Nucleic acid arrays and methods of synthesis

• US 20020076716A1
• Filed: 06/21/2001
• Published: 06/20/2002
• Est. Priority Date: 04/09/1998
• Status: Abandoned Application

• Current Assignee: Trustees of Boston University
• Original Assignee: Trustees of Boston University
• Inventors: Sano, Takeshi, Sabanayagam, Chandran R., Misasi, John, Cantor, Charles, Hatch, Anson
• Application Number:
  US09/886,779
• Publication Number:
  US 20020076716A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2531/101 Linear amplification, i.e. non exponential
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention generally relates to high density nucleic acid arrays and methods of synthesizing nucleic acid sequences on a solid surface. Specifically, the present invention contemplates the use of stabilized nucleic acid primer sequences immobilized on solid surfaces, and circular nucleic acid sequence templates combined with the use of isothermal rolling circle amplification to thereby increase nucleic acid sequence concentrations in a sample or on an array of nucleic acid sequences.

Claims

1. A method of generating an array, comprising:

a) providing;

i) a solid support comprising a plurality of positions for oligonucleotides, said positions defined by x and y coordinates;

ii) a plurality of identical oligonucleotides, each oligonucleotide comprising a sequence; and

iii) a plurality of unique circular DNA templates, each circular DNA template comprising a sequence of interest and a region complementary to at least a portion of said sequence of said oligonucleotides, said sequence of interest being different for each circular template;

b) immobilizing one oligonucleotide from said plurality of identical oligonucleotides in each of said positions on said solid support to create an ordered array comprising a plurality of identical immobilized oligonucleotides;

c) adding to each immobilized oligonucleotide of said ordered array a circular DNA template from said plurality of said unique circular DNA templates under conditions such that said immobilized oligonucleotide hybridizes to said circular DNA template to create a plurality of primed circular templates, each primed circular template comprising a different sequence of interest; and

d) extending each of said primed circular templates to create an extended immobilized oligonucleotide comprising at least two copies of said sequence of interest, thereby generating an ordered redundant array.

2. The method of claim 1, wherein said oligonucleotides are immobilized on a solid surface by a chemical linkage.

3. The method of claim 1, wherein said oligonucleotides are immobilized on said solid surface by the 5′

end of said oligonucleotides.

4. The method of claim 1, wherein said oligonucleotides are approximately 17 bases in length.

5. The method of claim 1 wherein said solid surface is selected from a group of materials comprising silicon, metal, and glass.

6. The method of claim 1 wherein said immobilized oligonucleotides are attached to a complimentary nucleic acid stabilizer sequence.

7. The method of claim 1, wherein said circular nucleic acid template is bacteriophage DNA.

8. The method of claim 1, wherein said circular nucleic acid template is non-bacteriophage DNA.

9. The method of claim 1, wherein said extending in step (d) is achieved with a polymerase.

10. The method of claim 9, wherein said polymerase is selected from a group comprising E. coli. DNA polymerase I, a fragment of E. coli. DNA polymerase I, or Φ

29 DNA polymerase.

11. An ordered redundant array of immobilized oligonucleotides produced according to the method of claim 1.

12. A method of hybridizing target nucleic acid fragments, comprising:

a) providing i) the ordered redundant array of extended immobilized oligonucleotides of claim 1;

and ii) a plurality of fragments of a target nucleic acid; and

b) bringing said fragments of said target nucleic acid into contact with said array under conditions such that at least one of said fragments hybridizes to one of said extended immobilized oligonucleotides on said array.

14. The method of claim 13, wherein said oligonucleotides are immobilized on a solid surface by a chemical linkage.

15. The method of claim 13, wherein said oligonucleotides are immobilized on said solid surface by the 5′

end of said oligonucleotides.

16. The method of claim 13, wherein said oligonucleotides are approximately 17 bases in length.

17. The method of claim 13 wherein said solid surface is selected from a group of materials comprising silicon, metal, and glass.

18. The method of claim 13 wherein said immobilized oligonucleotides are attached to a complimentary nucleic acid stabilizer sequence.

19. The method of claim 13, wherein said circular nucleic acid template is bacteriophage DNA.

20. The method of claim 13, wherein said circular nucleic acid template is non-bacteriophage DNA.

21. The method of claim 13, wherein said extending in step (d) is achieved with a polymerase.

22. The method of claim 21, wherein said polymerase is selected from a group comprising E. coli. DNA polymerase I, a fragment of E. coli. DNA polymerase I, or Φ

29 DNA polymerase.

23. An ordered redundant array of immobilized oligonucleotides produced according to the method of claim 13.

13. A method of generating an array capable of hybridizing to fragments of a target nucleic acid, comprising:

a) providing;

i) a solid support comprising positions for oligonucleotides, said positions defined by x and y coordinates;

ii) a plurality of oligonucleotides, each oligonucleotide comprising a sequence complementary to a different portion of the sequence of said target nucleic acid; and

iii) a plurality of corresponding circular DNA templates, each circular DNA template comprising a different portion of the sequence of said target;

b) immobilizing each of said oligonucleotides in one of said positions on said solid support to create an ordered array comprising a plurality of immobilized oligonucleotides;

c) adding to each immobilized oligonucleotide of said ordered array a corresponding circular DNA template under conditions such that said immobilized oligonucleotide hybridizes to said corresponding circular DNA template to create a plurality of primed circular templates; and

d) extending said primed circular templates to create an ordered redundant array of extended immobilized oligonucleotides, each extended immobilized oligonucleotide comprising at least two copies of said portion of said sequence of said target nucleic acid.

24. A method of hybridizing target nucleic acid fragments, comprising:

a) providing i) the ordered redundant array of extended immobilized oligonucleotides of claim 13; and

ii) a plurality of fragments of a target nucleic acid; and

b) bringing said fragments of said target nucleic acid into contact with said array under conditions such that at least one of said fragments hybridizes to one of said extended immobilized oligonucleotides on said array.