Sinicropi D. Mutation detection by real-time PCR: a simple, robust and highly selective method

Background: Molecular tests for diagnosis of disease, particularly cancer, are gaining increased acceptance by physicians and their patients for disease prognosis and selection of treatment options. Gene expression profiles and genetic mutations are key parameters used for the molecular characterization of tumors. A variety of methods exist for mutation analysis but the development of assays with high selectivity tends to require a process of trial and error, and few are compatible with real-time PCR. We sought to develop a real-time PCR-based mutation assay methodology that successfully addresses these issues. Methodology/Principal Findings: The method we describe is based on the widely used TaqManH real-time PCR technology, and combines Allele-Specific PCR with a Blocking reagent (ASB-PCR) to suppress amplification of the wildype allele. ASB-PCR can be used for detection of germ line or somatic mutations in either DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor specimens. A set of reagent design rules was developed enabling sensitive and selective detection of single point substitutions, insertions, or deletions against a background of wild-type allele in thousand-fold or greater excess. Conclusions/Significance: ASB-PCR is a simple and robust method for assaying single nucleotide mutations and polymorphisms within the widely used TaqManH protocol for real time RT-PCR. The ASB-PCR design rules consistentl