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

• US 20100036110A1
• Filed: 03/19/2009
• Published: 02/11/2010
• 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: Greenleaf, William J., Sims, Peter A., Xie, Xiaoliang Sunney
• Application Number:
  US12/407,486
• Publication Number:
  US 20100036110A1
• Time in Patent Office: Days
• US Class Current: 536/26.220
• 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 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

The invention features compositions, methods, and systems for single-molecule sequencing of nucleic acids based on the continuous measurement of the incorporation of fluorogenic nucleotides in microreactors. In particular, the invention features fluorogenic compounds for use in nucleic acid sequencing. The invention provides numerous advantages over previous systems such as unambiguous determination of sequence, continuous sequencing, long read lengths, low overall cost, and ease of sample preparation.

Claims

1-28. -28. (canceled)

29. 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.

30. The compound of claim 29, wherein Sugar is ribose or 2′

-deoxyribose.

31. The compound of claim 29, wherein Base is cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thiomnosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil.

32. The compound of claim 29, wherein Phosphate is a triphosphate.

33. The compound of claim 29, wherein [Self-reacting Component] comprises a self-immolative linker.

34. The compound of claim 29, wherein [Self-reacting Component] comprises a moiety that undergoes an intramolecular reaction to form a fluorophore upon removal of the phosphate.

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

36. The compound of claim 33 or 35, wherein said self-immolative

linker is

37. The compound of claim 36, wherein X—

NH has the formula

38. The compound of claim 34, having the formula:

39. The compound of claim 34 or 38, wherein said moiety that undergoes an intramolecular reaction to form a fluorophore upon removal of the phosphate has the formula:

40. A compound having the formula:

41. The compound of claim 40, having the formula:

42. The compound of claim 40, wherein R is cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil.

43. A compound having the formula:

44. The compound of claim 43, wherein R is cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6-dihydrouracil, and 5-bromouracil.

45. The compound of claim 43, having the formula:

46-47. -47. (canceled)