Nucleotide analogs

• US 20070117103A1
• Filed: 11/22/2005
• 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:
  US11/286,626
• Publication Number:
  US 20070117103A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone

Abstract

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

Claims

1. A nucleotide analog having the structure:

wherein X₁ is OH or PO₄;

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 between about 5 carbon atoms and about 1 carbon atom between a cleavable bond and the base B; and

X₄ is O or S.

2. The nucleotide analog of claim 1, wherein said linker X₃ is attached to a label.

3. The nucleotide analog of claim 2, wherein said label is an optically-detectable label.

4. The nucleotide analog of claim 3, wherein said optically-detectable label is a fluorescent label.

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

6. The nucleotide analog of claim 1, wherein X₃ comprises a cleavable bond.

7. The nucleotide analog of claim 6, wherein said cleavable bond X₃ is a chemically cleavable bond.

8. The nucleotide analog of claim 6, wherein said cleavable bond X₃ is a photochemically cleavable bond.

9. The nucleotide analog of claim 8, wherein said photochemically cleavable bond is selected from the group consisting of o-nitrobenzyl and derivatives thereof.

10. The nucleotide analog of claim 6, wherein said cleavable bond is selected from the group consisting of:

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

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

13. 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 a labeled nucleotide analog having a linker comprising between about 5 carbon atoms and about 1 carbon atom between a cleavable bond and a base;

(c) identifying said incorporated labeled nucleotide;

(d) cleaving said cleavable bond;

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

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

14. The method of claim 13 further comprising the step of:

(g) capping said cleavable bond.

15. The method of claim 13, wherein the label is a detectable label.

16. The method of claim 15, wherein said detectable label is selected from the group consisting of cyanine, rhodamine, fluorescien, coumarin, BODIPY, alexa, and conjugated multi-dyes.

17. The method of claim 13, wherein said template is individually optically resolvable.

18. The method of claim 13, wherein said template is attached to a surface.

19. The method of claim 13, wherein the polymerase is Klenow with reduced exonuclease activity.

20. 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 a labeled nucleotide analog having a linker greater than 5 carbon atoms between a base and a label;

(c) identifying said incorporated labeled nucleotide;

(d) removing the label and at least a portion of said linker, wherein a remaining portion of said linker has 5 or fewer carbon atoms;

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

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

21. 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 a labeled nucleotide analog having a linker comprising between about 5 carbon atoms and about 1 carbon atom;

(c) identifying said incorporated labeled nucleotide by a label;

(d) removing said label;

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

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