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Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR  Jennifer.

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Presentation on theme: "Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR  Jennifer."— Presentation transcript:

1 Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR  Jennifer B. Jackson, Daniel S. Choi, James D. Luketich, Arjun Pennathur, Anders Ståhlberg, Tony E. Godfrey  The Journal of Molecular Diagnostics  Volume 18, Issue 2, Pages (March 2016) DOI: /j.jmoldx Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 Figure 1 KRAS G12S assay sensitivity using either standard (top panels) or high-fidelity (bottom panels) polymerase enzyme during preamplification of 10 ng of gDNA. Preamplification was performed on water alone [no template control (NTC)], wild-type genomic DNA (gDNA), and gDNA containing 4% KRAS G12S allele frequency. After preamplification, digital PCR was performed on each sample. The numbers shown in the bottom right of each plot indicate the number of KRAS G12S mutants detected for each condition. Arb., arbitrary. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 Figure 2 Detection of 0.05% mutant allele frequency with and without multiplexed preamplification before digital PCR. Water alone [no template control (NTC)], 50 ng wild-type DNA, and 50 ng tumor DNA containing 0.05% mutant allele frequency were either analyzed by digital PCR alone (no preamplification) or preamplified before digital PCR (multiplexed preamplification). Non-preamplified samples (top panels) and samples subjected to multiplexed preamplification (bottom panels) were analyzed for the presence of KRAS G12S (A) or TP53 R73C (B) mutation. The numbers shown in the bottom right of each plot indicate the number of mutant alleles detected for each condition. P values are denoted in the top right of each plot. Arb., arbitrary. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 Figure 3 Detection of 0.05% and 0.01% allele frequency of SMAD4 R361G mutation. A: Detection of 0.05% SMAD4 R361G mutation with and without multiplexed preamplification before digital PCR. Water alone [no template control (NTC)], 50 ng wild-type DNA, and 50 ng tumor DNA containing 0.05% mutant allele frequency were either analyzed by digital PCR alone (no preamplification) or preamplified before digital PCR (multiplexed preamplification). Non-preamplified samples (top panels) and samples subjected to multiplexed preamplification (bottom panels) were analyzed for the presence of SMAD4 R361G. B: Detection of 0.01% SMAD4 R361G mutation with multiplexed preamplification before digital PCR. The numbers shown in the bottom right of each plot indicate the number of mutant alleles detected for each condition. P values are denoted in the top right of each plot. Arb., arbitrary. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5 Figure 4 Detection of low-frequency mutant alleles in serum-derived, circulating, cell-free DNA (ccfDNA) from cancer patients using multiplexed preamplification in digital PCR. Water alone [no template control (NTC)], wild-type ccfDNA, and ccfDNA from three cancer patients with the KRAS (patient MS1079; A), TP53 (patient MS310; B), or SMAD4 (patient 4873; C) mutation were subjected to multiplexed preamplification and assessed for presence of the mutation using digital PCR. A single digital PCR was performed on each patient ccfDNA sample to assess presence of mutation without preamplification (right panels). The numbers shown in the bottom right of each plot indicate the number of mutant alleles detected for each condition. P values are denoted in the top right of each plot. Arb., arbitrary. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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