Methods for Sequencing DNA

• US 20090318298A1
• Filed: 03/27/2009
• Published: 12/24/2009
• Est. Priority Date: 09/28/2006
• Status: Abandoned Application

• Current Assignee: Brigham and Women's Hospital Incorporated, President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Brigham and Women's Hospital Incorporated, President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Kim, Jae B., Seidman, Jonathan G., Porreca, Gregory J., Church, George M.
• Application Number:
  US12/412,471
• Publication Number:
  US 20090318298A1
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The invention is directed to methods for using sequence by ligation to sequence DNA immobilized on miniaturized, high density bead-based arrays. Methods are provided to fabricate an array of beads where the beads are coupled directly to a solid support. Methods are also provided to improve signal and reduce background in ligation-mediated DNA sequencing. In addition, methods are provided to improve the accuracy of reported tag counts when performing DNA sequencing by fluorescent nonamer ligation.

Claims

1. An array comprising a plurality of beads and a solid support, wherein each bead comprises oligonucleotides immobilized on the surface of the bead, and wherein each bead is connected to the solid support by at least one molecule on the bead which can bind to a corresponding molecule on a surface of the solid support.

2. The array of claim 1, wherein the beads are polony beads.

3. The array of claim 1, wherein the beads are arranged in a uniform layer of one bead thickness.

4. The array of claim 1, wherein the solid support is glass, metal, ceramic, or plastic.

5. The array of claim 1, wherein the each bead is connected to the solid support by at least one tether molecule covalently attached to both the surface of the solid support and to an oligonucleotide immobilized on the bead.

6. The array of claim 5, wherein the tether molecule is a linker selected from the group consisting of amino esters, bis(sulfosuccinimidyl)suberate (BS3), N-hydroxysuccinimidyl (NHS), N-(κ

-maleimidoundecanoyloxy)sulfosuccinimidyl (KMUS), N-ε

-maleimidocaproyloxy)succinimidyl (EMCS), iodoacetamide, dithiol, nitrobenzyl, and mixtures of any of them.

7. The array of claim 5,

wherein the tether molecule is selected from the group consisting of polyethylene glycol, poly(N-vinyl lactams), polysaccharides, polyacrylates, polyacrylamides, polyalkylene oxides, and copolymers of any of them;

and

wherein the tether molecule is covalently attached to at least one oligonucleotide immobilized on the bead through a linker selected from the group consisting of amino esters, bis(sulfosuccinimidyl)suberate (BS3), N-hydroxysuccinimidyl (NHS), N-(κ

-maleimidoundecanoyloxy)sulfosuccinimidyl (KMUS), N-(ε

-maleimidocaproyloxy)succinimidyl (EMCS), iodoacetamide, dithiol, nitrobenzyl, and mixtures of any of them.

8. The array of claim 1, wherein each bead is bound to an amino-silylated glass support through at least one oligonucleotide having an —

NH₂ group at its 3′

terminus, the —

NH₂ group at 3′

terminus being covalently attached to bis(sulfosuccinimidyl)suberate (BS3), and the BS3 being covalently attached to an —

NH₂ group on a surface of the amino-silylated glass support.

9. The array of claim 1, wherein each bead is bound to a glass support through at least one oligonucleotide having an —

NH₂ group at its 3′

terminus, the —

NH₂ group at the 3′

terminus being covalently attached to N-hydroxysuccinimide (NHS), the NHS being covalently attached to polyethylene glycol (PEG), and the PEG being covalently attached to a surface of the glass support.

10. A method for producing an array comprising the steps of:

a) providing a plurality of beads, wherein each bead comprises oligonucleotides immobilized on the surface of the bead,

b) providing a solid support;

c) connecting the plurality of beads to the solid support by binding at least one molecule on a bead to a corresponding molecule on a surface of the solid support.

11. The method of claim 10, wherein the beads are polony beads.

12. The method of claim 10, wherein the connecting step of step c) comprises covalently attaching a tether molecule to a surface of the solid support and to an oligonucleotide immobilized on a bead.

13. The method of claim 10,

wherein the solid support of step b) comprises a glass support comprising tether molecules covalently attached to the surface of the glass support, wherein the tether molecules comprise linker functional groups;

and

wherein the connecting step of step c) comprises covalently attaching at least one oligonucleotide immobilized on a bead to a linker functional group.

14. A method for DNA sequencing, comprising the steps of:

a) providing a plurality of beads, wherein each bead has immobilized on the surface thereof single-stranded template DNA having a 3′

terminus;

b) annealing a first oligonucleotide to the 3′

terminus of the template DNA, wherein the first oligonucleotide comprises a 5′

overhanging sequence;

c) annealing a second oligonucleotide to the 5′

overhanging sequence, wherein the second oligonucleotide comprises a 3′

blocking moiety; and

d) ligating the second oligonucleotide to the template DNA;

e) providing a solid support;

f) forming an array by connecting the plurality of beads to the solid support;

g) performing DNA sequencing on the array.

15. The method of claim 14, wherein the beads are polony beads.

16. The method of claim 14, wherein the DNA sequencing step of step g) comprises the steps of

h) annealing to the template DNA a sequencing primer and a degenerate oligonucleotide comprising a fluorescent tag;

and

i) ligating the sequencing primer to the degenerate oligonucleotide.

17. The method of claim 16, wherein step i) further comprises ligating with the inclusion of polyethylene glycol in the ligation reaction.

18. The method of claim 16, wherein step i) further comprises ligating at incrementally increasing temperatures from about 20°

C. to about 40°

C.

19. The method of claim 16, wherein step i) further comprises ligating at 18°

C. for five minutes, then at 25°

C. for five minutes, then at 30°

C. for five minutes, and then at 37°

C. for five minutes.

20. The method of claim 16, wherein step i) further comprises ligating with the inclusion of at least one compound having the property of decreasing the difference in melting temperature between A/T and G/C base pairs.

21. The method of claim 20, wherein the compound is betaine.

22. The method of claim 14, wherein step d) further comprises ligating with the inclusion of polyethylene glycol in the ligation reaction.

23. The method of claim 14, further comprising the step of removing free forward primer after step f).

24. The method of claim 14, wherein the template DNA, or the degenerate oligonucleotide comprising a fluorescent tag, further comprises nucleotide analogs having the property of decreasing the difference in melting temperature between A/T and G/C base pairs.

25. The method of claim 24, wherein the nucleotide analogs are selected from the group consisting of a 2-aminopurine, a 2,6-diaminopurine, bromodeoxyuridine, deoxyinosine, 5-nitroindole, locked nucleic acids, and mixtures of any of them.

26. The method of claim 14, wherein the 3′

blocking moiety is selected from the group consisting of an amino-modifier, a dideoxycytidine, a non-ribose, a covalent blocking group, a steric blocking group, a reversible blocking group, and mixtures of any of them.

27. The method of claim 14,

wherein the 3′

blocking moiety is an amino-modifier;

wherein the solid support is an amino-silylated glass substrate; and

wherein the connecting step of step f) comprises covalently attaching a tether molecule to the amino-silylated glass substrate and to an amino-modified template DNA resulting from step d).

28. The method of claim 14,

wherein the 3′

blocking moiety is an amino-modifier;

wherein the solid support is a glass support comprising tether molecules covalently attached to the surface of the glass support, wherein the tether molecules comprise linker functional groups; and

wherein the connecting step of step f) comprises covalently attaching a linker functional group to an amino-modified template DNA resulting from step d).