Table 1.
Kras mutations and their frequency in Colon Cancer.
Figure 1.
Diagram to illustrate the assay method.
Figure 2.
Optimization of allele-specific primer and blocker Tm.
CT values are for the Kras Mut1.1 assay (G216T). A mutant-specific primer was used to assay either a wild type or mutant RNA template. Circles represent response to wild type synthetic RNA. Squares represent response to mutant synthetic RNA. Error bars represent 95% confidence intervals. The final version of this assay included a blocker oligonucleotide with a Tm of 60.8°C and a variant-specific primer with a Tm of 50°C. A. The effect of primer Tm on variant-specific assay CT. B. The effect of blocker oligonucleotide Tm on variant-specific assay CT.
Table 2.
Effect of Reagent Design on Mutation Assay Performance in Cell Line RNA or DNA.
Table 3.
ASB-PCR Design Rules.
Table 4.
Performance of Mutation Assays using RNA or DNA Extracted from Cell Lines.
Figure 3.
Sensitivity and selectivity of the Mut1.2 assay.
A. Detection of cell line RNA containing Kras G216T mutant diluted into wild type cell line RNA using the Kras Mut1.2 assay. ΔRn is the difference between the normalized fluorescence of the TaqMan reporter probe at each PCR cycle and the background fluorescence measured during the first 15 PCR cycles. Each curve represents the time course of PCR assays (average of triplicate measurements) at each dilution. The horizontal line at ΔRn = 0.2 represents the threshold for determination of CT for the individual amplification curves. B. Serial-dilutions of RNA extracted from wild type COLO320 (filled squares) and mutant SW480 (filled circles) cell lines submitted to the Kras Mut1.2 assay. Error bars represent 2 times the standard deviation of triplicate determinations.
Figure 4.
Comparison of mutation detection by ASB-PCR and nucleic acid sequencing.
ASB-PCR results are shown for the 44 randomly-selected FFPE colorectal tumor specimens for which sequencing data were available. Genomic DNA extracted from 44 randomly-selected FFPE colorectal tumor specimens was submitted to ASB-PCR assays A) Kras G216T, assay Mut1.1, B) Kras G216A, assay Mut2.1, and C) Kras G219A, assay Mut3.1. In each graph the CT values measured in the wild type Kras assay (x-axis) is plotted vs. the CT values measured in the specified Kras variant allele assay (y-axis). Samples were assayed at 0.4 ng of DNA per well. The solid line represents the classification boundary, which was derived as the lower 95% prediction limit of a linear regression of variant-specific assay CT response on a titration of wild type samples submitted to the variant-specific assay. Error bars represent 95% confidence limits based on a pooled estimate of standard error for all samples with a mean CT less than 35. Note that samples for which the 95% confidence intervals overlapped were designated as wild type. Circles: (Ο) Samples called wild type by both PCR and sequencing; Squares (□): samples called mutant by both PCR and sequencing; Triangles (▵): samples called mutant by PCR but wild type by sequencing.
Figure 5.
Concordance of mutation assay results in DNA and RNA from 72 FPE tissue specimens for which both RNA and DNA data was available.
Circles: (Ο) Samples called wild type by both PCR and DNA sequencing; Squares (□): samples called mutant by both PCR and DNA sequencing; Triangles (▵): samples called mutant by ASB-PCR but wild type by sequencing. A: Kras G216T, assay Mut1.1. B: Kras G216A, assay Mut2.1. C: Kras G219A, assay Mut3.1.