a discriminating reverse primer, specific to the tail sequence of the mutation-specific reverse transcription primer (Fig. 1B). The wildtype-specific cDNA product is not amplified due to the lack of a binding site for the reverse primer. The ART-PCR assay was able to detect various mutant RNA transcripts of KRAS and BRAF genes in a 1000 - 10,000 fold excess of wild type transcripts and the quantitative detection was linear over a broad dynamic range. We demonstrate that the exceptional sensitivity of the ART-PCR assay is dependent on competitive primer extension during cDNA synthesis and is lost if the competitive wildtype-specific primer is either excluded from the reaction or replaced by a non-extendable oligonucleotide with the same sequence. Download : Download full-size image Fig. 1 . Schematic of ART-PCR assay.