Methods for identifying modified bases in nucleic acid templates
AssigneePacific Biosciences of California, Inc.
InventorsBenjamin Flusberg, Stephen Turner, Jon Sorenson, Kevin Travers, Cheryl Heiner
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Abstract
Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.
Claims19
A method for identifying one or more modified bases in a nucleic acid template, the method comprising:
- a) providing a circular nucleic acid template that may have one or more modified bases;
- b) providing a polymerase enzyme having strand displacement activity and capable of processing the circular nucleic acid template;
- c) contacting the circular nucleic acid template with the polymerase enzyme;
- d) monitoring processing of the circular nucleic acid template by the polymerase enzyme, wherein the polymerase enzyme makes multiple passes around the circular nucleic acid template, whereby the processing results in the synthesis of a nascent nucleic acid strand, and wherein the monitoring:
- (i) occurs during the processing, and (ii) detects incorporation of single nucleotides into the nascent nucleic acid strand, thereby generating multiple sequence reads complementary to the circular nucleic acid template;
- e) measuring the kinetics of single nucleotide incorporations;
- and
- f) identifying, if present, one or more modified bases within the circular nucleic acid template using the measured kinetics of nucleotide incorporations for the multiple sequence reads.
Dependencies are inferred from the claim text.