METHODS AND COMPOSITIONS FOR CONTINUOUS SINGLE-MOLECULE NUCLEIC ACID SEQUENCING BY SYNTHESIS WITH FLUOROGENIC NUCLEOTIDES

• US 20100227327A1
• Filed: 03/05/2010
• Published: 09/09/2010
• Est. Priority Date: 08/08/2008
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Xie, Xiaoliang Sunney, Greenleaf, William J., Sims, Peter A.
• Application Number:
  US12/718,421
• Publication Number:
  US 20100227327A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/207 the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

Disclosed herein are methods and compositions for continuous single-molecule nucleic acid sequencing by synthesis with fluorogenic nucleotides.

Claims

1. A method for sequencing a nucleic acid, said method comprising the steps of:

a) disposing in an optionally sealed microreactor a mixture in solution phase comprising a single copy of a target nucleic acid, a nucleic acid replicating catalyst, and a mixture of nucleotides, wherein said mixture of nucleotides comprises a first nucleotide comprising a first label that is substantially non-fluorescent until after incorporation of said first nucleotide into a nucleic acid based on complementarity to said target nucleic acid;

b) allowing continuous template-dependent replication of said target nucleic acid; and

c) sequencing said target nucleic acid by detecting in real time the individual incorporation of said first nucleotide during template-dependent replication by monitoring fluorescence emission resulting from said first label.

2. The method of claim 1, wherein said mixture in solution phase further comprises an activating enzyme that renders said first label fluorescent.

3. The method of claim 2, wherein said activating enzyme is an alkaline phosphatase, acid phosphatase, galactosidase, horseradish peroxidase, phosphodiesterase, phosphotriesterase, pyruvate kinase, lactic dehydrogenase, maltose phosphorylase, glucose oxidase, lipase, or combination thereof.

4. The method of claim 1, wherein said first label is photobleached after step (c).

5. The method of claim 1, wherein said first label is a phosphate label that is cleaved from said first nucleotide during replication.

6. The method of claim 1, wherein said mixture of nucleotides further comprises a second nucleotide comprising a second label that is substantially non-fluorescent until incorporation of said second nucleotide into said nucleic acid based on complementarity to said target nucleic acid.

7. The method of claim 6, wherein said mixture of nucleotides further comprises a third nucleotide comprising a third label that is substantially non-fluorescent until incorporation of said third nucleotide into said nucleic acid based on complementarity to said target nucleic acid.

8. The method of claim 7, wherein said mixture of nucleotides further comprises a fourth nucleotide comprising a fourth label that is substantially non-fluorescent until incorporation of said fourth nucleotide into said nucleic acid based on complementarity to said target nucleic acid.

9. The method of claim 1, further comprising repeating steps (b)-(c) at least once.

10. The method of claim 1, wherein said mixture in a solution phase has a volume of 0.0001 fL-1000 fL.

11. The method of claim 1, wherein said nucleic acid replicating catalyst is DNA polymerase, RNA polymerase, ligase, reverse transcriptase, or RNA-dependent RNA polymerase.

12. The method of claim 1, wherein said target nucleic acid is DNA, and said mixture in solution phase further comprises a primer.

13. The method of claim 1, wherein said target nucleic acid is RNA.

14. The method of claim 1, wherein steps (a)-(c) are repeated to obtain the sequence for 10, 25, 100, 300, 1000, or 10,000 base pairs of said target nucleic acid.

15. The method of claim 1, wherein said sequencing occurs continuously.

16. The method of claim 1, wherein said nucleic acid is immobilized on a surface of said microreactor.

17. The method of claim 1, wherein said nucleic acid is immobilized on a bead disposed in said microreactor.

18. The method of claim 2, wherein said activating enzyme is immobilized on a surface of said microreactor.

19. The method of claim 2, wherein said nucleic acid is immobilized on a bead disposed in said microreactor.

20. A compound having the formula:

Base-Sugar-Phosphate-[Self-reacting Component],

where Base is a nucleotide base, Sugar is selected from the group consisting of ribose, 2′

-deoxyribose, 2′

-O-methyl-ribose, ribose comprising a methylene connecting the 2′

oxygen and 4′

carbon, glycerol, 2-methyl morpholine, or threose, Phosphate is a polyphosphate, and Self-reacting Component is a moiety that undergoes an intramolecular reaction upon cleavage of the phosphate to which it is connected to form a fluorophore.

21. The compound of claim 20, wherein Sugar is ribose or 2′

-deoxyribose.

22. The compound of claim 20, wherein Base is cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil.

23. The compound of claim 20, wherein Phosphate is a triphosphate.

24. The compound of claim 20, wherein [Self-reacting Component] comprises a self-immolative linker.

25. The compound of claim 20, wherein [Self-reacting Component] comprises a moiety that undergoes an intramolecular reaction to form a fluorophore upon removal of the phosphate.

26. The compound of claim 20, having the formula:

27. The compound of claim 24, wherein said self-immolative linker is

28. The compound of claim 27, wherein X—

NH has the formula

29. The compound of claim 25, having the formula:

30. The compound of claim 25, wherein said moiety that undergoes an intramolecular reaction to form a fluorophore upon removal of the phosphate has the formula:

31. A compound having the formula:

32. The compound of claim 31, wherein R is cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil.

33. A compound having the formula:

34. The compound of claim 33, wherein R is cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil.

35. The compound of claim 33, having the formula: