THERMOSTABLE BLUNT-END LIGASE AND METHODS OF USE
Thermostable blunt-end ligases suitable for use in nucleic acid ligation reactions at elevated temperatures are provided. The ligases comprise fusion proteins including a DNA ligase and a DNA binding protein, e.g., a T4 DNA ligase with an N-terminal p50 fusion. The fusion proteins may include peptide linkers, peptide mimetics, terminal additions, tag peptides, D-amino acids, sugars, non-amino acid organic moieties, and polymers. The ligases are suitable for use in ligation reactions, including uniform-temperature ligation reactions, performed at about 60° C. or higher, e.g., at about 75° C. The ligases are suitable for use in nucleic acid amplification schemes with temperature cycling, e.g., temperature cycles to about 60° C. or higher, or temperature cycles from about 94° C. to about 60° C. Such nucleic acid amplification schemes may include one, two, three, or more temperature cycles. Methods of using the ligases, and articles of manufacture comprising the ligases are provided.
1 . A thermostable blunt-end DNA ligase comprising a fusion protein comprising, in N-terminal to C-terminal order, a leader sequence comprising the amino acid sequence SEQ ID NO: 4, a DNA binding protein, a glycine-rich amino acid sequence comprising SEQ ID NO: 5, and a DNA ligase, wherein said thermostable blunt-end DNA ligase is suitable for use in a blunt-ended DNA ligation reaction performed at about 60° C. or higher.
2 . The thermostable blunt-end DNA ligase of claim 1 , comprising a T4 DNA ligase with an N-terminal p50 fusion.
3 . The thermostable blunt-end DNA ligase of claim 1 , comprising an amino acid sequence selected from the group of amino acid sequences consisting of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
4 . A method of ligating blunt-end DNA at elevated temperature, comprising ligating blunt-end DNA using a thermostable blunt-end DNA ligase suitable for use in a blunt-ended DNA ligation reaction performed at about 60° C. or higher, wherein said thermostable blunt-end DNA ligase comprises a thermostable blunt-end DNA ligase of claim 1 .
5 . An article of manufacture, comprising a thermostable blunt-end DNA ligase suitable for use in a blunt-ended DNA ligation reaction performed at about 60° C. or higher and a container, wherein said thermostable blunt-end DNA ligase comprises a thermostable blunt-end DNA ligase of claim 1 .
6 . The article of manufacture of claim 5 , further comprising a buffer.
7 . A device for analyzing a sample containing DNA, comprising a thermostable blunt-end DNA ligase suitable for use in a blunt-ended DNA ligation reaction performed at about 60° C. or higher, wherein said thermostable blunt-end DNA ligase comprises a thermostable blunt-end DNA ligase of claim 1 .
8 . The method of claim 4 , wherein said thermostable blunt-end DNA ligase is suitable for use in a blunt-ended DNA ligation reaction performed at about 75° C., and said ligating is performed at about 75° C.
9 . The thermostable blunt-end DNA ligase of claim 1 , wherein said thermostable blunt-end DNA ligase is capable of making concatamers upon multiple ligation events in a blunt-ended DNA ligation reaction performed at about 60° C. or higher.
10 . The thermostable blunt-end DNA ligase of claim 1 , wherein said thermostable blunt-end DNA ligase is suitable for use in a nucleic acid amplification scheme which operates at a uniform temperature of about 60° C. or higher.
11 - 15 . (canceled)
16 . The thermostable blunt-end DNA ligase of claim 1 , wherein said fusion protein comprises a component selected from a peptide linker, an N-terminal addition, a C-terminal addition, a tag peptide, a D-amino acid, and a peptide mimetic.
17 . The thermostable blunt-end DNA ligase of claim 1 , wherein said fusion protein comprises a component selected from a sugar and a polymer.
18 - 21 . (canceled)
22 . A method of ligating blunt-end nucleic acids at an elevated temperature, comprising using a thermostable blunt-end nucleic acid ligase of claim 3 at a temperature of about 60° C. or higher.
23 . A device for analyzing a sample containing nucleic acids, comprising a thermostable blunt-end nucleic acid ligase of claim 1 .
24 . The device of claim 23 , wherein said thermostable blunt-end ligase comprises an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
25 . A fusion protein comprising the amino acid sequence SEQ ID NO: 20 and a nucleic acid ligase.
26 . The fusion protein of claim 25 , wherein the nucleic acid ligase is a DNA ligase.
27 . The fusion protein of claim 25 , wherein the nucleic acid ligase is a RNA ligase.
28 . The fusion protein of claim 25 , wherein the nucleic acid ligase is a thermostable DNA ligase.
29 . The fusion protein of claim 25 , wherein the nucleic acid ligase is a thermostable RNA ligase.