mutation detection by real-time pcr a simple, robust and highly selective method突变检测通过实时聚合酶链反应一个简单的、健壮的、高度选择性的方法.pdfVIP

Mutation Detection by Real-Time PCR: A Simple, Robust and Highly Selective Method John Morlan, Joffre Baker, Dominick Sinicropi* Genomic Health, Inc., Redwood City, California, United States of America Abstract 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 consistently produce highly selective mutation assays while obviating the need for redesign and optimization of the assay reagent