Methods and compositions for clonal amplification of nucleic acid

• US 20060008824A1
• Filed: 05/19/2005
• Published: 01/12/2006
• Est. Priority Date: 05/20/2004
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Leland Stanford Junior University (Stanford University)
• Inventors: Ronaghi, Mostafa, Mashayekhi, Foad
• Granted Patent: US 7,622,281 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00547 Bar codes
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Described herein are methods and compositions relating to amplifying nucleic acid. In certain embodiments, the invention provides methods for labeling and amplifying a nucleic acid molecule.

Claims

1. A method of labeling and amplifying a nucleic acid molecule, comprising attaching a unique primer to the nucleic acid molecule to be amplified and amplifying said labeled nucleic acid molecule.

2. The method of claim 1, wherein the unique primer is an adapter comprising a unique barcode.

3. The method of claim 2, wherein the unique primer is attached to the nucleic acid molecule by ligating the adapter comprising a unique barcode to the nucleic acid molecule.

4. The method of claim 2, wherein the unique barcode comprises a unique sequence of five nucleotides.

5. The method of claim 1, wherein the unique primer identifies a source of nucleic acid.

6. The method of claim 2, wherein the unique barcode identifies a source of nucleic acid.

7. The method of claim 5 or claim 6, wherein the source of nucleic acid is selected from the group consisting of:

a BAC clone, a bacterium, and a tissue sample.

8. The method of claim 1, wherein the labeled nucleic acid molecule is amplified by a sequencing-by-synthesis method.

9. The method of claim 8, wherein the labeled nucleic acid molecule is amplified by a method selected from the group consisting of:

massively parallel signature sequencing, BEAM, and polony technology.

10. A method of amplifying a nucleic acid molecule, comprising

(a) labeling the nucleic acid molecule with a unique primer;

(b) hybridizing said nucleic acid molecule to one or more anti-primers immobilized on a solid surface;

(c) incubating the solid surface of (b) with polymerase and nucleotides under conditions permitting extension of said one or more anti-primers;

(d) incubating the solid surface of (c) under conditions permitting denaturing of the nucleic acid molecules from the extended anti-primers;

removing the supernatant;

incubating the solid surface with polymerase, labeled nucleotides, and one or more oligonucleotides that hybridize to the immobilized extended anti-primers of (c), under conditions permitting extension of said hybridized oligonucleotides; and

identifying said solid surfaces comprising the extended hybridized oligonucleotides;

(e) immobilizing additional anti-primers on the solid surfaces of (d); and

(f) incubating the solid surfaces of (e) with polymerase, nucleotides, and one or more oligonucleotides that hybridize to the immobilized anti-primers of (e), under conditions permitting extension of said hybridized oligonucleotides, wherein the nucleic acid molecule is thereby amplified.

11. The method of claim 10, wherein the unique primer of (a) is an adapter comprising a unique barcode.

12. The method of claim 11, wherein the unique barcode comprises a unique sequence of five nucleotides.

13. The method of claim 10, further comprising:

(g) sequencing the immobilized amplified nucleic acid molecules of (f).

14. The method of claim 13, wherein the nucleic acid molecules are sequenced by pyrosequencing.

15. The method of claim 10, wherein the unique primer identifies a source of nucleic acid.

16. The method of claim 11, wherein the unique barcode identifies a source of nucleic acid.

17. The method of claim 15 or claim 16, wherein the source of nucleic acid is selected from the group consisting of:

a BAC clone, a bacterium, and a tissue sample.

18. The method of claim 10, wherein the solid surface is a bead.

19. The method of claim 18, wherein the bead is selected from the group consisting of:

a magnetic bead and a Sepharose bead.

20. A nucleic acid molecule attached to a solid surface and comprising a unique primer.

21. The method of claim 20, wherein the unique primer is an adapter that comprises a unique barcode.

22. The method of claim 21, wherein the unique barcode comprises a unique sequence of five nucleotides.

23. The nucleic acid molecule of claim 20, wherein the solid surface is a bead.

24. A solid surface comprising one or more nucleic acid molecules prepared by a method comprising:

(a) labeling the nucleic acid molecule with a unique primer;

(b) hybridizing said nucleic acid molecule to one or more anti-primers immobilized on a solid surface;

(c) incubating the solid surface of (b) with polymerase and nucleotides under conditions permitting extension of said one or more anti-primers;

(d) incubating the solid surface of (c) under conditions permitting denaturing of the nucleic acid molecules from the extended anti-primers;

removing the supernatant;

incubating the solid surface with polymerase, labeled nucleotides, and one or more oligonucleotides that hybridize to the immobilized extended anti-primers of (c), under conditions permitting extension of said hybridized oligonucleotides; and

identifying said solid surfaces comprising the extended hybridized oligonucleotides;

(e) immobilizing additional anti-primers on the solid surfaces of (d); and

(f) incubating the solid surfaces of (e) with polymerase, nucleotides, and one or more oligonucleotides that hybridize to the immobilized anti-primers of (e), under conditions permitting extension of said hybridized oligonucleotides, wherein the nucleic acid molecule is thereby amplified.

25. A kit comprising:

(a) a unique primer;

(b) a solid surface comprising immobilized anti-primers thereon;

(c) oligonucleotides that hybridize to the immobilized anti-primers of (b); and

(d) instructions for amplifying a nucleic acid molecule.

26. The kit of claim 25, wherein the unique primer of (a) is an adapter comprising a unique barcode.

27. The kit of claim 26, wherein the unique barcode comprises a unique sequence of five nucleotides.

28. The kit of claim 25, wherein the instructions of (d) include instructions for amplifying a nucleic acid molecule according to a method comprising:

(a) labeling the nucleic acid molecule with a unique primer;

(b) hybridizing said nucleic acid molecule to one or more anti-primers immobilized on a solid surface;

(c) incubating the solid surface of (b) with polymerase and nucleotides under conditions permitting extension of said one or more anti-primers;

(d) incubating the solid surface of (c) under conditions permitting denaturing of the nucleic acid molecules from the extended anti-primers;

removing the supernatant;

incubating the solid surface with polymerase, labeled nucleotides, and one or more oligonucleotides that hybridize to the immobilized extended anti-primers of (c), under conditions permitting extension of said hybridized oligonucleotides; and

identifying said solid surfaces comprising the extended hybridized oligonucleotides;

(e) immobilizing additional anti-primers on the solid surfaces of (d); and

(f) incubating the solid surfaces of (e) with polymerase, nucleotides, and one or more oligonucleotides that hybridize to the immobilized anti-primers of (e), under conditions permitting extension of said hybridized oligonucleotides, wherein the nucleic acid molecule is thereby amplified.