DNA ligation on RNA template

• US 10,648,021 B2
• Filed: 10/30/2017
• Issued: 05/12/2020
• Est. Priority Date: 01/16/2009
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Namsaraev, Eugeni A., Miao, Xin, Blume, John E.
• Primary Examiner(s): Forman, Betty J
• Application Number:
  US15/798,238
• Publication Number:
  US 20180223346A1
• Time in Patent Office: 925 Days
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2537/162 Helper probe

Abstract

Disclosed are methods and compositions for detection and amplification of nucleic acids, wherein two DNA strands hybridized to an RNA strand are ligated. In one aspect, the disclosed methods include removal of an energy source, such as ATP, upon charging a ligase to form an enzyme-AMP intermediate, and then adding substrate, which results in one complete round of RNA-templated DNA ligation. In another aspect, the ligation reaction is accomplished by use of a mixture of at least two different ligase enzymes. The disclosed methods and compositions for RNA-templated DNA ligation may be particularly useful for detection of RNA sequence variants, for example RNA splice variants, and for quantitative expression analysis.

Claims

1. A method for detecting a plurality of target nucleic acids from a nucleic acid sample, comprising:

a) contacting a DNA array-bound probe with;

(i) a DNA interrogation probe, and

(ii) a nucleic acid sample comprising at least one target nucleic acid of RNA,

wherein the array-bound probe and the interrogation probe hybridize to the target nucleic acid such that the array-bound probe and the interrogation probe are directly adjacent to one another, and wherein the interrogation probe contains a detectable label;

b) adding an effective amount of ATP to a solution of at least two different DNA ligase enzymes,

wherein the at least two different DNA ligase enzymes comprise a first ligase, which can form a DNA ligase-AMP intermediate complex, and a second ligase, which is a mutant ligase unable to form a DNA ligase-AMP intermediate complex;

c) depleting ATP from the solution of at least two different DNA ligase enzymes;

d) adding the solution of at least two different DNA ligase enzymes to step a), thereby ligating the array-bound probe and the interrogation probe; and

e) detecting the ligated product.

2. The method according to claim 1, wherein the first ligase is T4 DNA ligase.

3. The method according to claim 1, wherein the second ligase is E. coli NAD+-dependent ligase LigA which comprises a point mutant or an amino terminal truncation.

4. The method according to claim 1, wherein the second ligase is an E. coli NAD+-dependent ligase LigA mutant selected from the group consisting of:

Y22A, D32A and D36A.

5. The method of claim 1, wherein ATP depletion is accomplished by adding an effective amount of apyrase.

6. The method of claim 1, wherein ATP depletion is accomplished by adding an effective amount of hexokinase.

7. A method of detecting a plurality of target nucleic acids from a nucleic acid sample, comprising:

a) contacting an open circle DNA probe with a nucleic acid sample comprising at least one target nucleic acid of RNA, wherein the open circle probe comprises a 5′

end and a 3′

end, and wherein the open circle probe contains a detectable label;

b) adding an effective amount of ATP to a solution of at least two different DNA ligase enzymes,

wherein the at least two different DNA ligase enzymes comprise at least a first ligase, which can form a DNA ligase-AMP intermediate complex, and at least a second ligase, which is a mutant ligase unable to form a DNA ligase-AMP intermediate complex;

c) depleting ATP from the solution of at least two different DNA ligase enzymes;

d) adding the solution of DNA ligase-AMP intermediate to step a), thereby ligating the 5′

end of the open circle probe to the 3′

end of the open circle probe; and

e) detecting the ligated product.

8. The method according to claim 7, wherein ATP depletion is accomplished by adding an effective amount of apyrase.

9. The method according to claim 8, wherein the effective amount of apyrase is about 10 mU apyrase per μ

l.

10. The method according to claim 7, wherein ATP depletion is accomplished by adding an effective amount of hexokinase.

11. The method according to claim 10, wherein the effective amount of hexokinase is about 0.4 U hexokinase per μ

l.

12. The method according to claim 7, wherein the first ligase is T4 DNA ligase.

13. The method according to claim 7, wherein the second ligase is E. coli NAD+-dependent ligase LigA which comprises a point mutant or an amino terminal truncation.

14. The method according to claim 7, wherein the second ligase is an E. coli NAD+-dependent ligase LigA mutant selected from the group consisting of:

Y22A, D32A and D36A.

15. A method for detecting a plurality of target nucleic acids from a nucleic acid sample, comprising:

a) contacting a DNA array-bound probe with;

(i) a DNA interrogation probe, and

(ii) a nucleic acid sample comprising at least one target nucleic acid of DNA,

wherein the array-bound probe and the interrogation probe hybridize to the target nucleic acid such that the array-bound probe and the interrogation probe are directly adjacent to one another, and wherein the interrogation probe contains a detectable label;

b) adding an effective amount of ATP to a solution of at least two different DNA ligase enzymes,

wherein the at least two different DNA ligase enzymes comprise at least a first ligase, which can form a DNA ligase-AMP intermediate complex, and at least a second ligase, which is a mutant ligase unable to form a DNA ligase-AMP intermediate complex;

c) depleting ATP from the solution of at least two different DNA ligase enzymes;

d) adding the solution of at least two different DNA ligase enzymes to step a), thereby ligating the array-bound probe to the interrogation probe; and

e) detecting the ligated product.

16. The method according to claim 15, wherein the first ligase is T4 DNA ligase.

17. The method according to claim 15, wherein the second ligase is E. coli NAD+-dependent ligase LigA which comprises a point mutant or an amino terminal truncation.

18. The method according to claim 15, wherein the second ligase is an E. coli NAD+-dependent ligase LigA mutant selected from the group consisting of:

Y22A, D32A and D36A.

19. The method of claim 15, wherein ATP depletion is accomplished by adding an effective amount of apyrase.

20. The method of claim 15, wherein ATP depletion is accomplished by adding an effective amount of hexokinase.