Methods for determining base locations in a polynucleotide

Abstract

Disclosed are methods for polynucleotide sequencing that detect the location of selected nucleobases with greater precision. The methods can be used to determine the location and nature of modified bases in a polynucleotide, that is, non-canonical bases, or to improve accuracy of sequencing of “problem” regions of DNA sequencing such as homopolymers, GC rich areas, etc. The sequencing method exemplified is nanopore sequencing. Nanopore sequencing is used to generate a unique signal at a point in a polynucleotide sequence where an abasic site (AP site, or apurinic or apyrimidinic site) exists. As part of the method, an abasic site is specifically created enzymatically using a DNA glycosylase that recognizes a pre-determined nucleobase species and cleaves the N-glycosidic bond to release only that base, leaving an AP site in its place.

1 - 28 . (canceled) 
     
     
         29 . A method of detecting a sequence in an RNA polynucleotide molecule, the method comprising:
 (a) treating the RNA polynucleotide molecule with an RNA glycosylase that creates an abasic site corresponding to pseudouridine (Ψ), diydrouridine (D), inosine (I), and 7-methylguanosine (m7g) species in the polynucleotide;   (b) conducting single molecule sequencing on the polynucleotide prepared in step (a) where the sequencing indicates the abasic site within the polynucleotide sequence; and   (c) using the sequence from step (b) to identify the abasic site and correlating said abasic site to pseudouridine ('P), diydrouridine (D), inosine (I), and 7-methylguanosine (m7g).   
     
     
         30 . The method of  claim 29 , wherein the RNA glycosylase is EC 3.2.2.22. 
     
     
         31 . The method of  claim 29 , wherein the RNA glycosylase lacks beta lyase activity. 
     
     
         32 . The method of  claim 31 , wherein the RNA glycosylase is engineered to lack beta lyase activity. 
     
     
         33 . The method of  claim 29 , wherein conducting single molecule sequencing comprises nanopore-based sequencing comprises measuring an ionic current that identifies an abasic site. 
     
     
         34 . The method of  claim 33 , wherein the nanopore-based sequencing includes detecting an ionic current through a nanopore through which the polynucleotide passes. 
     
     
         35 . The method of  claim 31 , wherein conducting single molecule sequencing comprises nanopore-based sequencing comprises measuring an ionic current that identifies an abasic site. 
     
     
         36 . The method of  claim 35 , wherein the nanopore-based sequencing includes detecting an ionic current through a nanopore through which the polynucleotide passes. 
     
     
         37 . The method of  claim 29 , wherein the RNA polynucleotide molecule is mRNA. 
     
     
         38 . The method of  claim 29 , wherein the RNA polynucleotide molecule is tRNA. 
     
     
         39 . The method of  claim 29 , wherein the RNA polynucleotide molecule is genomic RNA.