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Multiplex Enrichment and Detection of Rare KRAS Mutations in Liquid Biopsy Samples using Digital Droplet Pre-Amplification.
- Source :
-
Analytical chemistry [Anal Chem] 2019 Jun 18; Vol. 91 (12), pp. 7516-7523. Date of Electronic Publication: 2019 May 24. - Publication Year :
- 2019
-
Abstract
- Oncology research is increasingly incorporating molecular detection of circulating tumor DNA (ctDNA) as a tool for cancer surveillance and early detection. However, noninvasive monitoring of conditions with low tumor burden remains challenging, as the diagnostic sensitivity of most ctDNA assays is inversely correlated with total DNA concentration and ctDNA abundance. Here we present the Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp) method, which combines single-molecule emulsification and short-round polymerase chain reaction (PCR) preamplification with digital droplet PCR detection of mutant DNA template. The MED-Amp assay increased mutant signal by over 50-fold with minimal distortion in allelic frequency. We demonstrate detection of as few as three mutant copies in wild-type DNA concentrations ranging from 5 to 50 ng. The MED-Amp assay successfully detected KRAS mutant ctDNA in 86% plasma samples obtained from patients with metastatic pancreatic ductal adenocarcinoma. This assay for high-sensitivity rare variant detection is appropriate for liquid biopsy samples or other limited clinical biospecimens.
- Subjects :
- Aged
Aged, 80 and over
Case-Control Studies
Female
Fluorescent Dyes chemistry
Gene Frequency
Humans
Limit of Detection
Liquid Biopsy
Male
Middle Aged
Mutation
Pancreatic Neoplasms genetics
Pancreatic Neoplasms pathology
Polymerase Chain Reaction
Circulating Tumor DNA blood
Lipid Droplets chemistry
Proto-Oncogene Proteins p21(ras) genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1520-6882
- Volume :
- 91
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- Analytical chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 31072097
- Full Text :
- https://doi.org/10.1021/acs.analchem.8b01605