1. A Multiplexed Fragment Analysis-Based Assay for Detection of JAK2 Exon 12 Mutations 
Larissa V. Furtado, Helmut C. Weigelin, Kojo S.J. Elenitoba-Johnson, Bryan L. Betz 
The Journal of Molecular Diagnostics 
Volume 15, Issue 5, Pages (September 2013)
DOI: /j.jmoldx
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. Figure 1
Assay design: JAK2 exon 12 mutation detection by fragment analysis. The location and amino acid numbering of the JAK2 exon 12 mutation region is indicated with respect to the assay design. The test combines a length mutation assay to detect deletion and duplication mutations with an allele-specific PCR assay to detect K539L substitution mutations. One primer set (e12-F/e12-R) amplifies a 281 bp fragment containing the entire JAK2 exon 12 coding sequence. This permits detection of deletion and duplication mutations presenting as abnormally sized amplification products. The second primer set (K539L-F/e12-R) provides allele-specific detection of K539L nucleotide substitution mutations. The forward primer is designed to specifically amplify only from specimens containing the c.1615_1616delinsTT mutation (numbering based on NM_ ). Cross-reactivity for the alternate K539L mutation c.1615_1616delinsCT was also observed. Hence, this 131 bp amplification product is expected only in specimens harboring one of these K539L substitution mutations. It should be noted that the 281 bp and 131 bp fragments migrate at 280 bp and 127 bp, respectively, by capillary electrophoresis.
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
Case examples. A: JAK2 exon 12 wild-type case. B: Deletion mutation. C: Duplication mutation. D: K539L (c.1615_1616delinsTT) mutation. Pink analysis bins represent the locations of expected mutant fragment sizes (Mut). The gray bin indicates the wild-type (WT) JAK2 exon 12 control fragment size. A small peak at 260 bp is frequently present outside the deletion bin, but does not interfere with assay interpretation.
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
Comparison of the fragment analysis assay, direct sequencing, and HRM for detection of JAK2 exon 12 mutations in four positive cases. A: Fragment analysis assay. B: Direct sequencing. C: HRM difference plots (left panel) and normalized melt curves (right panel). Positive cases have colored plot lines different from the green negative controls. Each method accurately identified and distinguished the four evaluated mutations.
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 the fragment analysis assay, direct sequencing, and HRM for detection of a JAK2 exon 12 mutation in a case with low-level mutant allele (case 13). A: Fragment analysis assay unequivocally detects the 6 bp deletion mutation (Mut). B: Direct sequencing shows very low level background peaks suggestive of this mutation, but the level is below what can unequivocally be called positive. C: HRM difference plots; negative controls are represented by green plot lines. This low level mutation clusters with negative controls (arrow) and is challenging to identify by HRM due to minor variance from negative cases. WT, wild type.
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
Sensitivity of fragment analysis, direct sequencing, and HRM for detection of JAK2 exon 12 mutations. Mutation positive DNA was diluted into wild-type DNA and tested by each assay. A: Fragment analysis assay of K539L (c.1615_1616delinsTT), R541_E543delinsK,I540S, and V536_L546dup mutations diluted down to 0.5% mutant allele. B: Direct sequencing of K539L (top panel) and N542_E543del (bottom panel) mutations diluted to 10% and 5%. The asterisks indicate the mutant sequence trace. C: HRM difference plots of K539L and N542_E543del mutations diluted to 10% and 5%. Analytic sensitivity for the robust detection of mutations was determined to be 2% for fragment analysis, 10% for direct sequencing, and 10% for HRM.
The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx )
Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions