Enhanced ligation reactions

• US 9,765,388 B2
• Filed: 03/15/2016
• Issued: 09/19/2017
• 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): PRIEST, AARON A
• Application Number:
  US15/071,086
• Publication Number:
  US 20160186224A1
• Time in Patent Office: 553 Days
• Field of Search: None
• 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 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.