title: 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, BRAF, 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.
authors: Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
authors: Seung-Tae
authors: ST
authors: Lee
authors: Ji-Youn
authors: JY
authors: Kim
authors: Min-Jung
authors: MJ
authors: Kown
authors: Sun Wook
authors: SW
authors: Kim
authors: Jae Hoon
authors: JH
authors: Chung
authors: Myung-Ju
authors: MJ
authors: Ahn
authors: Young Lyun
authors: YL
authors: Oh
authors: Jong-Won
authors: JW
authors: Kim
authors: Chang-Seok
authors: CS
authors: Ki
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D052138:Genes, Neoplasm; D025202:Molecular Diagnostic Techniques; D009154:Mutation; D000090243:Nucleophosmin; D009841:Oligonucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; C000716967:NPM1 protein, human; D009841:Oligonucleotides; D000090243:Nucleophosmin; D004247:DNA; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf