Improving FFPE DNA Purification Workflows with Chemical Catalysts
In this webinar, you will learn how:
- Decrosslinking is necessary but inherently problematic for FFPE nucleic acid purification workflows
- Promega has developed novel chemical approaches to catalyze the reversal of formalin crosslinks in FFPE DNA
- Decrosslinking incubations can impact FFPE DNA quality for NGS, particularly C-to-T transitions originating from cytosine deamination
Summary
Formalin-fixed paraffin-embedded (FFPE) tissues are an important sample type. Before DNA is purified from FFPE, formalin crosslinks must be reversed using long incubations at high temperatures. This decrosslinking step can compromise nucleic acid quality and pose challenges for laboratory workflows. This webinar will share novel chemical methods that catalyze the reversal of formalin-induced DNA-DNA/protein crosslinks. Chemical screens to identify decrosslinking reagents (catalysts) that selectively reverse formalin crosslinks were conducted. Compounds were identified that reduce the necessary FFPE tissue decrosslinking time from 4 hours to 30 minutes. The quality of FFPE DNA purified using the catalyst technology was evaluated using qPCR, absorbance, fluorescence, and hybridization applications. In addition, Next-generation sequencing (NGS) using targeted amplification was employed to assess DNA quality. NGS analyses indicate that rates of mutational artifacts in FFPE, such as C-to-T transitions from deamination, increase with longer decrosslinking times. However, DNA purified using the optimized catalyst workflow exhibited relatively lower rates of C-to-T transitions, suggesting that the shortened catalytic-driven decrosslinking incubation improves DNA quality for sequencing applications. The methods developed in this study significantly shortens FFPE tissue DNA purification workflows and has the potential to improve DNA sequencing quality.
Speaker
Kevin Mayer, PhD
Senior Research Scientist
Kevin Mayer is a Senior Research Scientist in the Protein and Nucleic Acid Group at Promega Corporation. Kevin has a diverse background spanning molecular pathology, genetics, and biotechnology. He began his career as a research assistant in Mayo Clinic’s Division of Experimental Pathology and Laboratory Medicine, where he studied HPV viral integration events in head and neck cancers. While at Mayo, he also interned as a laboratory technician in the Molecular Hematopathology group at Mayo Medical Labs. Before joining Promega, Kevin received his PhD in Genetics from the University of Wisconsin-Madison, where he studied genetic pathways underlying plant flowering time. In addition to these activities, Kevin has provided consulting services with the non-profit WiSolve to assist Madison-area biotech businesses.