Identification of 5-methyl-C in nucleic acid templates

摘要

A method for identifying a 5-MeC in a template nucleic is provided. The method comprises providing a template having 5-MeC, converting the 5-MeC into a further modification selected from 5-caC and 5-FC. The converted template is then sequenced, and a change in sequencing is detected that is indicative of the further modification, allowing for identifying the 5-MeC in the template nucleic acid.

主权项

1. A method for identifying a 5-methylcytosine (5-MeC) in a template nucleic acid, the method comprising:
a) providing a template nucleic acid comprising the 5-MeC; b) converting the 5-MeC into a modified base by treating the template nucleic acid with a Tet protein, wherein the modified base is selected from 5-carboxylcytosine (5-caC) and 5-formylcytosine (5-fC), thereby generating a modified template nucleic acid; c) sequencing the modified template nucleic acid in the presence of fluorescently labeled nucleotides in a single-molecule sequencing reaction; d) monitoring said single-molecule sequencing reaction, wherein said monitoring comprises measuring interpulse duration, which is the length of time between fluorescent pulses, and pulse width, which is the width of fluorescent pulses, during said single-molecule sequencing reaction, and the fluorescent pulses are generated during incorporation of single bases of said fluorescently labeled nucleotides into a nascent nucleic acid strand synthesized during said single molecule sequencing reaction; and e) identifying the 5-methylcytosine (5-MeC) in the template nucleic acid by detecting a kinetic change during said sequencing the modified template nucleic acid, wherein said kinetic change is a kinetic change relative to a control template nucleic acid and comprises at least one change selected from the group consisting of a change in the interpulse duration and a change in the pulse width, wherein the presence of said kinetic change indicates the presence of the 5-MeC in the template nucleic acid.