Methods for identifying nucleic acid modifications

摘要

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.

主权项

1. A method for identifying a modification, the method comprising:
a) providing a template nucleic acid comprising the modification, wherein the modification is a base modification; b) providing a polymerase capable of processing the template nucleic acid; c) contacting the template nucleic acid comprising the modification with the polymerase; d) monitoring processing of the template nucleic acid by the polymerase, wherein the processing results in the synthesis of a nascent nucleic acid strand, and further wherein the monitoring (i) occurs in real time during the processing and (ii) detects incorporation of single nucleotides into the nascent nucleic acid strand both upstream and downstream of the modification to generate a sequence read that is complementary to the template nucleic acid; and e) during the monitoring, detecting a change in the processing, wherein the change comprises a kinetic difference during the incorporation of the single nucleotides, and further wherein the change is indicative of the modification, thereby identifying the modification.