Thermostable blunt-end ligase and methods of use

Abstract

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.