Presentation is loading. Please wait.

Presentation is loading. Please wait.

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

Published byJerome Stephens Modified over 5 years ago

Similar presentations

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

1 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 (September 2016) DOI: /j.jmoldx Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 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  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 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  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 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  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5 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  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6 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  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7 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  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

8 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  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

9 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  , DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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

Similar presentations

Keyur P. Patel, Bedia A. Barkoh, Zhao Chen, Deqin Ma, Neelima Reddy, L

Keyur P. Patel, Bedia A. Barkoh, Zhao Chen, Deqin Ma, Neelima Reddy, L
An Enhanced Polymerase Chain Reaction Assay to Detect Pre- and Full Mutation Alleles of the Fragile X Mental Retardation 1 Gene  Alessandro Saluto, Alessandro.

An Enhanced Polymerase Chain Reaction Assay to Detect Pre- and Full Mutation Alleles of the Fragile X Mental Retardation 1 Gene  Alessandro Saluto, Alessandro.
Large Pathogenic Expansions in the SCA2 and SCA7 Genes Can Be Detected by Fluorescent Repeat-Primed Polymerase Chain Reaction Assay  Claudia Cagnoli,

Large Pathogenic Expansions in the SCA2 and SCA7 Genes Can Be Detected by Fluorescent Repeat-Primed Polymerase Chain Reaction Assay  Claudia Cagnoli,
Triplet Repeat Primed PCR Simplifies Testing for Huntington Disease

Triplet Repeat Primed PCR Simplifies Testing for Huntington Disease
Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR–Based Multicolor Melting Curve Analysis  Qiuying Huang,

Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR–Based Multicolor Melting Curve Analysis  Qiuying Huang,
Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1  Grace X.Y. Lim, Yu.

Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1  Grace X.Y. Lim, Yu.
Todd S. Laughlin, Michael W. Becker, Jane L. Liesveld, Deborah A

Todd S. Laughlin, Michael W. Becker, Jane L. Liesveld, Deborah A
Rapid Detection of the Epidermal Growth Factor Receptor Mutation in Non-Small-Cell Lung Cancer for Analysis of Acquired Resistance Using Molecular Beacons 

Rapid Detection of the Epidermal Growth Factor Receptor Mutation in Non-Small-Cell Lung Cancer for Analysis of Acquired Resistance Using Molecular Beacons 
Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA  Christina Wood-Bouwens, Billy T. Lau, Christine.

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA  Christina Wood-Bouwens, Billy T. Lau, Christine.
Laila C. Schenkel, Charles Schwartz, Cindy Skinner, David I

Laila C. Schenkel, Charles Schwartz, Cindy Skinner, David I
A Rapid Polymerase Chain Reaction-Based Screening Method for Identification of All Expanded Alleles of the Fragile X (FMR1) Gene in Newborn and High-Risk.

A Rapid Polymerase Chain Reaction-Based Screening Method for Identification of All Expanded Alleles of the Fragile X (FMR1) Gene in Newborn and High-Risk.
Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480  Anthony Y.Y. Hsieh, Sara.

Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480  Anthony Y.Y. Hsieh, Sara.
LightCycler Technology in Molecular Diagnostics

LightCycler Technology in Molecular Diagnostics
Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR  Jennifer.

Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR  Jennifer.
A Multiplex SNaPshot Assay as a Rapid Method for Detecting KRAS and BRAF Mutations in Advanced Colorectal Cancers  Sandrine Magnin, Erika Viel, Alice.

A Multiplex SNaPshot Assay as a Rapid Method for Detecting KRAS and BRAF Mutations in Advanced Colorectal Cancers  Sandrine Magnin, Erika Viel, Alice.
Expansion of an FMR1 Grey-Zone Allele to a Full Mutation in Two Generations  Isabel Fernandez-Carvajal, Blanca Lopez Posadas, Ruiqin Pan, Christopher Raske,

Expansion of an FMR1 Grey-Zone Allele to a Full Mutation in Two Generations  Isabel Fernandez-Carvajal, Blanca Lopez Posadas, Ruiqin Pan, Christopher Raske,
Loss of Heterozygosity Studies Revisited

Loss of Heterozygosity Studies Revisited
Methylation-Specific Multiplex Ligation-Dependent Probe Amplification Enables a Rapid and Reliable Distinction between Male FMR1 Premutation and Full-Mutation.

Methylation-Specific Multiplex Ligation-Dependent Probe Amplification Enables a Rapid and Reliable Distinction between Male FMR1 Premutation and Full-Mutation.
Application of Single-Molecule Amplification and Resequencing Technology for Broad Surveillance of Plasma Mutations in Patients with Advanced Lung Adenocarcinoma 

Application of Single-Molecule Amplification and Resequencing Technology for Broad Surveillance of Plasma Mutations in Patients with Advanced Lung Adenocarcinoma 
Rapid Assessment of the Heterogeneous Methylation Status of CEBPA in Patients with Acute Myeloid Leukemia by Using High-Resolution Melting Profile  Tsung-Chin.

Rapid Assessment of the Heterogeneous Methylation Status of CEBPA in Patients with Acute Myeloid Leukemia by Using High-Resolution Melting Profile  Tsung-Chin.

Similar presentations

Ads by Google