A Single-Molecule Real-Time (SMRT) sequencing assay for DNA replication fidelity in PCR

12th June 2015
Versione stampabile

Venue: Edificio Povo 2, via Sommarive nr. 9, Povo (Tn) - Room B101
 at 2:00 p.m.

  • Jennifer L. Ong -  New England Biolabs, DNA Enzymes Division, Ipswich, MA

The ability of DNA polymerases to accurately copy DNA has many implications for both biology and biotechnology.  The fidelity of DNA polymerases vary widely, from 10^-1 per base for lesion bypass polymerases to accurate replicative polymerases with error rates as low as 10^-6 per base.  We describe a next-generation sequencing assay to directly characterize misincorporation events made by polymerases, specifically during PCR.  Amplification products are directly sequenced using Pacific Biosciences Single-Molecule Real-Time (SMRT) sequencing technology, and replication errors are distinguished from sequencing errors using consensus accuracy.  We have validated this assay by measuring the fidelity of several wild-type and commercially available high-fidelity PCR enzymes.