Presentation is loading. Please wait.

Presentation is loading. Please wait.

Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma  Cynthia Spittle, M. Renee Ward, Katherine.

Published byHarry Chambers Modified over 5 years ago

Similar presentations

Presentation on theme: "Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma  Cynthia Spittle, M. Renee Ward, Katherine."— Presentation transcript:

1 Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma  Cynthia Spittle, M. Renee Ward, Katherine L. Nathanson, Phyllis A. Gimotty, Eric Rappaport, Marcia S. Brose, Angelica Medina, Richard Letrero, Meenhard Herlyn, Robin H. Edwards  The Journal of Molecular Diagnostics  Volume 9, Issue 4, Pages (September 2007) DOI: /jmoldx Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Detection of V600E in melanoma cell lines. Pyrograms generated for BRAF wild-type (WM3208) (A), heterozygous mutant (WM 902B) (B), and homozygous mutant (WM39) (C) cell lines distinguish T versus A peaks (yellow shading). Nucleotide dispensation order is given below each pyrogram. Numerical position for each nucleotide is indicated at the top. Dispensations of C at positions 1 and 6 are included as controls for misincorporation. Dideoxy cycle sequence tracings for each cell line are shown for comparison. The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx ) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Detection of BRAF variant mutations. Pyrograms for two variant mutations, V600R (B) and V600K (C), show distinct patterns compared with V600E (A). Changes in relative A/G peak heights at pyrogram positions 2, 3, 5, 7, and/or 8 reflect dinucleotide substitutions given at right. The specific variants are confirmed by dideoxy cycle sequencing. The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx ) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 Applicability of pyrosequencing assay to FFPE tissue. Pyrograms generated from a V600E mutant tumor (A) and a V600K mutant tumor (B) are identical for paired frozen and FFPE tumors. The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx ) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

5 Figure 4 Cell line DNA mixing study. Theoretical peak heights, calculated from initial percent mutant A peak in undiluted heterozygous V600E mutant tumor, are correlated with actual peak heights generated for each dilution (Pearson's correlation = 0.96), indicating a linear relationship. The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx ) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

6 Figure 5 Analytic sensitivity for V600E detection in a heterozygous tumor. Pyrograms generated from dilution series for two separate experiments (day 1 and day 2) are shown with corresponding dideoxy sequence tracings for day 1 data. The percent tumor corresponding to each data set is indicated at left. The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx ) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

7 Figure 6 Comparison of pyrosequencing data with SNP microarrays. A: SNP XbaI 50K microarray data for chromosome 7 displayed with CNAT 3.0 software. Melanoma cell lines analyzed and location of BRAF gene are indicated. For each cell line, genome smoothed chromosome copy number data are shown at the top and the LOH score is shown at the bottom. Chromosome location in megabases is given at the bottom of the data set for each cell line. Scale for copy number and LOH score indicated at right. B: Corresponding BRAF pyrosequencing data including percent T and A peaks are indicated for each cell line. The Journal of Molecular Diagnostics 2007 9, DOI: ( /jmoldx ) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Download ppt "Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma  Cynthia Spittle, M. Renee Ward, Katherine."

Similar presentations

Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis  Philippe Szankasi, David.

Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis  Philippe Szankasi, David.
A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β- Thalassemia Mutations  Fu Xiong, Qiuying Huang, Xiaoyun Chen, Yuqiu Zhou, Xinhua.

A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β- Thalassemia Mutations  Fu Xiong, Qiuying Huang, Xiaoyun Chen, Yuqiu Zhou, Xinhua.
Detection of Exon 12 Mutations in the JAK2 Gene

Detection of Exon 12 Mutations in the JAK2 Gene
The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer  Irene Mancini,

The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer  Irene Mancini,
High-Throughput Homogeneous Mass Cleave Assay Technology for the Diagnosis of Autosomal Recessive Parkinson's Disease  Christopher Schroeder, Michael.

High-Throughput Homogeneous Mass Cleave Assay Technology for the Diagnosis of Autosomal Recessive Parkinson's Disease  Christopher Schroeder, Michael.
Detection of Low-Level KRAS Mutations Using PNA-Mediated Asymmetric PCR Clamping and Melting Curve Analysis with Unlabeled Probes  Ji Eun Oh, Hee Sun.

Detection of Low-Level KRAS Mutations Using PNA-Mediated Asymmetric PCR Clamping and Melting Curve Analysis with Unlabeled Probes  Ji Eun Oh, Hee Sun.
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.
A Method to Evaluate the Quality of Clinical Gene-Panel Sequencing Data for Single- Nucleotide Variant Detection  Chung Lee, Joon S. Bae, Gyu H. Ryu, Nayoung.

A Method to Evaluate the Quality of Clinical Gene-Panel Sequencing Data for Single- Nucleotide Variant Detection  Chung Lee, Joon S. Bae, Gyu H. Ryu, Nayoung.
High-Throughput, Multiplex Genotyping Directly from Blood or Dried Blood Spot without DNA Extraction for the Screening of Multiple G6PD Gene Variants.

High-Throughput, Multiplex Genotyping Directly from Blood or Dried Blood Spot without DNA Extraction for the Screening of Multiple G6PD Gene Variants.
Hendrikus J. Dubbink, Peggy N. Atmodimedjo, Ronald van Marion, Niels M

Hendrikus J. Dubbink, Peggy N. Atmodimedjo, Ronald van Marion, Niels M
A Quantitative Allele-Specific PCR Test for the BRAF V600E Mutation Using a Single Heterozygous Control Plasmid for Quantitation  Philippe Szankasi, N.

A Quantitative Allele-Specific PCR Test for the BRAF V600E Mutation Using a Single Heterozygous Control Plasmid for Quantitation  Philippe Szankasi, N.
Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift.

Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift.
Kirby Siemering, Shehnaaz S. M. Manji, Wendy M

Kirby Siemering, Shehnaaz S. M. Manji, Wendy M
High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations  Bjørnar Gilje, Reino Heikkilä, Satu.

High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations  Bjørnar Gilje, Reino Heikkilä, Satu.
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.
A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β- Thalassemia Mutations  Fu Xiong, Qiuying Huang, Xiaoyun Chen, Yuqiu Zhou, Xinhua.

A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β- Thalassemia Mutations  Fu Xiong, Qiuying Huang, Xiaoyun Chen, Yuqiu Zhou, Xinhua.
Gene Dysregulations Driven by Somatic Copy Number Aberrations-Biological and Clinical Implications in Colon Tumors  Manny D. Bacolod, Francis Barany 

Gene Dysregulations Driven by Somatic Copy Number Aberrations-Biological and Clinical Implications in Colon Tumors  Manny D. Bacolod, Francis Barany 
Philippe Szankasi, Mohamed Jama, David W. Bahler 

Philippe Szankasi, Mohamed Jama, David W. Bahler 
Single Nucleotide Polymorphism-Based System Improves the Applicability of Quantitative PCR for Chimerism Monitoring  Egle Gineikiene, Mindaugas Stoskus,

Single Nucleotide Polymorphism-Based System Improves the Applicability of Quantitative PCR for Chimerism Monitoring  Egle Gineikiene, Mindaugas Stoskus,
A High-Resolution Melting Protocol for Rapid and Accurate Differential Diagnosis of Thyroid Nodules  Irene Mancini, Pamela Pinzani, Cinzia Pupilli, Luisa.

A High-Resolution Melting Protocol for Rapid and Accurate Differential Diagnosis of Thyroid Nodules  Irene Mancini, Pamela Pinzani, Cinzia Pupilli, Luisa.

Similar presentations

Ads by Google