title: Selective amplification of rare mutations using locked nucleic acid oligonucleotides that competitively inhibit primer binding to wild-type DNA.
abstract: Detection of mutated genomic DNA from cancer cells circulating in blood may improve tumor staging and patient selection for targeted therapy. However, the task of detecting a few mutated cells in the presence of a large excess of wild-type cells requires a sensitive and selective assay. We describe a novel approach to detect circulating melanoma cells harboring a common point mutation in the BRAF kinase. In the first step, primer binding to wild-type BRAF is competitively blocked by a locked nucleic acid (LNA) oligonucleotide. In the second step, the LNA-blocking approach is combined with a mutant-specific forward primer. This two-step approach easily detected 10 BRAF g[1799T>A]-mutated melanoma cells mixed with 10(5) wild-type cells. To determine the clinical utility of this method, we tested its ability to detect human blood spiked with a defined number of BRAF1799T>A-mutated melanoma cells. Blood was first enriched for melanoma cells using an antibody-mediated negative selection procedure before whole genome amplification (WGA). Mutant BRAF in the WGA-amplified genomic DNA was further amplified by a two-step real-time PCR protocol. Using this approach, we could readily identify mutant DNA from as few as 10 melanoma cells in 1 ml of human blood.
authors.affiliation: Division of Dermatology, Department of Medicine, Harbor-UCLA Medical Center, Torrance, California, USA.
authors.forename: Reid P
authors.initials: RP
authors.lastname: Oldenburg
authors.forename: Monica S
authors.initials: MS
authors.lastname: Liu
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kolodney
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000097:blood / Q000302:isolation & purification; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology; D008545:Melanoma / Q000175:diagnosis / Q000235:genetics*; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009841:Oligonucleotides; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000175:diagnosis / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; D017931:DNA Primers; D004273:DNA, Neoplasm; D009841:Oligonucleotides; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf