HIERARCHICAL ASSEMBLY OF POLYNUCLEOTIDES

• US 20100047876A1
• Filed: 07/31/2009
• Published: 02/25/2010
• Est. Priority Date: 08/08/2008
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M.
• Application Number:
  US12/533,141
• Publication Number:
  US 20100047876A1
• Time in Patent Office: Days
• US Class Current: 435/91.200
• 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 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

Methods, compositions and apparatuses for hierarchical assembly of oligonucleotide sequences are provided.

Claims

1. A method of making a polynucleotide comprising the steps of:

a) providing an oligonucleotide array having a plurality of adjacent, discrete features attached thereto, wherein each feature comprises a substrate oligonucleotide;

b) contacting a first discrete feature having a first substrate attached thereto with an oligonucleotide primer;

c) allowing the oligonucleotide primer to hybridize to the first substrate oligonucleotide and extending the substrate oligonucleotide to generate an extended oligonucleotide;

d) releasing the extended oligonucleotide and allowing the extended oligonucleotide to contact an adjacent, second discrete feature having a second substrate attached thereto; and

e) allowing the extended oligonucleotide to hybridize to the second substrate oligonucleotide and extending the hybridized extended oligonucleotide and second substrate oligonucleotide to generate a first polynucleotide.

2. The method of claim 1, wherein the step of releasing is performed by contacting the extended oligonucleotide with a helicase, a strand displacement polymerase or heat.

3. The method of claim 1, wherein the oligonucleotide array comprises a chip, a slide or a plate.

4. The method of claim 1, wherein a pair of primers is provided in step a).

5. The method of claim 1, wherein contact occurs by diffusion.

6. The method of claim 1, wherein the first polynucleotide is amplified.

7. The method of claim 6, wherein amplification is performed by polymerase chain reaction or ligase chain reaction.

8. The method of claim 1, further comprising removing one or both of an extended oligonucleotide and a first polynucleotide having a mismatch.

9. The method of claim 8, wherein the one or both of the extended oligonucleotide and the first polynucleotide having a mismatch are removed by mismatch-sensitive hybridization, mutS binding, MutHSL cleavage near the mismatch or cleavage at the mismatch.

10. The method of claim 1, wherein the oligonucleotide primer is between 8 and 25 nucleotides in length.

11. The method of claim 1, wherein the first and second substrate oligonucleotides are between 50 and 100 nucleotides in length.

12. The method of claim 1, wherein the first polynucleotide is greater than 100 nucleotides in length.

13. The method of claim 1, wherein the first polynucleotide is between 100 and 150 nucleotides in length.

14. The method of claim 1, wherein the primer is added by ink-jet printing.

15. The method of claim 1, further comprising the steps of:

f) releasing the first polynucleotide and allowing the first polynucleotide to contact an adjacent, third discrete feature having a third substrate attached thereto; and

g) allowing the first polynucleotide to hybridize to the third substrate oligonucleotide and extending the hybridized first polynucleotide and third substrate oligonucleotide to generate a second polynucleotide.

16. The method of claim 15, wherein the second polynucleotide is greater than 200 nucleotides in length.

17. The method of claim 15, wherein the second polynucleotide is between 200 and 300 nucleotides in length.