NUCLEIC ACID ADAPTORS AND USES THEREOF

• US 20130261027A1
• Filed: 09/29/2011
• Published: 10/03/2013
• 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: Li, Bin, Chen, Zhoutao, Biorac, Tanya, Wei, Melvin
• Granted Patent: US 9,540,637 B2
• Time in Patent Office: Days
• US Class Current: 506/26
• 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 joining a first and a second nucleic acid, comprising:

a) providing a first nucleic acid overhang having a first nucleic acid sequence and a second nucleic acid overhang having a second nucleic acid sequence, the first and second nucleic acid sequences being at least partially complementary to each other, wherein the first overhang is hybridized to a first blocking oligonucleotide over at least a portion of its length and the second overhang is hybridized to a second blocking oligonucleotide over at least a portion of its length;

b) separating the first and the second blocking oligonucleotides from the first and the second nucleic acid overhangs; and

c) hybridizing the first nucleic acid sequence with the second nucleic acid sequence, thereby joining together the first and the second nucleic acid overhangs.

2. The method of claim 1, wherein the first nucleic acid overhang comprises a first double-stranded nucleic acid adaptor.

3. The method of claim 1, wherein the second nucleic acid overhang comprises a second double-stranded nucleic acid adaptor.

4. The method of claim 1, wherein the first blocking oligonucleotide comprises a single-stranded oligonucleotide.

5. The method of claim 1, wherein the second blocking oligonucleotide comprises a single-stranded oligonucleotide.

6. The method of claim 1, wherein the first blocking oligonucleotide and the second blocking oligonucleotide are not hybridized to each other.

7. The method of claim 1, wherein the first overhang is located at an end of a first nucleic acid duplex, the second overhang is located at an end of a second nucleic acid duplex, the hybridization of the first overhang to the first blocking oligonucleotide forms a third nucleic acid duplex, the hybridization of the second overhang to the second blocking oligonucleotide forms a fourth nucleic acid duplex, and wherein the melting point of the first nucleic acid duplex and of the second nucleic acid duplex are both substantially greater than the melting points of both the third and fourth nucleic acid duplexes.

10. The method of claim 7, wherein the first nucleic acid duplex comprises a first double-stranded nucleic acid adaptor.

11. The method of claim 7, wherein the second nucleic acid duplex comprises a second double-stranded nucleic acid adaptor.

12. The method of claim 1, wherein the first nucleic acid overhang comprises one end of a first polynucleotide and the second nucleic acid overhang comprises one end of a second polynucleotide.

13. The method of claim 12, wherein the first and second polynucleotides are double-stranded polynucleotides and form part of a single contiguous double-stranded nucleic acid molecule.

14. The method of claim 1, wherein joining in step (c) includes circularizing the first and second polynucleotides.

15. The method of claim 12, wherein the first and second polynucleotides are double stranded polynucleotides and form part of different double-stranded nucleic acid molecules.

16. The method of claim 1, wherein the separating in step (b) comprises denaturing the first single-stranded blocking oligonucleotide from the first overhang.

17. The method of claim 1, wherein the separating in step (b) comprises denaturing the second single-stranded blocking oligonucleotide from the second overhang.

18. The method of claim 1, further comprising ligating the hybridized first and the second nucleic acid overhangs with a ligase enzyme.

19. The method of claim 1, wherein the joining together the first and second overhangs in step (c) forms a junction on each nucleic acid strand between the joined first and the second nucleic acid overhangs.

20. The method of claim 19, wherein at least one junction comprises a nick.

21. The method of claim 20 further comprising the step:

moving the nick to a new position.

22. The method of claim 21, wherein the moving the nick to a new position comprises performing a nick translation reaction on the nick.

23. The method of claim 22, 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.

24. The method of claim 22, wherein the length of time of the nick translation reaction is modulated to increase or decrease the distance to move the nick to a new position within the polynucleotide of interest.

8-9. -9. (canceled)

25-40. -40. (canceled)