POLYMERASE IDLING METHOD FOR SINGLE MOLECULE DNA SEQUENCING

• US 20150132756A1
• Filed: 11/11/2014
• Published: 05/14/2015
• Est. Priority Date: 11/14/2013
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Peter, Brian Jon, Mannion, John, Myerson, Joel
• Granted Patent: US 9,546,398 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

A method for sequencing a nucleic acid is provided. In certain embodiments the method comprises obtaining a duplex comprising a nucleic acid and a primer, wherein the primer has a nuclease resistant 3′ end, combining the duplex with a chain terminator nucleotide and a proof-reading polymerase to produce a reaction in which the polymerase idles on the added chain terminator nucleotide, identifying the chain terminator nucleotide added to the end of the primer; and adding a nuclease-resistant nucleotide to the end of the primer after the polymerase has idled on and removed the added chain terminator nucleotide, thereby producing a duplex comprising the template and an extended primer that has a nuclease resistant 3′ end.

Claims

1. A method of sequencing, comprising:

(a) obtaining a duplex comprising a nucleic acid and a primer, wherein the primer has a nuclease resistant 3′

end;

(b) combining the duplex with a chain terminator nucleotide and a DNA polymerase to produce a reaction in which the polymerase idles on the added chain terminator nucleotide by repeatedly adding the chain terminator nucleotide onto the end of the primer using the nucleic acid as a template, and then removing the chain terminator nucleotide;

(c) identifying the chain terminator nucleotide added to the end of the primer; and

(d) adding a nuclease-resistant nucleotide to the end of the primer after the polymerase has idled on, and removed the added chain terminator nucleotide, thereby producing a duplex comprising said template and an extended primer that has a nuclease resistant 3′

end.

2. The method of claim 1, further comprising repeating steps (b) and (c) on the product of step (d), thereby determining the sequence of two consecutive nucleotides of the nucleic acid.

3. The method of claim 2, wherein the nuclease-resistant nucleotide of (d) is a reversible chain terminator nucleotide and wherein the method comprises, after step (d), removing a blocking moiety from the nuclease resistant nucleotide before repeating step (b).

4. The method of claim 1, further comprising repeating steps (b), (c) and (d) at least 10 times, thereby determining the sequence of at least 10 consecutive nucleotides in the nucleic acid.

5. The method of claim 1, wherein the method comprises reaction conditions chosen such that the chain terminator nucleotide has a higher probability of incorporation relative to the nuclease-resistant nucleotide.

6. The method of claim 5, wherein the method comprises quantifying how much chain terminator nucleotide is added to the end of the primer by the polymerase.

7. The method of claim 1, wherein the combining step (b) comprises combining the duplex with a single chain terminator nucleotide corresponding to G, A, T or C.

8. The method of claim 7, wherein the identifying step comprises detecting pyrophosphate release.

9. The method of claim 7, wherein the identifying step comprises direct or indirect detection of protons.

10. The method of claim 7, wherein the chain terminator nucleotide is a phosphate labeled fluorescent nucleotide and the identifying step comprises reading fluorescence.

11. The method of claim 7, wherein the chain terminator nucleotide comprises a charge blockade label, and the identifying step comprises use of a nanopore.

12. The method of claim 1, wherein:

the combining step (b) comprises combining the duplex with four chain terminators corresponding to G, A, T and C, wherein the four chain terminators comprise different distinguishable labels; and

the identifying step (c) comprises identifying which of the four chain terminator nucleotides is added to the end of the primer.

13. The method of claim 1, wherein the nuclease-resistant nucleotide contains a phosphorothioate.

14. The method of claim 1, wherein the duplex is DNA that comprises single stranded regions and double stranded regions, that has been stretched on a substrate.

15. The method of claim 14, wherein the duplex is in a nanofluidic channel on a substrate.

16. The method of claim 14, wherein the duplex is made by:

nicking a double stranded DNA at one or more sites, making single stranded gaps in the double stranded DNA using an exonuclease, adding an nuclease resistant reversible terminator onto 3′

ends adjacent to the gaps, and then deprotecting the added nuclease resistant reversible terminator nucleotides.

17. The method of claim 1, wherein the DNA polymerase is a proofreading polymerase.

18. The method of claim 12, wherein the identifying step (c) comprises use of a nanopore.

19. The method of claim 1, wherein the nucleic acid or the primer is immobilized on a solid support.

20. The method of claim 1, wherein the combining step (b) comprises a pyrophosphorolysis reaction.