Enhanced ligation reactions

Patent metadata

Field	Value
Publication number	US-10093969-B2
Application number	US-201715695244-A
Country	US
Kind code	B2
Filing date	Sep 5, 2017
Priority date	Dec 17, 2010
Publication date	Oct 9, 2018
Grant date	Oct 9, 2018

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.

First claim

What is claimed:

1. A method for ligating nucleic acids, comprising:
a) forming a plurality of polynucleotide duplexes each having a nick, by (i) hybridizing a first template polynucleotide with a first and second probe oligonucleotide so that the first and second probe oligonucleotides abut each other to form a first nick, wherein the second probe oligonucleotide is labeled with a first distinctive reporter moiety, and wherein the 5′ end of the first template polynucleotide is attached to a support, and (ii) hybridizing a second template polynucleotide with a third and fourth probe oligonucleotide so that the third and fourth oligonucleotides abut each other to form a second nick wherein, the fourth probe oligonucleotide is labeled with a second distinctive reporter moiety, wherein the first and second template polynucleotides include different sequences, wherein the second and fourth probe oligonucleotides include different sequences, wherein the first and second distinctive reporter moieties distinguish the different sequences of the second and fourth probe oligonucleotides respectively, and wherein the 5′ end of the second template polynucleotide is attached to the support;
b) closing the first and second nick to form a first and second ligation product respectively by conducting a nucleic acid ligation reaction with a ligase and an aprataxin enzyme that catalyzes removal of an adenylate group from a terminal 5′ phosphate of a nucleic acid; and
c) detecting the first distinctive reporter moiety thereby detecting the sequence of a portion of the first template polynucleotide that hybridizes to the second probe oligonucleotide, and detecting the second distinctive reporter moiety thereby detecting the sequence of a portion of the second template polynucleotide that hybridizes to the fourth probe oligonucleotide.

2. The method of claim 1 , wherein the support comprises a planar support.

3. The method of claim 1 , wherein the planar support comprises a plurality of wells arranged in an array.

4. The method of claim 1 , wherein the planar support comprises 100-10 7 wells.

5. The method of claim 4 , wherein each well is capacitively coupled to a field effect transistor (FET).

6. The method of claim 5 , wherein the FET detects protons.

7. The method of claim 1 , wherein the support comprises a bead.

8. The method of claim 7 , further comprising depositing the bead onto an array.

9. The method of claim 8 , wherein the array comprises a plurality of wells.

10. The method of claim 8 , wherein the array comprises 100-10 7 wells.

11. The method of claim 8 , wherein each well is capacitively coupled to a field effect transistor (FET).

12. The method of claim 11 , wherein the FET detects pyrophosphate, hydrogen ions, charge transfer or heat.

13. The method of claim 1 , wherein the aprataxin enzyme comprise the amino acid sequence according to SEQ ID NO:1.

14. The method of claim 1 , wherein the ligase comprises a small footprint ligase comprising the amino acid sequence according to any one of SEQ ID NOS: 6-8.

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

16. The method of claim 1 , wherein the first and second distinctive report moieties comprise different fluorophores.

17. The method of claim 1 , further comprising: denaturing the first and second ligation product from the first and second polynucleotides respectively.

18. The method of claim 1 , further comprising:
c) forming a plurality of polynucleotide duplexes each having a nick, by (i) hybridizing the first template polynucleotide with a fifth probe oligonucleotide so that the first ligation product and the fifth probe oligonucleotide abut each other to form a third nick, wherein the fifth probe oligonucleotide is labeled with a third distinctive reporter moiety, and (ii) hybridizing the second template polynucleotide with a sixth probe oligonucleotide so that the second ligation product and the sixth probe oligonucleotide abut each other to form a fourth nick, wherein the sixth probe oligonucleotide is labeled with a fourth distinctive reporter moiety, wherein the third and fourth reporter moieties distinguish the different sequences of the fifth and sixth probe oligonucleotides respectively;
d) closing the third and fourth nick to form a third and fourth ligation product respectively, by conducting a nucleic acid ligation reaction with a ligase and an aprataxin enzyme that catalyzes removal of an adenylate group from a terminal 5′ phosphate of a nucleic acid;
e) detecting the third distinctive reporter moiety thereby detecting the sequence of a portion of the first template polynucleotide that hybridizes to the fifth oligonucleotide, and detecting the fourth distinctive reporter moiety thereby detecting the sequence of a portion of the second template polynucleotide that hybridizes to the sixth oligonucleotide; and
f) denaturing the third and fourth ligation products from the first and second polynucleotides respectively.

19. The method of claim 18 , wherein the first, second, third and fourth distinctive reporter moieties comprise different fluorophores.

Assignees

• Life Technologies Corp

Inventors

• Hendricks Stephen
• King David
• Xi Lei
• Peris Marian

Classifications

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2531/119

  Strand displacement amplification [SDA] · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/46

  involving cholinesterase · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title