Nucleic acid adaptors and uses thereof

• US 10,450,608 B2
• Filed: 12/08/2016
• Issued: 10/22/2019
• 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
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/372,938
• Publication Number:
  US 20170088893A1
• Time in Patent Office: 1,048 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: 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 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. Also provided herein are compositions generated using the blocking oligonucleotide adapters, including circularized polynucleotides, that may be manipulated, for example, through nick translation, to yield a linear mate pair construct.

Claims

1. A method for generating a circular double-stranded polynucleotide, comprising:

a) hybridizing a first double-stranded nucleic acid adaptor having a first target-joining end and a first overhang end to a first single-stranded blocking oligonucleotide over at least a portion of the length of the first overhang end, wherein the first overhang end contains a first overhang nucleic acid sequence, and wherein the sequence of the first single-stranded blocking oligonucleotide is complementary to at least a portion of the first overhang nucleic acid sequence;

b) hybridizing a second double-stranded nucleic acid adaptor having a second target joining end and a second overhang end to a second single-stranded blocking oligonucleotide over at least a portion of the length of the second overhang end, wherein (i) the second overhang end contains a second overhang nucleic acid sequence, (ii) the sequence of the second single-stranded blocking oligonucleotide is complementary to at least a portion of the second overhang nucleic acid sequence and (iii) the first and second overhang nucleic acid sequences are at least partially complementary to each other;

c) joining a linear double-stranded target polynucleotide having a first end and a second end to the first double-stranded nucleic acid adaptor and the second double-stranded nucleic acid adaptor, wherein the first end is joined to the first target joining end of the first double-stranded nucleic acid adaptor, and the second end is joined to the second target joining end of the second double-stranded nucleic acid adaptor; and

d) subjecting the linear double-stranded target polynucleotide that is joined to the first and second double-stranded nucleic acid adaptors to conditions that decrease the stability of the hybridization between the first overhang end and the first single-stranded blocking oligonucleotide and between the second overhang end and the second single-stranded blocking oligonucleotide such that the first overhang end of the first double-stranded nucleic acid adaptor and the second overhang end of the second double-stranded nucleic acid adaptor are exposed and the first overhang end of the first double-stranded nucleic acid adaptor is hybridized to the second overhang end of the second double-stranded nucleic acid adaptor to form a double-stranded adaptor joining region and resulting in a nick at the end of each overhang end, thereby forming a circular double-stranded polynucleotide having two nicks in the adaptor joining region.

2. The method of claim 1, wherein the first and second overhang ends are 5′

overhang ends or wherein the first and second overhang ends are 3′

overhang ends.

3. The method of claim 1, further comprising subjecting the circular double-stranded polynucleotide to nick translation.

4. The method of claim 3, wherein the nick translation is conducted in a reaction mixture comprising (i) a coupled 5′

to 3′

DNA polymerization/degradation reaction mixture, or (ii) a coupled 5′

to 3′

DNA polymerization/strand displacement reaction mixture.

5. The method of claim 3, wherein the nick translation is conducted in a reaction mixture comprising a DNA polymerase enzyme.

6. The method of claim 3, wherein the nick translation is conducted in a reaction mixture comprising an enzyme selected from the group consisting of E. coli DNA polymerase I, Taq DNA polymerase, Vent DNA polymerase, Klenow DNA polymerase I, Tfi DNA polymerase, Bst DNA polymerase, and phi29 DNA polymerase.

7. The method of claim 3, wherein the nick translation is conducted in a reaction mixture comprising deoxyribonucleoside triphosphates.

8. 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 both the first and second double-stranded nucleic acid adaptors comprise biotin.

9. The method of claim 1, wherein the first single-stranded blocking oligonucleotide and/or the second single-stranded blocking oligonucleotide is shorter than the overhang end to which it hybridizes.

10. The method of claim 3, further comprising cleaving the circular double-stranded polynucleotide with a single-strand specific endonuclease enzyme at the position of the nicks to release a linear polynucleotide which includes a mate pair construct.

11. The method of claim 1, wherein a nucleotide at the position of at least one of the nicks lacks a 5′

phosphate group.

12. The method of claim 1, wherein the conditions that decrease the stability of the hybridization between the first overhang end and the first single-stranded blocking oligonucleotide and between the second overhang end and the second single-stranded blocking oligonucleotide include altering the ionic strength, pH and/or temperature of a medium in which the linear double-stranded target polynucleotide that is joined to the first and second double-stranded nucleic acid adaptors is contained.