Nucleic acid adaptors and uses thereof

• US 9,540,637 B2
• Filed: 09/29/2011
• Issued: 01/10/2017
• Est. Priority Date: 01/09/2008
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Wei, Melvin Shiwade, Li, Bin, Chen, Zhoutao, Biorac, Tanya
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US13/877,203
• Publication Number:
  US 20130261027A1
• Time in Patent Office: 1,930 Days
• Field of Search: 435/6.12, 435/91.2, 435/91.1
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/20 Triglyceride splitting, e.g. by means of lipase
• 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 1/6832 Enhancement of hybridisation reaction
• 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 2521/131 Terminal transferase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2537/163 blocking probe
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 301/01003 Triacylglycerol lipase (3.1.1.3)

Abstract

Provided herein are compositions, systems, methods, and kits for joining together the ends of one or more polynucleotides using at least one pair of blocking oligonucleotide adaptors. Blocking oligonucleotide adaptors can be used to reduce the formation of adaptor dimers or trimers (or higher-order concatemers) which can improve the yield of desirable polynucleotide-adaptor products in any recombinant nucleic acid workflow. Blocking oligonucleotide adaptors can comprise a double-stranded oligonucleotide adaptor (duplex) having an overhang cohesive portion that anneals with a blocking oligonucleotide which can be a separate single-stranded oligonucleotide. A blocking oligonucleotide, when annealed to an overhang portion, can prevent undesirable hybridization of the overhang portion to another nucleic acid, such as the overhang portion from another blocking oligonucleotide adaptor or a polynucleotide of interest.

Claims

1. A method for circularizing a polynucleotide, comprising:

a) providing (i) a linear target polynucleotide having a first end and a second end, (ii) a first double-stranded nucleic acid adaptor with a target joining end and a first overhang end wherein the first overhang end comprises a first overhang nucleic acid sequence, (iii) and a second double-stranded nucleic acid adaptor with a target-joining end and a second overhang end wherein the second overhang end comprises a second overhang nucleic acid sequence, wherein the first and second overhang nucleic acid sequences being at least partially complementary to each other, wherein the first overhang is hybridized to a first single-stranded blocking oligonucleotide over at least a portion of its length and the second overhang is hybridized to a second single-stranded blocking oligonucleotide over at least a portion of its length, and wherein the target-joining end of the first double-stranded nucleic acid adaptor is joined to the first end of the linear target polynucleotide and the target joining end of the second double-stranded nucleic acid adaptor is joined to the second end of the linear target polynucleotide;

b) removing the first and the second single-stranded blocking oligonucleotides from the first and the second overhangs; and

c) hybridizing the first overhang having a first nucleic acid sequence with the second overhang having a second nucleic acid sequence, thereby circularizing the polynucleotide, wherein the first and second overhangs are not enzymatically ligated together.

2. The method of claim 1, wherein the first and the second single-stranded blocking oligonucleotides are not hybridized to each other.

3. The method of claim 1, wherein the removing comprises denaturing the first single-stranded blocking oligonucleotide from the first overhang and denaturing the second single-stranded blocking oligonucleotide from the second overhang.

4. The method of claim 1, wherein circularizing the polynucleotide includes forming a junction on each nucleic acid strand between the first and second overhangs.

5. The method of claim 4, wherein at least one junction comprises a nick.

6. The method of claim 4, wherein the circularized polynucleotide includes two nicks.

7. The method of claim 6, further comprising the step:

moving each of the two nicks to a new position within the circularized polynucleotide.

8. The method of claim 7, wherein the moving the two nick comprises performing a nick translation reaction on the two nicks.

9. The method of claim 8, wherein the nick translation reaction is a coupled 5′

to 3′

DNA polymerization/degradation reaction, or is a coupled 5′

to 3′

DNA polymerization/strand displacement reaction.

10. The method of claim 8, wherein the length of time of the nick translation reaction is modulated to increase or decrease the distance to move the two nick to a new position within the circularized polynucleotide.

11. The method of claim 7, further comprising cleaving the circularized polynucleotide with a single-strand specific endonuclease enzyme at the new position of the two nicks to release a linear polynucleotide which includes a mate pair construct.

12. The method of claim 1, wherein the first double-stranded nucleic acid adaptor comprises biotin, or the second double-stranded nucleic acid adaptor comprises biotin, or the first and second double-stranded nucleic acid adaptors comprise biotin.

13. The method of claim 12, further comprising the step of reacting the biotin with streptavidin.

14. The method of claim 11, further comprising joining the ends of the released linear polynucleotide to additional adaptors to generate an adaptor-mate pair construct.

15. The method of claim 14, further comprising attaching the adaptor-mate pair construct to a surface.

16. The method of claim 15, further comprising sequencing the adaptor-mate pair construct.