Nucleotide analogs

• US 20070117104A1
• Filed: 12/05/2005
• Published: 05/24/2007
• Est. Priority Date: 11/22/2005
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Buzby, Philip
• Application Number:
  US11/295,406
• Publication Number:
  US 20070117104A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2533/101 Primer extension

Abstract

The invention provides nucleotide analogs for use in sequencing nucleic acid molecules.

Claims

1. A nucleotide analog having the structure:

wherein X₁ is ONH₂;

X₂ is H or OH;

B is selected from the group consisting of a purine, a pyrimidine and derivatives thereof;

X₃ is a linker comprising a cleavable bond, wherein said linker further comprises between about 25 atoms and about 1 atom between the base B and the label; and

X₄ is O or S.

2. The nucleotide analog of claim 1, wherein said cleavable bond is a disulfide bond.

3. The nucleotide analog of claim 1, wherein said cleavable bond is an ester.

4. The nucleotide analog of claim 1, wherein said label is an optically-detectable label.

5. The nucleotide analog of claim 2, wherein said optically-detectable label is a fluorescent label.

6. The nucleotide analog of claim 1, wherein B is selected from the group consisting of cytosine, uracil, thymine, adenine, guanine, and analogs thereof.

7. The nucleotide analog of claim 5, wherein said cleavable bond X₃ is a chemically cleavable bond.

8. The nucleotide analog of claim 5, wherein said cleavable bond X₃ is a photochemically cleavable bond.

9. The nucleotide analog of claim 7, wherein said photochemically cleavable bond is selected from the group consisting of o-nitrobenzyl and derivatives thereof.

10. The nucleotide analog of claim 1, wherein X₃ comprises a triple bond.

11. The nucleotide analog of claim 1, wherein said linker X₃ has between about 5 atoms and about 1 atom.

12. A method for sequencing a nucleic acid template, the method comprising the steps of:

(a) exposing a nucleic acid template to a primer capable of hybridizing to said template and a polymerase capable of catalyzing nucleotide addition to said primer;

(b) incorporating the nucleotide analog of claim 1 to said primer;

(c) identifying the incorporated nucleotide analog;

(d) cleaving the cleavable bond; and

,

(e) unblocking the hydroxyl amine located at the 3′

position of the nucleotide analog.

13. The method of claim 11, further comprising the steps of:

(f) repeating steps b, c, and d at least once; and

(g) determining a sequence of said template based upon the order of incorporation of said labeled nucleotides.

14. The method of claim 11, wherein the unblocking step comprises exposing the nucleotide analog to an acid that cleaves the amine group from the nucleotide analog.

15. The method of claim 11 further comprising the step of:

(h) capping said cleavable bond.

16. The method of claim 11, wherein said template is individually optically resolvable.

17. The method of claim 11, wherein said template is attached to a surface.

18. The method of claim 11, wherein the polymerase is Klenow with reduced exonuclease activity.

19. The nucleotide analog of claim 1, wherein said label is a detectable label selected from the group consisting of cyanine, rhodamine, fluorescien, coumarin, BODIPY, alexa, and conjugated multi-dyes.

20. A nucleotide analog having the structure:

wherein X₁ is selected from the group consisting of hydroxyl amines, aldehydes, allylamines, alkenes, alkynes, alcohols, amines, aryls, esters, ethers, carbonitriles, phosphates, carbonates, carbamates, borates, nitrates, sugars, phosphoramidates, phenylsulfanates, sulfates, sulfones and heterocycles;

X₂is H or OH;

B is selected from the group consisting of a purine, a pyrimidine and derivatives thereof;

X₃ is a linker comprising a cleavable bond, wherein said linker further comprises between about 25 atoms and about 1 atom between the base B and the label; and

X₄ is O or S.