Methods and devices for sequencing nucleic acids

• US 20050260609A1
• Filed: 05/24/2004
• Published: 11/24/2005
• Est. Priority Date: 05/24/2004
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Lapidus, Stanley N.
• Application Number:
  US10/852,028
• Publication Number:
  US 20050260609A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

The invention provides methods and devices for high throughput single molecule sequencing of a plurality of target nucleic acids using a universal primer. Devices of the invention comprise a plurality of oligonucleotides, each having the same sequence, bound to a solid support, and ligated to a plurality of target nucleic acids.

Claims

1. A substrate for use in sequencing nucleic acids, the substrate comprising:

a solid support; and

a plurality of oligonucleotides, each having the same sequence, attached to said solid support in a spatial arrangement such that each of said oligonucleotides is individually optically resolvable,

wherein each of said oligonucleotides comprises

at least five nucleotides;

a primer attachment site; and

a terminal attachment site for attaching a target polynucleotide.

2. The substrate of claim 1, wherein each of said oligonucleotides comprises between about 7 nucleotides and about 100 nucleotides.

3. The substrate of claim 1, further comprising a plurality of target polynucleotides, each being attached to said terminal attachment site of a different one of said oligonucleotides.

4. The substrate of claim 1, further comprising a plurality of primers, each having the same sequence and being capable of hybridizing to said oligonucleotides.

5. The substrate of claim 1, wherein each of said oligonucleotides is attached to said solid support via a linker.

6. The substrate of claim 5, wherein said linker is a biotin/avidin couple.

7. The substrate of claim 5, wherein said linker is digoxigenin/anti-digoxigenin.

8. The substrate of claim 3, wherein said substrate comprises between about 50 and about 100,000 target polynucleotides, each being attached to said terminal attachment site of a different one of said oligonucleotides.

9. A kit comprising the substrate of claim 4 and a polymerase enzyme capable of adding nucleotides to said primers in a template-dependent manner.

10. The substrate of claim 3, wherein each of said target polynucleotides is attached to said terminal attachment site of a different one of said oligonucleotides through blunt-end or cohesive-end ligation.

11. A method for sequencing a target nucleic acid, the method comprising:

exposing the substrate of claim 3 to a plurality of primers, each having the same sequence and capable of hybridizing to said oligonucleotides;

extending said primer in the presence of one or more nucleotides comprising a detectable label; and

detecting label incorporated into said extended primer, thereby to determine the sequences of said target nucleic acids.

12. A method for sequencing nucleic acids, the method comprising:

attaching a plurality of oligonucleotides, each having the same sequence, to a surface of a solid support in a spatial arrangement such that each of said oligonucleotides is individually optically resolvable,

attaching each of a plurality of target polynucleotides to a different one of said oligonucleotides, producing a plurality of chimeric polynucleotides;

exposing said chimeric polynucleotides to a primer capable of hybridizing to said oligonucleotides;

extending said primer in the presence of one of more nucleotides comprising a detectable label; and

detecting label incorporated into said extended primer, thereby to determine the sequences of said target nucleic acids.

13. The method of claim 12, wherein said extending step comprises extending said primer in the presence of a single species of labeled nucleotide and said detecting step comprises detecting said labeled nucleotide if it is incorporated into said extended primer.

14. The method of claim 13, further comprising repeating said extending and detecting steps sequentially.

15. The method of claim 13, wherein said single species of labeled nucleotide is selected from the group consisting of dUTP, dATP, dCTP and dGTP.

16. The method claim 12, wherein said label is an optically-detectable label.

17. The method of claim 16, wherein said optically-detectable label is a fluorescent label.

18. The of claim 17, wherein said fluorescent label is selected from the group consisting of a fluorescein, a rhodamine, a phosphor, a polymethadine dye derivative, a fluorescent phosphoramidite, a texas red dye, a green fluorescent protein, an acridine, a cyanine, a cyanine 5 dye, a cyanine 3 dye, a 5-(2′

-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), a BODIPY, an ALEXA, and a derivative or modification of any of the foregoing.

19. The method of claim 12, wherein said step of attaching each of a plurality of target nucleic acids occurs prior to said step of attaching a plurality of oligonucleotides.

20. The method of claim 12, wherein said providing step comprises

attaching said oligonucleotides to said surface of said solid support;

and

attaching each of a plurality of target polynucleotides to a different one of said oligonucleotides.

21. The method of claim 20, wherein said step of attaching each of said plurality of target polynucleotides occurs prior to said step of attaching said oligonucleotides.

22. The method of claim 12, wherein said step of attaching each of said plurality of target nucleic acids comprises blunt-end or cohesive-end ligation.

23. The method of claim 12, further comprising the step of compiling a sequence of a complement of each of said target nucleic acids based upon sequential incorporation of said nucleotides into said extended primer.