Compositions and methods for preparing oligonucleotide solutions

• US 9,745,573 B2
• Filed: 08/15/2016
• Issued: 08/29/2017
• 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
• Primary Examiner(s): CROW, ROBERT THOMAS
• Application Number:
  US15/237,490
• Publication Number:
  US 20160348098A1
• Time in Patent Office: 379 Days
• Field of Search: None
• 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 target nucleic acids comprising:

(i) generating a pool of oligonucleotides comprising;

(a) providing a first substrate and at least 50 different single-stranded oligonucleotides linked to said first substrate through cleavable linkers; and

(b) cleaving said linkers, thereby releasing said single-stranded oligonucleotides from said first substrate as a pool of oligonucleotides comprising said oligonucleotides;

(ii) hybridizing the target nucleic acids with the pool of oligonucleotides;

(iii) capturing the hybridized pool of single-stranded oligonucleotides on a second substrate; and

(iv) detecting the target nucleic acids.

2. The method of claim 1, wherein the at least 50 different single-stranded oligonucleotides are linked to said first substrate at discrete sites.

3. The method of claim 2, wherein the discrete sites are contiguous.

4. The method of claim 1, wherein step (i) comprises amplifying the released oligonucleotides to generate the pool of oligonucleotides.

5. The method of claim 4, wherein the amplifying comprises a polymerase chain reaction.

6. The method of claim 4, wherein the amplifying comprises rolling circle amplification.

7. The method of claim 1, wherein step (iii) comprises amplifying the target nucleic acids.

8. A method of detecting target nucleic acids comprising:

(i) generating a pool of oligonucleotides comprising;

(a) obtaining a first substrate comprising a plurality of single-stranded oligonucleotides linked to said first substrate, wherein the plurality of single-stranded oligonucleotides are different from one another, wherein the plurality of single-stranded oligonucleotides linked to said first substrate comprises at least 50 oligonucleotides,

(b) releasing the plurality of single-stranded oligonucleotides from the first substrate as a pool of single-stranded oligonucleotides;

(ii) hybridizing the pool of single-stranded oligonucleotides with the target nucleic acids;

(iii) capturing the hybridized pool of oligonucleotides on a second substrate; and

(iv) detecting the target nucleic acids.

9. The method of claim 8, wherein the plurality of single-stranded oligonucleotides is linked to said first substrate at discrete sites.

10. The method of claim 9, wherein the discrete sites are contiguous.

11. The method of claim 8, wherein the plurality of single-stranded oligonucleotides are labeled.

12. The method of claim 8, wherein step (i) comprises amplifying the released pool of single-stranded oligonucleotides.

13. The method of claim 12, wherein the amplifying comprises a polymerase chain reaction.

14. The method of claim 12, wherein the amplifying comprises rolling circle amplification.

15. The method of claim 8, wherein at least one single-stranded oligonucleotide of the plurality of single-stranded oligonucleotides is linked to said first substrate through a cleavable linker.

16. The method of claim 8, wherein step (iii) comprises amplifying the target nucleic acids.