Table 1.
Sequences of Oligonucleotides used in the present study*.
Fig 1.
Principle of real-time WTB-qPCR.
a. Real-time traditional PCR (i.e. without WTB) cannot distinguish WT- and MT-alleles of the KRAS gene as both forms release fluorescence upon amplification. b. Conversely, for real time WTB-PCR, only MT-alleles can be amplified because the amplification of WT-alleles is blocked by the WTB oligonucleotide. c. Demonstration of the overlapping sequences associated with WTB and reverse primer. The WT-allele template, WTB, and primer are shown at the top, middle, and bottom positions, respectively, in which the dot symbols indicate the complementary positions between the template and WTB or reverse primers, and the plus symbols indicate the overlapping sequences between WTB and reverse primers. The red and blue letters indicate WT-alleles in the template and LNA in WTB, respectively.
Fig 2.
Comparison of the FFPE DNA extraction features associated with various commercially available kits.
Panel a and b show the plot scatter and column scatter graphs generated based on the quantity of DNA isolated using four commercial kits, respectively. Panel c and d show the plot scatter and column scatter graphs of ΔCq between FFPE and reference samples, respectively.
Table 2.
Optimization of WTB concentration in real-time PCR.
Fig 3.
Gel images of amplicons generated after various cycle numbers.
Reaction mixtures containing serial concentrations of WTB (2.0, 1.0, 0.5, and 0 μM) and homozygous WT- and MT-gDNA were performed in quadruplicate reaction tubes. At the specified cycle numbers indicated on the left hand side of image, one of the quadruplex tubes was taken out and placed on ice. When the reactions were complete, all of the samples were subjected to gel electrophoresis. The gDNA type and the WTB concentrations are labeled above the image.
Table 3.
Summary of the WTB suppression effects on the amplifications of 12 missense mutations at KRAS codons 12 and 13.
Fig 4.
Enhanced sensitivity of the real-time WTB-PCR system.
Panel a to d show the amplification curves of real-time PCR systems with (indicated by blue lines) and without (indicated by red lines) WTB and specified concentrations of MT-alleles (indicated in each panel), in which the olive curves indicated NTC reactions. In a 20 μl reaction mixture, increasing quantities of AsPC-1 gDNA (containing a homozygous mutation at c.35G>A; 5, 10, 50, 500, 5,000, 12,500, 25,000, 50,000 pg) were spiked into samples containing WT-gDNA, giving a total of 50 ng of gDNA. These were used to prepare templates containing increasing mutant quantities (i.e. 0.01, 0.02, 0.1, 1, 10, 25, 50, 100%). This image shows amplification curves following the use of specific mutant concentrations in the template DNA. The amplification curves associated with all 12 missense mutations and the associated mutant quantities used as templates are available in S2 Fig. The Cq values of reaction mixtures without WTB were consistently lower than those of reaction mixtures with WTB, suggesting that the WTB-PCR is a sensitive assay.
Fig 5.
Sequencing results of WTB-PCR products from DNA samples containing various percentages of mutated KRAS alleles.
Sequencing chromatographs are shown for the products from the real-time traditional PCR (top row) and WTB-PCR (bottom row) reactions in Fig 4 and S3 Fig. The numbers at the bottom of each segment indicate the percentages of mutated KRAS c.35G>A allele in the PCR reaction mixtures. The arrows indicate the position of the G to A mutation of the KRAS c.35G>A allele.
Fig 6.
Quantitative curves of real-time WTB-qPCR.
a. A standard curve was generated by plotting the average Cq values from real-time WTB-PCR against the log concentrations of mutant KRAS alleles (i.e. 5, 10, 50, 500, 5,000, 12,500, 25,000, 50,000 pg). The total amount of template was 50 ng of genomic DNA containing increasing levels of the mutated c.35G>A allele (i.e. 0.01, 0.02, 0.1, 1, 10, 25, 50, and 100%). The average Cq values are shown in Fig 4 and S3 Fig. These were automatically determined with MxPro Software. The amplification efficiency of real-time WTB-PCR was 96.8% (slope, -3.401; R2 = 0.992). b. A standard curve was generated by plotting the average ΔCq values between real-time traditional PCR and WTB-PCR against minus log percentage concentrations of mutant c.35G>A alleles (i.e. 0.01, 0.02, 0.1, 1, 10, 25, 50, and 100%) in 50 ng of genomic DNA. The standard curves from panel b were used to calculate the percentage of KRAS mutant alleles in heterozygous genomic DNA.
Fig 7.
Examples of results obtained from clinical samples.
Sequencing chromatographs are shown for the products from the real-time traditional PCR (top row) and WTB-PCR (bottom row) for three clinical FFPE samples. The numbers at the bottom of each segment indicate the percentages of mutated KRAS allele in the PCR reaction mixtures. The arrows indicate the position of each mutated nucleotide in the KRAS allele.
Table 4.
Summary of KRAS mutation analysis of 49 clinical samples.