Detection of MPL Mutations by a Novel Allele-Specific PCR-Based Strategy  Larissa V. Furtado, Helmut C. Weigelin, Kojo S.J. Elenitoba-Johnson, Bryan L. Betz  The Journal of Molecular Diagnostics  Volume 15, Issue 6, Pages 810-818 (November 2013) DOI: 10.1016/j.jmoldx.2013.07.006 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Assay design: MPL exon 10 mutation detection by multiplexed allele-specific PCR. The location and amino acid numbering of the MPL mutation region is indicated with respect to the assay design. The test is performed in a two-tube format to detect the four most common MPL mutations. In each tube, one primer set [MPL-forward (MPL-Fwd) and MPL-reverse (MPL-Rev)] amplifies a 211-bp fragment containing the entire MPL exon 10 coding sequence, which serves as an amplification control. The tube A assay includes S505N and W515L mutation–specific primers along with outer MPL-forward and MPL-reverse primers. The tube B assay includes W515K and W515A mutation–specific primers along with the same outer primers. The allele-specific PCR primers are designed to specifically amplify only from specimens containing the particular mutations. Hence, amplification products are expected only in specimens harboring one of these MPL exon 10 mutations. The forward primers in both assays are labeled to permit detection by capillary electrophoresis: blue indicates FAM (tube A assay) and yellow indicates NED (tube B assay). Note: The 211-bp control product migrates at 206/207 bp by capillary electrophoresis. In tube A, the 94-bp (S505N) and 124-bp (W515L) fragments migrate at 90/91 bp and 116/117 bp, respectively, by capillary electrophoresis. In tube B, the 122-bp (W515K) and 126-bp (W515A) fragments migrate at 116 bp and 123 bp, respectively, by capillary electrophoresis. The Journal of Molecular Diagnostics 2013 15, 810-818DOI: (10.1016/j.jmoldx.2013.07.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Electropherograms of five case examples for tubes A and B. Negative: wild-type control. Pink bins represent the location of each of the mutation-specific amplification products. Gray bins indicate the control product location. The Journal of Molecular Diagnostics 2013 15, 810-818DOI: (10.1016/j.jmoldx.2013.07.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Comparison of direct sequencing and allele-specific PCR assay for detection of MPL W515L mutations. Sequencing effectively detects mutations with >10% to 15% mutant allele burden (case 14). A low-level mutation (case 9) shows a low-level background peak by direct sequencing; however, the level is below what could unequivocally be called positive (Pos) because the mutation could not be definitely confirmed on the opposite sequence trace. Cases 20, 47, and 59 are interpreted as negative (Neg) by Sanger sequencing because the mutation is completely absent in the electropherogram. The allele-specific PCR assay unequivocally detected the W515L mutation in all cases. Equiv. Pos, equivocal positive. The Journal of Molecular Diagnostics 2013 15, 810-818DOI: (10.1016/j.jmoldx.2013.07.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Analytic sensitivity. Mutation-positive DNA for each targeted mutation was diluted into wild-type DNA down to 0% mutant allele. Analytic sensitivity for the robust detection of each mutation was determined to be 2.5% mutant allele. The Journal of Molecular Diagnostics 2013 15, 810-818DOI: (10.1016/j.jmoldx.2013.07.006) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions