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Updated: Jul 14, 2026
Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
Published on: March 29, 2017
Selective amplification of rare mutations using locked nucleic acid oligonucleotides that competitively inhibit
Reid P Oldenburg1, Monica S Liu, Michael S Kolodney
1Division of Dermatology, Department of Medicine, Harbor-UCLA Medical Center, Torrance, California, USA.
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.
Insights
This study presents a novel method to detect rare mutated BRAF DNA from melanoma cells in blood. The assay effectively identifies mutated cells, aiding in cancer staging and targeted therapy selection.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- Detecting circulating tumor DNA (ctDNA) is crucial for cancer management.
- Sensitive and selective assays are needed to identify rare mutated cells amidst wild-type DNA.
Purpose of the Study:
- To develop a novel, highly sensitive assay for detecting BRAF-mutated melanoma cells in blood.
- To evaluate the clinical utility of this assay for cancer staging and therapy selection.
Main Methods:
- Utilized a two-step approach combining locked nucleic acid (LNA) competitive blocking with mutant-specific primers.
- Employed antibody-mediated negative selection and whole genome amplification (WGA) for blood enrichment.
- Applied a two-step real-time PCR protocol for mutant BRAF amplification and detection.
Main Results:
- The LNA-blocking assay detected 10 BRAF g[1799T>A]-mutated melanoma cells among 10(5) wild-type cells.
- Successfully identified mutant BRAF DNA from as few as 10 melanoma cells per ml of human blood.
- Demonstrated clinical utility in detecting mutated melanoma cells in spiked blood samples.
Conclusions:
- The developed assay offers high sensitivity and specificity for detecting circulating melanoma cells with BRAF mutations.
- This method holds promise for improving tumor staging and guiding targeted therapy decisions in melanoma patients.
- The assay's ability to detect rare mutated DNA in blood supports its potential for non-invasive cancer monitoring.
