1. A Quantitative Allele-Specific PCR Test for the BRAF V600E Mutation Using a Single Heterozygous Control Plasmid for Quantitation 
Philippe Szankasi, N. Scott Reading, Cecily P. Vaughn, Josef T. Prchal, David W. Bahler, Todd W. Kelley 
The Journal of Molecular Diagnostics 
Volume 15, Issue 2, Pages (March 2013)
DOI: /j.jmoldx
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Illustration of quantitation strategies. Amplification plots for both WT and V600E mutant allele-specific PCRs showing the calibration standards (red) and an unknown sample (blue). A: pBRAF-HET method. B: Conventional standard curve method.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. Figure 2
Comparison of different pBRAF-HET calibrator plasmid concentrations. The percentage mutant allele was determined as described in Materials and Methods for each HCL sample. Differently shaded bars indicate the concentration of calibrator plasmid used for allele quantitation.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. Figure 3
Quantitation using linearized versus circular pBRAF-HET calibrator plasmid. A: Mutant allele burden for 14 HCL samples was calculated using linear and circular pBRAF-HET calibrator (pHET method) and plotted against data from the same samples calculated with a standard curve–based calculation of mutant allele burden (STD method). Averages from three experiments are shown. B: Schematic of the circular and linear pBRAF-HET plasmid.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. Figure 4
Comparison of quantitation with the pBRAF-HET calibrator plasmid to pyrosequencing. Comparison of allele quantitation by allele-specific PCR and pyrosequencing for HCL specimens with R2 values shown without (and with) the circled discordant HCL case (A) and solid tumor specimens that were within the range of positivity (≥5% mutant allele) by pyrosequencing with R2 values shown (B).
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. Figure 5
Correlation of BRAF V600E allele burden using pBRAF-HET with HCL cell count. BRAF V600E mutant allele burden is compared with the HCL cell count estimated from flow cytometry data. Log-transformed data are shown.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions