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Nested High-Resolution Melting Curve Analysis

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1 Nested High-Resolution Melting Curve Analysis
Serge Carillo, Laurent Henry, Eric Lippert, François Girodon, Isabelle Guiraud, Céline Richard, Frédérique Dubois Galopin, Cedric Cleyrat, Eric Jourdan, Robert Kralovics, Sylvie Hermouet, Thierry Lavabre-Bertrand  The Journal of Molecular Diagnostics  Volume 13, Issue 3, Pages (May 2011) DOI: /j.jmoldx Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 Figure 1 Nested HRM setting (differential curves). The normalized and temperature-shifted difference plot (differential plot graph) magnifies high-resolution melting analysis, compared with the Tm graph. A: Typical Tm graphs. B: Corresponding normalized and temperature-shifted difference plot. A and B: Black lines: wild type (Ni002); gray lines: mutant c1627_1632del (Vi327). C: Large dispersion of differential plot curves from applying HRM directly to genomic DNA (12 negative controls). D: Nested HRM (post PCR HRM) allows reducing dispersion of the wild-type peak and defining a cutoff value. Black line: 9 negative controls; gray line: 1 negative control, 10 times replicated; dashed line: PCR positive control (Vi327). E: Reliability test: 30 points of wild type (black) or mutant (gray: c1614_1616CAA>GTT/Na571) 5 × 105 plasmids were used. To get heterodimers of mutant, we added 10% (5 × 104) of wild type. Positive control: c1627_1632del (Vi327) PCR (dotted line). The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 Figure 2 Mutant detection in a clinical sample. A: Normalized and temperature-shifted difference plot of the various kinds of mutation found. Wild-type control: Ni002 (line 1). New mutants: c1614_1616CAA>GTT*/Na571 (line 2); c1619T>C/Na141 (line 3); c1609T>A;1616A>T/Bo3 (line 4). Duplication: c1608_11640 dup/Di0871 (line 5). Mutants already reported: c1615_1616AA>TT/Bo4 (line 6); c1614_1616CAA>ATT/Di0121 (line 7); c1622_1627 del/Di0151 (line 8); c1624_1626 del/Bo5 (line 9); and c1624_1629del/Ni0846 (line 10). An asterisk indicates a novel nucleotidic mutation corresponding to an already described abnormal amino acid sequence. B: Chromatogram of wild type and the three new mutants. Arrows indicate mutation position. The asterisk in c1614_1616CAA_GTT*/Na571 indicates a novel nucleotidic mutation corresponding to an already described abnormal amino acid sequence. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 Figure 3 Sensitivity of nested HRM versus HRM. A: Comparison of serial dilution of c1609T>A; 1616A>T* (Bo3, allelic burden: 53%) analyzed with HRM or nested HRM and serial dilution of c1614_1616CAA>GTT (Na571, allelic burden: 27%) with nested HRM. B: Routine analysis of a case with low allelic burden, using nested HRM versus HRM. Dashed lines: wild-type (Ni002) control; gray lines: c1627_1632 del (Vi327) control; thick black lines: c1620_1621del; 1624_1627del/Cf705 with PCR; thin black lines: same, but without PCR; pairs of lines correspond to duplicate analysis for each case. For the sake of clarity, only one of the wild-type controls is shown. The Journal of Molecular Diagnostics  , DOI: ( /j.jmoldx ) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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