This does not alter their adherence to PLOS ONE policies on sharing data and materials. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors of this work are funded and employed by New England Biolabs, Inc, a manufacturer and vendor of molecular biology reagents, including DNA polymerases. įunding: This work was privately funded by New England Biolabs, of which all authors are employees. Custom scripts used in this study are publicly available at.
#Ag source pcr archive#
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: Sequencing data pertaining to this study is deposited into the Sequence Read Archive (SRA) with an accession number SRP095133. Received: SeptemAccepted: DecemPublished: January 6, 2017Ĭopyright: © 2017 Potapov, Ong. PLoS ONE 12(1):Įditor: Ruslan Kalendar, University of Helsinki, FINLAND In total, we analyzed PCR products at the single-molecule level and present here a more complete picture of the types of mistakes that occur during DNA amplification.Ĭitation: Potapov V, Ong JL (2017) Examining Sources of Error in PCR by Single-Molecule Sequencing. For very accurate polymerases, DNA damage introduced during temperature cycling, and not polymerase base substitution errors, appeared to be the major contributor toward mutations occurring in amplification products. Inverted repeat structural elements in lacZ caused polymerase template-switching between the top and bottom strands during replication and the frequency of these events were measured for different polymerases. PCR-mediated recombination by Taq polymerase was observed at the single-molecule level, and surprisingly found to occur as frequently as polymerase base substitution errors, suggesting it may be an underappreciated source of error for multiplex amplification reactions. In addition to well-characterized polymerase base substitution errors, other sources of error were found to be equally prevalent. In this report, a single-molecule sequencing assay was used to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. However, many next-generation sequencing technologies first rely on DNA amplification, via the Polymerase Chain Reaction (PCR), as part of sample preparation workflows. Next-generation sequencing technology has enabled the detection of rare genetic or somatic mutations and contributed to our understanding of disease progression and evolution.
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