METHODS FOR DETERMINING BASE LOCATIONS IN A POLYNUCLEOTIDE

• US 20180258474A1
• Filed: 04/05/2016
• Published: 09/13/2018
• Est. Priority Date: 04/06/2015
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Jain, Miten, Olsen, Hugh Edward, Akeson, Mark A.
• Granted Patent: US 10,760,117 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

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.

Claims

1. A method for detecting a sequence of a polynucleotide molecule, comprising:

(a) preparing a polynucleotide molecule which contains an abasic site; and

(b) conducting single molecule sequencing on the polynucleotide molecule prepared in step (a), including determining a sequence that comprises the abasic site, whereby the abasic site is identified within the sequence and may be correlated with a nucleotide in a reference sequence.

2. The method of claim 1, wherein preparing the polynucleotide molecule comprises treating the polynucleotide molecule with a reagent that specifically acts on a specific nucleobase species to create said abasic site.

3. The method of claim 2, wherein said reagent is a glycosylase that removes the specific nucleobase species from a sugar in the polynucleotide backbone.

4. The method of claim 1, wherein said abasic site is correlated to one of (a) a non-canonical base;

and (b) a location of an A, T, C, or G base in a sequence.

5. The method of claim 1, further comprising a step of correlating an abasic site to a location within a homopolymeric stretch in said polynucleotide molecule.

6. The method of claim 1, further comprising a step of correlating an abasic site to an epigenetic modification.

7. The method of claim 6, wherein said epigenetic modification is one of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formyl cytosine (5fC), and 5-carboxycytosine (5caC).

8. The method of claim 1, further comprising the step of correlating an abasic site to DNA damage of DNA adducts, 8-oxo-guanine, alkylated bases, ethenoadducts, and thymine dimers.

9. The method of claim 1, wherein the single molecule sequencing is nanopore-based sequencing comprising measuring an ionic current that identifies an abasic site.

10. The method of claim 9, wherein the nanopore-based sequencing includes detecting an ionic current through a nanopore through which the polynucleotide passes.

11. A method for detecting a sequence in a polynucleotide, comprising:

(a) treating a polynucleotide molecule with a glycosylase that creates an abasic site corresponding to a predetermined nucleobase species in the polynucleotide;

(b) conducting sequencing on the polynucleotide prepared in step (a), wherein the sequencing indicates an 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 said predetermined nucleobase.

12. The method of claim 11, wherein the predetermined nucleobase species is one of uracil, 5-methylcytosine (m5C), 5,6-dihydrouracil, 5-hydroxymethylcytosine, hypoxanthine and xanthine.

13. The method of claim 11, wherein the polynucleotide molecule is RNA and the predetermined nucleobase species is one or more of pseudouridine (LP), dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G).

14. The method of claim 11, further comprising the step of treating the polynucleotide with a reagent that modifies a specific predetermined nucleobase species to a different species of nucleobase by acting on the nucleobase, and then treating the polynucleotide molecule with said glycosylase, wherein said glycosylase acts on the different species of nucleobase.

15. The method of claim 14, wherein the reagent is 5 methyl-cytosine deaminase and the glycosylase is G/T(U)-mismatch DNA glycosylase.

16. The method of claim 14, wherein the reagent is KRuO₄ (potassium perruthenate).

17. The method of claim 11, wherein the glycosylase is one that lacks beta-lyase activity.

18. The method of claim 17, wherein the glycosylase is mutated to lack beta-lyase activity.

19. The method of claim 11, wherein the glycosylase and substrate converted to an abasic site is as follows:

20. The method of claim 11, wherein the polynucleotide molecule is a naturally occurring polynucleotide molecule comprising a non-canonical base.

21. The method of claim 20 wherein the glycosylase is uracil-DNA glycosylase or 5-methylcytosine DNA glycosylase.

22. The method of claim 20 further comprising the step of treating the polynucleotide molecule with an enzyme to modify a specific base species and then removing bases modified by the enzyme with said glycosylase.

23. The method of claim 11, wherein the polynucleotide molecule comprises a sequence of canonical bases, and wherein the glycosylase removes selected canonical bases from the polynucleotide molecule to create abasic sites within the polynucleotide molecule.

24. A method for improving sequence accuracy in a naturally occurring polynucleotide, comprising:

(a) conducting a first sequencing of the polynucleotide;

(b) treating a copy of the polynucleotide to remove a portion of bases having a predetermined structure of one of A, T, C, or G;

(c) conducting a second sequencing of a polynucleotide treated in step (b) and identifying abasic sites corresponding to said predetermined structure; and

(d) comparing results of the first sequencing and the second sequencing and correlating abasic sites found in the second sequencing with predetermined sites in the first sequencing, said comparison providing improved sequence accuracy.

25. The method of claim 24 comprising converting a number of cytosine bases to 5-methylcytosine and removing the 5-methyl cytosine with 5-methyl cytosine glycosylase to create abasic sites at the locations of cytosines.

26-28. -28. (canceled)