Polymerase idling method for single molecule DNA sequencing

• US 10,590,479 B2
• Filed: 11/16/2016
• Issued: 03/17/2020
• Est. Priority Date: 11/14/2013
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Peter, Brian Jon, Myerson, Joel
• Primary Examiner(s): Priest, Aaron A
• Application Number:
  US15/353,592
• Publication Number:
  US 20170067103A1
• Time in Patent Office: 1,217 Days
• Field of Search: None
• 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 DNA, comprising:

(a) obtaining a duplex comprising a nucleic acid and a primer;

(b) combining the duplex with one or more nucleoside polyphosphates, one or more chain terminator nucleotides, a DNA polymerase, and inorganic pyrophosphate to produce a reaction in which the polymerase repeatedly adds a chain terminator nucleotide onto the end of the primer using the nucleic acid as a template, and then removes the chain terminator nucleotide by pyrophosphorolysis; and

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

wherein;

i) the probability of incorporating a chain terminator nucleotide is greater than the probability of incorporating a nucleoside polyphosphate.

2. The method of claim 1, wherein identifying step (c) involves measuring the products of the pyrophosphorolysis reaction.

3. The method of claim 1, wherein the identifying step (c) occurs in real time.

4. The method of any claim 1, further comprising determining the sequence of at least 10 consecutive nucleotides in the nucleic acid.

5. The method of claim 1, wherein the chain terminator nucleotide comprises a label.

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 5, wherein the label is a fluorescent label.

8. The method of claim 1, wherein the polymerase lacks exonuclease activity.

9. The method of claim 1, wherein the identifying step comprises direct or indirect detection of protons.

10. The method of claim 5, wherein the label is attached to a terminal phosphate.

11. The method of claim 5, wherein the label comprises a charge blockade label, and the identifying step comprises use of a nanopore.

12. The method of any 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 chain terminator nucleotide comprises a fluorescence quencher.

14. The method of any claim 1, wherein the chain terminator nucleotide is a dideoxynucleotide.

15. The method of claim 1, wherein the primer is extended by more than one nucleotide in step (b).

16. The method of claim 1, wherein the nucleoside polyphosphates are nucleoside triphosphates.

17. The method of any claim 12, further comprising determining the sequence of 10 or more consecutive nucleotides.

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

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

20. The method of any claim 5, wherein the nucleoside polyphosphates comprise a label.