Defining the Performance Parameters of a Rapid Screening Tool for FMR1 CGG-Repeat Expansions Based on Direct Triplet-Primed PCR and Melt Curve Analysis

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Presentation transcript:

Defining the Performance Parameters of a Rapid Screening Tool for FMR1 CGG-Repeat Expansions Based on Direct Triplet-Primed PCR and Melt Curve Analysis Indhu-Shree Rajan-Babu, Mulias Lian, Anh H. Tran, Truong T. Dang, Huong T.-M. Le, Minh N. Thanh, Caroline G. Lee, Samuel S. Chong The Journal of Molecular Diagnostics Volume 18, Issue 5, Pages 719-730 (September 2016) DOI: 10.1016/j.jmoldx.2016.05.002 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 LightCycler 480 and Rotor-Gene Q HRM melt profiles of 1-step (A) and 2-step (B) direct triplet-primed PCR and melt curve analysis of fragile X reference DNA from the Coriell Cell Repositories (CCR). rpts, repeats. The Journal of Molecular Diagnostics 2016 18, 719-730DOI: (10.1016/j.jmoldx.2016.05.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Two-step LightCycler 480 melt profiles of plasmid DNA harboring inserts with normal, intermediate, and premutation size repeat segments. A: Direct triplet-primed PCR (dTP-PCR) melt curve analysis (MCA) of 9 plasmid DNA constructs were generated from Coriell Cell Repositories reference and cord blood DNA. Each plasmid DNA was assayed in triplicate. The inset panels display the electropherogram of matching plasmid DNA, with numbered black arrows and arrowheads indicating the number of CGG repeats and base pair size of the ROX-labeled internal size calibrator, respectively. B: dTP-PCR MCA of 8 plasmid DNA mixtures carrying borderline FMR1 alleles. Each plasmid DNA mixture was assayed in triplicate. Black and gray peaks represent the melt profiles of plasmid DNA mixtures and internal reference controls, respectively. Gray-shaded vertical bars indicate the indeterminate zone established by the controls NA20234 (31/46 repeats) and NA20236 (31/54 repeats). Melt peak temperatures are expressed as means ± SD. The Journal of Molecular Diagnostics 2016 18, 719-730DOI: (10.1016/j.jmoldx.2016.05.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Effect of well/rotor position on the melt peak temperatures (Tms) of sample replicates. Melt profiles followed by bar graphs showing the Tm of sample replicates assayed on LightCycler 480 (A) and Rotor-Gene Q High Resolution Melt (HRM) instrument (B). Vertical-dotted gray and black lines indicate the melt domains of NA20232 and NA20230, respectively. Mean Tms of LightCycler 480 and Rotor-Gene Q HRM–derived melt profiles of NA20232 and NA20230 are expressed as means ± SD (C). rpts, repeats. The Journal of Molecular Diagnostics 2016 18, 719-730DOI: (10.1016/j.jmoldx.2016.05.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Analytic sensitivity of male (left) and female (right) Coriell Cell Repositories reference DNA assessed by 2-step direct triplet-primed PCR melt curve analysis on the Rotor-Gene Q High Resolution Melt system. Each panel included the melt peaks of control DNA (gray nondotted peaks) generated using the standard 100-ng template. Gray-shaded vertical bars in the left panels indicate the indeterminate zone established by the controls NA20232 (46 repeats) and NA20230 (54 repeats); gray-shaded vertical bars in the right panels indicate the indeterminate zone established by the controls NA20234 (31/46 repeats) and NA20236 (31/54 repeats). rpts, repeats. The Journal of Molecular Diagnostics 2016 18, 719-730DOI: (10.1016/j.jmoldx.2016.05.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Two-step direct triplet-primed PCR melt curve analysis on the Rotor-Gene Q High Resolution Melt system showing the assay's analytic specificity. To 100 ng of the normal female DNA, 50, 100, and 150 ng of each of the myotonic dystrophy type 1 (DM1) or Huntington disease (HD) DNA were added, and the mixtures were assayed in the presence of control DNA. Melt profiles (gray-dotted peaks) and electropherograms (insets) of DM1 and HD DNA are shown. Gray-shaded vertical bars indicate the indeterminate zone established by the controls NA20234 (31/46 repeats) and NA20236 (31/54 repeats). Melt peak temperatures are expressed as means ± SD. The Journal of Molecular Diagnostics 2016 18, 719-730DOI: (10.1016/j.jmoldx.2016.05.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Two-step direct triplet-primed PCR melt curve analysis on the Rotor-Gene Q High Resolution Melt system showing the effect of altered assay reaction volume on melt peak temperatures. Three FMR1 reference DNA samples were tested, and each sample was assayed in triplicate for 5 different reaction volumes. Each panel also included the melt peaks of internal reference controls (gray peaks) generated from the standard 15-μL reaction volume. Gray-shaded vertical bars indicate the indeterminate zone established by the controls NA20232 (46 repeats) and NA20230 (54 repeats). rpts, repeats. The Journal of Molecular Diagnostics 2016 18, 719-730DOI: (10.1016/j.jmoldx.2016.05.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 7 Rotor-Gene Q High Resolution Melt 2-step direct triplet-primed PCR melt curve analysis of saliva, buccal swab, and blood spot DNA. A: Melt profiles and electropherograms of DNA extracted from saliva and buccal swab. B: Melt profiles of blood spot mimics prespiked with male permutation (+GM06891) or full-mutation (+GM07862) cell line DNA, presented alongside GM06891-only and GM07862-only DNA melt peaks. C: Melt profiles of dried blood spot DNA from 32 patients with clinically diagnosed autism spectrum disorder, with electropherograms of the expansion-positive sample and 1 expansion-negative sample. Gray-shaded vertical bars indicate the indeterminate zone established by the controls NA20232 (46 repeats) and NA20230 (54 repeats). rpts, repeats. The Journal of Molecular Diagnostics 2016 18, 719-730DOI: (10.1016/j.jmoldx.2016.05.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 8 Two-step direct triplet-primed PCR melt curve analysis on the Rotor-Gene Q High Resolution Melt system showing the effect of sodium acetate and glycogen on the melt profiles of 2 plasmid DNA samples. Each reaction was performed in triplicate. Melt profiles of plasmids and controls generated in the absence of sodium acetate and glycogen are shown in the first row to facilitate comparison. Gray-shaded vertical bars indicate the indeterminate zone established by the controls NA20232 (46 repeats) and NA20230 (54 repeats) Melt peak temperatures are expressed as means ± SD. rpts, repeats. The Journal of Molecular Diagnostics 2016 18, 719-730DOI: (10.1016/j.jmoldx.2016.05.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions