Reducing adapter dimer formation

• US 8,575,071 B2
• Filed: 11/23/2010
• Issued: 11/05/2013
• Est. Priority Date: 11/03/2010
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Lau, Patrick, Lee, Danny
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US12/952,563
• Publication Number:
  US 20120108440A1
• Time in Patent Office: 1,078 Days
• Field of Search: None
• US Class Current: 506/26
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/163 blocking probe
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Provided herein is a method of reducing adapter dimer formation comprising contacting a sample comprising target nucleic acid sequences with 5′ and 3′ adapters in the presence of one or more hairpin oligonucleotides. Also provided is a method of preparing a library of nucleic acid sequences comprising contacting first adapter oligonucleotides with a sample comprising target nucleic acid sequences under conditions to form first ligation products, contacting the sample with one or more hairpin oligonucleotides that binds to the first adapter oligonucleotides, and contacting the sample with second adapter oligonucleotides under conditions to bind to the first ligation products and form second ligation products, wherein the second ligation products form the library of nucleic acid sequences.

Claims

1. A method of preparing a library of nucleic acid sequences comprising:

(a) contacting first adapter oligonucleotides with a sample comprising target nucleic acid sequences under conditions to form first ligation products;

(b) after step (a), contacting the sample with one or more hairpin oligonucleotides having a 5′

overhang portion configured to bind unligated first adaptor oligonucleotides whereby said 5′

overhang portion is complementary to and anneals to at least a portion of said unligated first adapter oligonucleotides;

wherein the hairpin oligonucleotides do not anneal to the first ligation products;

(c) after step (b), contacting the sample with second adapter oligonucleotides under conditions to bind to the first ligation products and form second ligation products, wherein the second ligation products form the library of nucleic acid sequences; and

wherein the first and second adapters are linear and single-stranded.

2. The method of claim 1, wherein step (a) occurs in the presence of a ligase and in the absence of ATP.

3. The method of claim 1, further comprising isolating the second ligation products.

4. The method of claim 3, wherein the isolation comprises gel electrophoresis.

5. The method of claim 1, further comprising amplifying the second ligation products.

6. The method of claim 1, further comprising sequencing the second ligation products.

7. The method of claim 1, wherein the target nucleic acid sequences comprise RNA molecules.

8. The method of claim 7, wherein the RNA molecules comprise small RNA molecules.

9. The method of claim 8, wherein the small RNA molecules comprise molecules selected from the group consisting of miRNA molecules, siRNA molecules, tRNA molecules, rRNA molecules, and combinations thereof.

10. The method of claim 1, wherein the target nucleic acid sequence comprises single-stranded DNA.

11. The method of claim 1, wherein the first adapter is a 3′

-pre-adenylated adapter.

12. The method of claim 1, wherein the second adapter is a 5′

-RNA adapter.

13. The method of claim 1, wherein the hairpin oligonucleotide comprises RNA.

14. The method of claim 1, wherein the hairpin oligonucleotide comprises a label.

15. The method of claim 14, wherein the label binds a column or bead.

16. The method of claim 15, wherein the bead is a magnetic bead.