Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach
Author(s)
Type
Journal Article
Abstract
Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.
Date Issued
2015-09-28
Date Acceptance
2015-09-09
Citation
PLOS One, 2015, 10 (9)
ISSN
1932-6203
Publisher
Public Library of Science
Journal / Book Title
PLOS One
Volume
10
Issue
9
Copyright Statement
© 2015 Pender et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
License URL
Subjects
Carcinoma, Non-Small-Cell Lung
Cell Line, Tumor
Clone Cells
DNA, Neoplasm
Formaldehyde
Gene Frequency
Genotyping Techniques
High-Throughput Nucleotide Sequencing
Humans
Lung Neoplasms
Multiplex Polymerase Chain Reaction
Mutation
Paraffin Embedding
Proto-Oncogene Proteins p21(ras)
Temperature
Tissue Fixation
General Science & Technology
MD Multidisciplinary
Publication Status
Published
Article Number
e0139074