Method for making populations of defined nucleic acid molecules

• US 20040259146A1
• Filed: 06/02/2004
• Published: 12/23/2004
• Est. Priority Date: 06/13/2003
• Status: Active Grant

• Current Assignee: Cold Spring Harbor Laboratory
• Original Assignee: Cold Spring Harbor Laboratory, Rosetta Inpharmatics LLC (Merck & Co., Inc.)
• Inventors: Kilian, Kristopher A., Coffey, Ernest M., Hannon, Gregory J., Cleary, Michele A., Friend, Stephen H.
• Granted Patent: US 8,133,670 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: Y10S 977/792 Nucleic acid array, e.g. human genome array

Abstract

The present invention provides methods of making a population of nucleic acid molecules, wherein each nucleic acid molecule comprises a predetermined nucleic acid sequence, each of said methods comprising the steps of: (a) synthesizing, on a substrate, a population of nucleic acid molecules wherein: i) each synthesized nucleic acid molecule comprises a predetermined nucleic acid sequence; and ii) each synthesized nucleic acid molecule is localized to a defined area of said substrate; (b) harvesting said population of synthesized nucleic acid molecules from said substrate to yield harvested nucleic acid molecules; and (c) introducing said harvested nucleic acid molecules into vector molecules.

Claims

1. A method of making a population of nucleic acid molecules, wherein each nucleic acid molecule comprises a predetermined nucleic acid sequence, said method comprising the steps of:

(a) synthesizing a population of nucleic acid molecules on a substrate, wherein;

i) each synthesized nucleic acid molecule comprises a predetermined nucleic acid sequence, and

ii) each synthesized nucleic acid molecule is localized to a defined area of said substrate;

(b) harvesting said population of synthesized nucleic acid molecules from said substrate to yield harvested nucleic acid molecules; and

(c) introducing said harvested nucleic acid molecules into vector molecules.

2. The method of claim 1 further comprising the step of amplifying a member of the group consisting of said synthesized nucleic acid molecules and said harvested nucleic acid molecules prior to introducing said harvested nucleic acid molecules into vector molecules.

3. The method of claim 2 wherein said amplification is by polymerase chain reaction (PCR).

4. The method of claim 1 wherein said synthesized nucleic acid molecules have an average length of from about 20 nucleotides to about 100 nucleotides.

5. The method of claim 1 wherein said synthesized nucleic acid molecules have an average length of from about 50 nucleotides to about 100 nucleotides.

6. The method of claim 1 wherein said synthesized nucleic acid molecules have an average length of from about 60 nucleotides to about 100 nucleotides.

7. The method of claim 1 wherein said synthesized nucleic acid molecules have an average length of from about 80 nucleotides to about 100 nucleotides.

8. The method of claim 1 wherein said population of synthesized nucleic acid molecules is synthesized using an ink-jet printer.

9. The method of claim 1 wherein said substrate is selected from the group consisting of glass, plastic, polyacrylamide and nitrocellulose.

10. The method of claim 9 wherein said substrate consists essentially of glass.

11. The method of claim 9 wherein said substrate consists essentially of plastic.

12. The method of claim 1 wherein said population of synthesized nucleic acid molecules is harvested from said substrate by contacting said substrate with an amount of ammonium hydroxide sufficient to remove said synthesized nucleic acid molecules from said substrate.

13. The method of claim 1 wherein said substrate comprises a multiplicity of defined areas.

14. The method of claim 13 wherein said substrate comprises from about 1,000 to about 30,000 defined areas.

15. The method of claim 13 wherein each of said defined areas comprises a population of synthesized nucleic acid molecules, wherein each synthesized nucleic acid molecule localized to a defined area comprises a nucleic acid sequence that is essentially identical to the nucleic acid sequence of every other synthesized nucleic acid molecule localized to the same defined area of said substrate.

16. The method of claim 15 wherein said substrate comprises a first defined area and a second defined area wherein each of said synthesized nucleic acid molecules localized to the first defined area of said substrate comprises a nucleic acid sequence essentially identical to the nucleic acid sequence of every synthesized nucleic acid molecule localized to the second defined area of said substrate.

17. The method of claim 1 wherein each of said synthesized nucleic acid molecules comprises a 5′

primer binding region, a target sequence, and a 3′

primer binding region, wherein said target sequence is located between the 5′

primer binding region and the 3′

primer binding region.

18. The method of claim 17 wherein a member of the group consisting of the 5′

primer binding region and the 3′

primer binding region of each of the synthesized nucleic acid molecules comprises a restriction endonuclease recognition site.

19. The method of claim 17 wherein each synthesized nucleic acid molecule comprises a 5′

primer binding region that is essentially identical to the 5′

primer binding region of every synthesized nucleic acid molecule within the population of nucleic acid molecules on the substrate.

20. The method of claim 17 wherein the population of synthesized nucleic acid molecules comprises a first sub-population of synthesized nucleic acid molecules and a second sub-population of synthesized nucleic acid molecules, wherein:

(a) each synthesized nucleic acid molecule within the first sub-population of synthesized nucleic acid molecules comprises a 5′

primer binding region that is essentially identical to every other 5′

primer binding region within the first sub-population of synthesized nucleic acid molecules, but that is different from every 5′

primer binding region within the second sub-population of synthesized nucleic acid molecules;

(b) each synthesized nucleic acid molecule within the second sub-population of synthesized nucleic acid molecules comprises a 5′

primer binding region that is essentially identical to every other 5′

primer binding region within the second sub-population of synthesized nucleic acid molecules, but that is different from every 5′

primer binding region within the first sub-population of synthesized nucleic acid molecules; and

(c) said first sub-population of synthesized nucleic acid molecules and said second sub-population of synthesized nucleic acid molecules are localized to separate defined areas of said substrate.

21. The method of claim 17 wherein each synthesized nucleic acid molecule comprises a 3′

primer binding region that is essentially identical to the 3′

primer binding region of every other synthesized nucleic acid molecule within the population of nucleic acid molecules synthesized on the substrate.

22. The method of claim 17 wherein the population of synthesized nucleic acid molecules comprises a first sub-population of synthesized nucleic acid molecules and a second sub-population of synthesized nucleic acid molecules, wherein:

(a) each synthesized nucleic acid molecule within the first sub-population of synthesized nucleic acid molecules comprises a 3′

primer binding region that is essentially identical to every other 3′

primer binding region within the first sub-population of synthesized nucleic acid molecules, but that is different from every 3′

primer binding region within the second sub-population of synthesized nucleic acid molecules;

(b) each synthesized nucleic acid molecule within the second sub-population of synthesized nucleic acid molecules comprises a 3′

primer binding region that is essentially identical to every other 3′

primer binding region within the second sub-population of synthesized nucleic acid molecules, but that is different from every 3′

primer binding region within the first sub-population of synthesized nucleic acid molecules; and

(c) said first sub-population of synthesized nucleic acid molecules and said second sub-population of synthesized nucleic acid molecules are localized to separate defined areas of said substrate.

23. The method of claim 13 wherein each of said synthesized nucleic acid molecules localized to a defined area of said substrate comprises a target sequence essentially identical to the target sequence of every other synthesized nucleic acid molecule localized to the same defined area of said substrate.

24. The method of claim 23 wherein said target sequence of each synthesized nucleic acid molecule localized to a defined area of said substrate is different from the target sequence of each synthesized nucleic acid molecule localized to every other defined area of said substrate.

25. The method of claim 23 wherein said substrate comprises a first defined area and a second defined area wherein each of said synthesized nucleic acid molecules localized to the first defined area of said substrate comprises a nucleic acid sequence essentially identical to the nucleic acid sequence of every synthesized nucleic acid molecule localized to the second defined area of said substrate.

26. The method of claim 17 wherein each of said synthesized nucleic acid molecules comprises a 5′

end and a 3′

end, and a target identifier sequence located immediately adjacent to said 5′

end or immediately adjacent to said 3′

end.

27. The method of claim 26 wherein each of said synthesized nucleic acid molecules comprises a target identifier sequence located immediately adjacent to said 5′

end.

28. The method of claim 26 wherein each of said synthesized nucleic acid molecules comprises a target identifier sequence located immediately adjacent to said 3′

end.

29. The method of claim 26 wherein said target identifier sequence has an average length of from about 4 to about 8 base pairs.

30. The method of claim 17 wherein said synthesized nucleic acid molecule further comprises an RNA polymerase promoter.

31. The method of claim 1 wherein said vector molecules are expression vector molecules.

32. The method of claim 1 wherein said population of nucleic acid molecules is synthesized on a glass substrate using an ink-jet printer and wherein the nucleic acid molecules of said population of synthesized nucleic acid molecules have an average length of from about 80 to 100 nucleotides.

33. The method of claim 1 wherein the population of synthesized nucleic acid molecules comprises a first sub-population of synthesized nucleic acid molecules and a second sub-population of synthesized nucleic acid molecules, wherein:

(a) each member of the first sub-population of synthesized nucleic acid molecules is identical to substantially every other member of the first sub-population of synthesized nucleic acid molecules;

(b) each member of the second sub-population of synthesized nucleic acid molecules is identical to substantially every other member of the second sub-population of synthesized nucleic acid molecules;

(c) each member of the first sub-population of synthesized nucleic acid molecules is complementary to substantially all members of the second sub-population of synthesized nucleic acid molecules;

(d) the first sub-population of synthesized nucleic acid molecules is annealed to the second sub-population of synthesized nucleic acid molecules after the first and second sub-populations of synthesized nucleic acid molecules are harvested from the substrate, to produce a population of double stranded nucleic acid molecules; and

(e) the population of double stranded nucleic acid molecules are cloned into a population of vector molecules.

34. The method of claim 1 wherein the harvested nucleic acid molecules are single stranded DNA molecules that are converted to double stranded DNA molecules, wherein each double stranded DNA molecule comprises one of the single stranded DNA molecules hybridized to a complementary nucleic acid molecule, and wherein the double stranded DNA molecules are introduced into vector molecules.