Quantification of DNA methylation using methylation-sensitive restriction enzymes & digital PCR
Yu Zhao & Stephanie Barbari
Monday, September 30, 2024
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Chapters
Introduction
Background
Sodium bisulfite treatment for DNA methylation IS NOT compatible with UNG PCR system
Methylation-sensitive restriction enzymes (MSREs)
Principle of digital PCR
Benefit of digital PCR > DNA methylation analysis
Workflow Overview
Roche Digital LightCycler Nanowell Plate Types
DNA Methylation Analysis
Target of Interest – RASSF1A Promoter
High GC enhancers improve the accuracy of quantification
Digital PCR detection of RASSF1A promoter methylation
Example of methylated fraction data analysis
3D clustering
Methylation status of RASSF1A in cancer cell lines
Highlights
Speaker: Stephanie Barbari
Disclaimer
Outline
GT Molecular, Inc.
Liquid biopsy can revolutionize cancer care
Minimal Residual Disease (MRD)
Proposed MRD strategy
Digital PCR can detect and quantify very low amounts of DNA
Multiplexing on Roche Digital LightCyler®
NPM1 mutations in AML
IDH1 and IDH2 mutations
Co-occurring mutations in NPM1 and IDH1/2 in AML
Co-occurring mutations in NPM1 and IDH1/2 in AML
MRD status at hematopoietic stem cell transplantation (HSCT)
Example Data: NPM1 – 288 insertion
Example Data: IDH1 – R132C/H
Example Data: IDH2 – R140Q & R172K
Limit Of Detection: Assay can detect at least 0.03% VAF for all targets
Compatibility: Assay is compatible with DNA from several matrix types
Mutations Detected in Liquid Biopsy
Conclusions and Next Steps
Acknowledgements
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