T7 DNA Ligase Variants R129D with Increased Specificity for Cohesive End Polynucleotides
Disclosed is a novel T7 DNA ligase mutant with the mutation R129D, that had higher specificity toward cohesive end-ligation even in the presence of polyethylene glycol. Controlling the composition of final ligation products is important and thus, a variant which improves the specificity of T7 DNA ligase to cohesive ends will generate more reliable results.
1 . A mutant T7 DNA ligase or a biologically active fragment thereof, comprising: the mutation R129D.
2 . The mutant T7 DNA ligase or a biologically active fragment of claim 1 further including a histidine tag at its C-terminus.
4 . The mutant T7 DNA ligase or a biologically active fragment of claim 2 wherein the histidine tag has six histidine residues.
5 . The mutant T7 DNA ligase or a biologically active fragment of claim 2 further including alternating Serine and Glycine amino acid residues at the N-terminal end of the histidine tag.
6 . A mutant T7 DNA ligase or a biologically active fragment having at least 95% identity to SEQ ID NO: 1 and having only conservative amino acid substitutions.
7 . The mutant T7 DNA ligase or a biologically active fragment of claim 6 further including a histidine tag at its C-terminus.
8 . The mutant T7 DNA ligase or a biologically active fragment of claim 6 wherein the histidine tag has six histidine residues.
9 . The mutant T7 DNA ligase or a biologically active fragment of claim 7 further including alternating Serine and Glycine amino acid residues at the N-terminal end of the histidine tag.
10 . A polynucleotide encoding an amino acid sequence of the mutant T7 DNA ligase or the biologically active fragment thereof of claim 1 .
11 . A vector incorporating a polynucleotide of claim 10 .
12 . A cell transformed with and expressing a polynucleotide of claim 10 .
13 . The polynucleotide of claim 10 which is DNA or RNA.
14 . A method for performing a ligation reaction between a nucleic acid template and one or more nucleotides, comprising:
contacting a mutant T7 DNA ligase or a biologically active fragment thereof of claim 1 with the nucleic acid template in the presence of the nucleotides to form a reaction mixture, and
providing ligation conditions for the reaction mixture whereby the nucleotides are ligated with the nucleic acid template.
15 . The method of claim 14 wherein the mutant T7 DNA ligase or a biologically active fragment further includes a 6-membered histidine tag at its C-terminus.
16 . The method of claim 15 wherein the histidine tag has six histidine residues.
17 . The method of claim 15 further including alternating Serine and Glycine amino acid residues are at the N-terminal end of the histidine tag.