期刊
JOURNAL OF INVESTIGATIVE DERMATOLOGY
卷 128, 期 2, 页码 398-402出版社
ELSEVIER SCIENCE INC
DOI: 10.1038/sj.jid.5700920
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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 BRAF 1799T > 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 1ml of human blood.
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