Enhanced ligation reactions

Patent metadata

Field	Value
Publication number	US-9765388-B2
Application number	US-201615071086-A
Country	US
Kind code	B2
Filing date	Mar 15, 2016
Priority date	Dec 17, 2010
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

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 system for ligating nucleic acids comprising:
a) a single-stranded polynucleotide template;
b) first and second oligonucleotide probes configured to abut each other while they are hybridized to the polynucleotide template, thereby forming a nick, wherein one end of the polynucleotide template, the first oligonucleotide probe, or the second oligonucleotide probe, is attached to a surface comprising a bead or a flowcell;
c) a modified small footprint ligase having the amino acid sequence according to SEQ ID NO:6 or 7; and
d) an agent that catalyzes removal of an adenylate group from the terminal 5′ phosphate of nucleic acid.

2. The system 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 system of claim 2 , wherein the aprataxin enzyme comprises the amino acid sequence according to SEQ ID NO:1.

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

5. The system of claim 1 , wherein the first or second oligonucleotide is labeled with a distinctive detectable reporter moiety.

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

7. The system of claim 1 , wherein the flowcell comprises a microwell array, wherein at least one microwell in the array is capacitively coupled to a sensor that detects a change in a nucleotide incorporation byproduct.

8. The system of claim 7 , wherein the nucleotide incorporation byproduct includes phyrophosphate, hydrogen ions, charge transfer or heat.

Assignees

• Life Technologies Corp

Inventors

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

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q2565/537

  characterised by the capture oligonucleotide acting as a primer · CPC title

• C12Q1/46

  involving cholinesterase · CPC title

• C12Q1/6874

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

• C12Q2531/119

  Strand displacement amplification [SDA] · CPC title