Methods and apparatus for preparing arrays comprising features having degenerate biopolymers

• US 7,504,213 B2
• Filed: 11/25/2003
• Issued: 03/17/2009
• Est. Priority Date: 07/09/1999
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Leproust, Eric M., Wolber, Paul K., Sana, Theodore R.
• Primary Examiner(s): Kim; Young J
• Application Number:
  US10/722,155
• Publication Number:
  US 20050019786A1
• Time in Patent Office: 1,939 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6813 Hybridisation assays

Abstract

Methods and apparatus are disclosed for synthesizing a plurality of biopolymers at predetermined feature locations on a surface of a substrate. One or more of the feature locations comprises degenerate biopolymers. One or more biopolymer subunit precursors are added, in multiple rounds of subunit additions, at each of multiple feature locations on the surface to form the plurality of biopolymers on the surface. For each feature location comprising degenerate biopolymers, the biopolymer subunit precursors comprise a mixture of biopolymer subunit precursors for forming the degenerate biopolymers at the feature location.

Claims

1. A method for synthesizing a plurality of biopolymers comprising nucleotides at predetermined feature locations on a surface of a substrate wherein a feature location of said feature locations comprises degenerate biopolymers comprising nucleotides, said method comprising:

producing a mixture of two or more different biopolymer subunit precursors; and

providing said mixture to a predetermined feature location on said surface of said substrate in a round of multiple rounds of subunit additions to produce the feature location comprising degenerate biopolymers.

2. A method according to claim 1 wherein said biopolymers are polynucleotides.

3. A method according to claim 1 wherein said degenerate biopolymers comprise a contiguous stretch of 1 to 5 degenerate nucleotides.

4. The method of claim 1, wherein said biopolymer subunit precursors are nucleotide precursors.

5. The method of claim 4, wherein said mixture of different biopolymer subunit precursors comprises nucleotide precursors corresponding to C, G, A and T.

6. The method of claim 1, wherein said biopolymer subunit precursors are monomers.

7. The method of claim 1, wherein said degenerate biopolymer feature location comprises degenerate biopolymers having less than 10 sites of degeneracy.

8. A method for synthesizing a plurality of biopolymers comprising nucleotides at predetermined feature locations on a surface of a substrate wherein a feature location of said feature locations comprises degenerate biopolymers comprising nucleotides, said method comprising, in each round of multiple rounds of subunit additions, providing biopolymer subunit precursors at each of multiple feature locations on said surface to form said plurality of said biopolymers on said surface, wherein, for the feature location comprising said degenerate biopolymers, said biopolymer subunit precursors for a round of said multiple rounds are provided as a mixture of two or more different biopolymer subunit precursors for forming said degenerate biopolymers, each round of subunit additions comprising

(a) dispensing from a dispensing system said biopolymer subunit precursors to said predetermined feature locations,

(b) dispensing activator to said predetermined feature locations, and

(c) repeating steps (a)-(b).

9. A method according to claim 8 wherein said biopolymers are polynucleotides.

10. A method according to claim 8 wherein said degenerate biopolymers comprise a contiguous stretch of 1 to 5 degenerate nucleotides.

11. A method according to claim 8 wherein said biopolymers are oligonucleotides.

12. A method according to claim 8 wherein said dispensing system comprises at least one droplet dispensing device.

13. The method of claim 12, wherein said droplet dispensing device comprises a reservoir comprising said mixture.

14. A method according to claim 8, which is a computer based method wherein steps (a) through (c) are carried out under computer control.

15. The method of claim 8, wherein said biopolymer subunit precursors are nucleotide precursors.

16. The method of claim 15, wherein said mixture of different biopolymer subunit precursors comprises nucleotide precursors corresponding to C, G, A and T.

17. The method of claim 8, wherein said biopolymer subunit precursors are monomers.

18. A method for synthesizing a plurality of biopolymers comprising nucleotides at predetermined feature locations on a surface of a substrate, wherein a feature location of said feature locations comprises degenerate biopolymers comprising nucleotides, said method comprising, in each round of multiple rounds of subunit additions, providing biopolymer subunit precursors at each of multiple feature locations on said surface to form said plurality of said biopolymers on said surface, wherein, for the feature location comprising said degenerate biopolymers, said biopolymer subunit precursors for a round of said multiple rounds are provided as a mixture comprising a predetermined ratio of two or more different biopolymer subunit precursors for forming said degenerate biopolymers, each round of subunit additions comprising

(a) dispensing from a dispensing system said biopolymer subunit precursors to said predetermined feature locations in a droplet manner,

(b) dispensing activator to said predetermined feature locations, and

(c) repeating steps (a)-(b) to form said plurality of biopolymers comprising nucleotides at predetermined feature locations on said surface.

19. A method according to claim 18 wherein said biopolymers are polynucleotides.

20. A method according to claim 18 wherein said degenerate biopolymers comprise a contiguous stretch of 1 to 5 degenerate nucleotides.

21. A method according to claim 18 wherein said biopolymers are oligonucleotides.

22. A method according to claim 18 wherein said dispensing system comprises at least one droplet dispensing device.

23. The method of claim 22, wherein said droplet dispensing device comprises a reservoir comprising said mixture.

24. A method according to claim 18, which is a computer based method wherein steps (a) through (c) are carried out under computer control.

25. The method of claim 18, wherein said biopolymer subunit precursors are nucleotide precursors.

26. The method of claim 25, wherein said mixture comprising a predetermined ratio of different biopolymer subunit precursors comprises nucleotide precursors corresponding to C, G, A and T.

27. The method of claim 18, wherein said biopolymer subunit precursors are monomers.

28. A method for synthesizing a plurality of biopolymers comprising nucleotides at predetermined feature locations on a surface of a substrate, wherein a feature location of said feature locations comprises degenerate biopolymers comprising nucleotides, said method comprising, in each round of multiple rounds of subunit additions, providing biopolymer subunit precursors at each of multiple feature locations on said surface to form said plurality of said biopolymers on said surface, wherein, for the feature location comprising said degenerate biopolymers, said biopolymer subunit precursors for a round of said multiple rounds are provided as a mixture of two or more different biopolymer subunit precursors for forming said degenerate biopolymers.