Fig 1.
SensiScreen® KRAS exon 2 features 7 mutation-specific assays as well as a common reference assay. All assays include a common reverse primer and a green fluorescent HydrolEasy™ probe. The reference assay features an allele-independent forward primer (not shown), while the mutation-specific assays include allele-specific forward primers and a BaseBlocker™. The allele-specific forward primers include the particular sequence variation in the 3´-end (green) and can include an additional sequence variation in position +3 or +4 relative to the 3´-end (purple). The BaseBlocker™ is complementary to the wild type sequence (yellow) and specifically blocks amplification of the wild-type allele.
Fig 2.
Addition of a BaseBlocker™ strongly increases SensiScreen® assay specificity.
(A) Normalized real-time PCR amplification plot showing the specificity of SensiScreen® G12S simplex assay in the absence and presence of a wild type blocking BaseBlocker™. (B+C) The inclusion of a BaseBlocker™ (+) impairs amplification of the wild-type allele by 99.95% (B) and increases the difference in threshold cycle (Ct) value between wild-type (WT) and mutant (G12S) DNA samples from -0.7 to >11.2 (C). SensiScreen® KRAS G12S Simplex real-time PCR ±2000 nM of BaseBlocker™ was performed on a Corbett Rotor-Gene 6000 instrument using 50 ng of wild type (WT) human genomic DNA and approximately 500 copies of KRAS G12S template. PCR amplification plot (A) is representative of three independent experiments. Bars (B) represent the mean +S.D. of three independent experiments.
Table 1.
KRAS mutations detected by SensiScreen® KRAS exon 2 simplex and multiplex.
Fig 3.
Rotor-Gene 6000 PCR amplification plots of SensiScreen® KRAS exon 2 simplex assays using serial dilutions of mutated DNA in a wild type background.
50 ng and/or approximately 16,000 copies of DNA was added to each reaction. The threshold was set at 10% of the average fluorescence signal of the reference assay at cycle 45. Legend describes the fraction of cell line DNA and/or mutated copies of KRAS exon 2 templates.
Fig 4.
Rotor-Gene 6000 PCR amplification plots of SensiScreen® KRAS exon 2 multiplex assays using serial dilutions of mutated DNA in a wild type background.
50 ng and/or approximately 16,000 copies of DNA was added to each reaction. The threshold was set at 10% of the average fluorescence signal of the reference assay at cycle 45. Legend describes the fraction of cell line DNA and/or mutated copies of KRAS exon 2 templates.
Table 2.
Sensitivity and PCR efficiency of SensiScreen® simplex and multiplex assays determined by serial dilutions of mutated DNA in a wild type background.
50 ng and/or approximately 16,000 copies of DNA was added to each real-time PCR mixture. The threshold was set at 10% of the average fluorescence signal of the reference assay at cycle 45.
Fig 5.
Dot plots of SensiScreen® clinical data.
(A+C) Patient samples were analysed for KRAS exon 2 mutations using SensiScreen® simplex. (B+D) Patients were also analysed for KRAS exon 2 mutations using SensiScreen® multiplex. Samples were regarded as mutant if Delta Ct (DCt) values were ≤9 and Ct values were ≤38. Samples with no Ct values for the mutation assay were plotted as Ct = 45. Novel mutant indicates samples that were not identified as mutant by other methods.
Table 3.
Samples analysed with SensiScreen® were regarded as mutant if Delta Ct (ΔCt) values were ≤9 and Ct values were ≤38.