journal.pone.0139074.PDF (2.3 MB)
Efficient genotyping of KRAS mutant non-small cell lung cancer using a multiplexed droplet digital PCR approach
journal contributionposted on 2020-09-03, 13:59 authored by Alexandra Pender, Isaac Garcia-Murillas, Sareena Rana, Rosalind J Cutts, Gavin Kelly, Kerry Fenwick, Iwanka Kozarewa, David Gonzalez de Castro, Jaishree Bhosle, Mary O'Brien, Nicholas C Turner, Sanjay Popat, Julian Downward
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.
Carcinoma, Non-Small-Cell LungCell Line, TumorClone CellsDNA, NeoplasmFormaldehydeGene FrequencyGenotyping TechniquesHigh-Throughput Nucleotide SequencingHumansLung NeoplasmsMultiplex Polymerase Chain ReactionMutationParaffin EmbeddingProto-Oncogene Proteins p21(ras)TemperatureTissue FixationDownwardCBGeneral Science & Technology