Mutant Enrichment with 3 '-Modified Oligonucleotides A Practical PCR Method for Detecting Trace Mutant DNAs

Abstract

Many clinical situations necessitate highly sensitive and reliable molecular assays; however, the achievement of such assays remains a challenge due to the inherent limitations of molecular testing methods. Here, we describe a simple and inexpensive enrichment technique that we call mutant enrichment with 3'-modified oligonucleotides (MEMO). The method is based on the use of a 3'-modified oligonucleotide primer that blocks extension of the normal allele but enables extension of the mutated allele. The performance of the technique was evaluated with respect to its ability to detect common cancer mutations in the EGFR, KRAS, BRAE, TP53, JAK2, and NPM1 genes. We achieved sensitivities of 10(-2) to 10(-6) using downstream Sanger sequencing, depending on the concentrations and thermodynamics of the primers. MEMO may be applicable to the quantitative real-time PCR platform and other downstream assays. This technique may be practically applicable to various medical situations. (J Mol Diagn 2011, 13:657-664. DOI: 10.1016/j.jmoldx.2011.07.003)