Enhanced ligation reactions

• US 9,315,861 B2
• Filed: 11/28/2012
• Issued: 04/19/2016
• Est. Priority Date: 12/17/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Hendricks, Stephen, King, David, Xi, Lei, Peris, Marian
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US14/360,892
• Publication Number:
  US 20150031026A1
• Time in Patent Office: 1,238 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/46 involving cholinesterase
• 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 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2537/1376 Displacement by an enzyme
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of at least one agent that generates a ligatable terminal 5′ phosphate group by removing an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, an aprataxin enzyme can catalyze removal of an adenylate group from a terminal 5′ phosphate of a nucleic acid. In some embodiments, methods for ligating nucleic acid ends comprise: conducting a nucleic acid ligation reaction in the presence of an aprataxin enzyme under conditions suitable for ligating nucleic acid ends.

Claims

1. A method for ligating nucleic acids comprising:

(a) forming a first single-stranded nick on a nucleic acid duplex by hybridizing a polynucleotide to a first and second oligonucleotide, wherein the first and second oligonucleotides abut each other, and wherein one end of the polynucleotide, the first oligonucleotide or the second oligonucleotide is attached to a surface;

(b) closing the first single-stranded nick by conducting a nucleic acid ligation reaction in the presence of a ligase and an agent that catalyzes removal of an adenylate group from a terminal 5′

phosphate of a nucleic acid.

2. The method of claim 1, wherein the agent that catalyzes removal of an adenylate group from a terminal 5′

phosphate of a nucleic acid comprises an aprataxin enzyme.

3. The method of claim 2, wherein the aprataxin enzyme comprises the amino acid sequence according to SEQ ID NO:

1.

4. The method of claim 1, wherein the ligase comprises a mesophilic or thermostable ligase enzyme.

5. The method of claim 1, wherein the ligase comprises a small footprint ligase having the amino acid sequence according to any one of SEQ ID NOS:

3-8.

10. The method of claim 1, wherein the nucleic acid ligation reaction further comprises:

conducting at least one cycle of a repetitive cycle ligation reaction in the presence of a ligase and an aprataxin enzyme under conditions suitable for ligating nucleic acid ends, by (a) forming a second single-stranded nick which includes hybridizing a third oligonucleotide to the polynucleotide so that the third and second oligonucleotides abut each other, and (b) closing the second single-stranded nick by conducting a nucleic acid ligation reaction in the presence of a ligase and an agent that catalyzes removal of an adenylate group from a terminal 5′

phosphate of a nucleic acid.

11. The method of claim 10, wherein repetitive cycle ligation reaction comprises:

a ligase chain reaction (LCR), gap LCR, or ligation detection reaction (LDR).

12. The method of claim 10, wherein the agent that catalyzes removal of an adenylate group from a terminal 5′

phosphate of a nucleic acid comprises an aprataxin enzyme.

13. The method of claim 1, wherein the first or the second oligonucleotide is labeled with a detectable reporter moiety.

14. The method of claim 1, wherein the first or the second oligonucleotide includes an internal or terminal scissile linkage which is selected from a group consisting of a phosphoramidate, phosphorothioate, or phosphorodithiolate linkage.

6. A method for sequencing comprising:

a) hybridizing a template polynucleotide to a first and second oligonucleotide probe so that the first and second oligonucleotide probes abut each other to form a nick, wherein the second oligonucleotide probe is labeled with a distinctive detectable reporter moiety and wherein the polynucleotide is attached to a surface;

b) contacting the nick with at least one aprataxin enzyme and at least one ligase enzyme to close the nick; and

c) detecting the distinctive detectable reporter moiety thereby determining the sequence of the second oligonucleotide probe hybridized to the template polynucleotide.

7. The method of claim 6, comprising:

a) hybridizing a template polynucleotide to a first and second oligonucleotide probe so that the first and second oligonucleotide probes abut each other to form a first nick, wherein the second oligonucleotide probe is labeled with a first distinctive detectable reporter moiety;

b) contacting the first nick with at least one aprataxin enzyme and at least one ligase enzyme to close the first nick;

c) detecting the first distinctive detectable reporter moiety thereby determining the sequence of the second oligonucleotide probe hybridized to the template polynucleotide;

d) hybridizing the template polynucleotide to a third and fourth oligonucleotide probe so that the third and fourth oligonucleotide probes abut each other to form a second nick, wherein the fourth oligonucleotide probe is labeled with a second distinctive detectable reporter moiety;

e) contacting the second nick with at least one aprataxin enzyme and at least one ligase enzyme to close the second nick; and

f) detecting the second distinctive detectable reporter moiety thereby determining the sequence of the fourth oligonucleotide probe hybridized to the template polynucleotide.

8. The method of claim 6, wherein the aprataxin enzyme comprises the amino acid sequence according to SEQ ID NO:

1.

9. The method of claim 6, wherein the ligase comprises a small footprint ligase having the amino acid sequence according to any one of SEQ ID NOS:

3-8.