Compositions and methods for preparing oligonucleotide solutions

• US 8,669,053 B2
• Filed: 09/11/2009
• Issued: 03/11/2014
• Est. Priority Date: 08/18/1999
• Status: Expired due to Fees

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Stuelpnagel, John R., Chee, Mark S.
• Primary Examiner(s): CROW, ROBERT THOMAS
• Application Number:
  US12/558,440
• Publication Number:
  US 20100151464A1
• Time in Patent Office: 1,642 Days
• Field of Search: 435/6, 435/6.1, 435/6.11, 435/6.12
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: G11C 7/1048 Data bus control circuits, e.g. precharging, presetting, equalising
• CPC Class Codes: H03K 19/0966 Self-timed logic
• CPC Class Codes: Y10S 977/792 Nucleic acid array, e.g. human genome array

Abstract

The present invention is directed to methods and compositions for generating a pool of oligonucleotides. The invention finds use in preparing a population or subpopulations of oligonucleotides in solution. The pool of oligonucleotides finds use in a variety of nucleic acid detection and/or amplification assays.

Claims

1. A method of detecting the presence of target nucleic acids comprising:

a) obtaining a pool of greater than 50 different single stranded oligonucleotides, wherein said pool of oligonucleotides has been produced by;

(i) synthesizing greater than 50 different single stranded oligonucleotides on discrete sites of a first substrate, wherein each of the different single stranded oligonucleotides has a different sequence, and

(ii) releasing said greater than 50 different single stranded oligonucleotides from said first substrate to generate said pool of greater than 50 different single stranded oligonucleotides;

b) contacting said pool of greater than 50 different single stranded oligonucleotides with a composition comprising target nucleic acids, whereby said target nucleic acids hybridize with greater than 50 different single stranded oligonucleotides in said pool of oligonucleotides;

c) capturing said greater than 50 different single stranded oligonucleotides in said pool of oligonucleotides hybridized with said target nucleic acids on a second substrate;

d) contacting said target nucleic acids with a primer, wherein said target nucleic acids are amplified upon binding to said primer; and

e) detecting the presence of said target nucleic acids.

2. The method of claim 1, wherein said pool of greater than 50 different single stranded oligonucleotides has been produced by:

(i) synthesizing greater than 50 different single stranded oligonucleotides on discrete sites of a first substrate,

(ii) releasing said greater than 50 different single stranded oligonucleotides from said first substrate, and

(iii) amplifying said greater than 50 different single stranded oligonucleotides that are released from said first substrate, wherein said releasing and amplifying generates said pool of oligonucleotides.

3. The method of claim 1, wherein said pool of greater than 50 different single stranded oligonucleotides has been produced by:

(i) synthesizing greater than 50 different single stranded oligonucleotides on discrete sites of a first substrate,

(ii) releasing said greater than 50 different single stranded oligonucleotides from said first substrate, and

(iii) modifying said greater than 50 different single stranded oligonucleotides that are released from said first substrate, wherein said releasing and modifying generates said pool of oligonucleotides.

4. The of claim 1, wherein the discrete sites are contiguous with other sites.

5. The method of claim 1, wherein the discrete sites are non-contiguous with other sites.

6. The method of claim 1, wherein said greater than 50 different single stranded oligonucleotides that are linked to said discrete sites on the first substrate are linked through a cleavable linker.

7. The method of claim 6, wherein said releasing of said greater than 50 single stranded oligonucleotides from said first substrate comprises cleavage of the cleavable linker with light.

8. The method of claim 6, wherein said releasing of said greater than 50 single stranded oligonucleotides from said first substrate comprises cleavage of the cleavable linker with a chemical.

9. The method of claim 6, wherein said releasing of said greater than 50 single stranded oligonucleotides from said first substrate comprises cleavage of the cleavable linker with an enzyme.

10. The method of claim 1, wherein said greater than 50 different single stranded oligonucleotides in said pool of oligonucleotides are labeled.

11. The method of claim 1, wherein said first substrate is selected from the group consisting of glass, plastics, polysaccharides, nylon, nitrocellulose, resins, silica, silicon, carbon, and metals.

12. The method of claim 2, wherein said amplifying comprises polymerase chain reaction.

13. The method of claim 2, wherein said amplifying comprises rolling circle amplification.