Enzymatic ligation of nucleic acids

Abstract

Methods, assays, compositions and kits for the ligation of short polynucleotides are presented herein. The short polynucleotides are optionally no more than 7 nucleotides in length, and can be as short as 3 or 4 nucleotides in length. The ligation is optionally performed by CV ligase.

1 - 53 . (canceled) 
     
     
         54 . An artificial small footprint ligase derived from a Hin ( Haemophilus Influenzae ) DNA ligase or any fragment or variant thereof that comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, and/or has one or more C-terminal amino acids deleted. 
     
     
         55 . An artificial small footprint ligase derived from a Hin DNA ligase or any fragment or variant thereof that consists of the sequence of SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. 
     
     
         56 . An artificial small footprint ligase derived from a Hin DNA ligase that has at least 70% identity to SEQ ID NO: 5, said ligase comprising an amino acid mutation at position 193 of SEQ ID NO: 5, optionally wherein said amino acid mutation consists of changing the glycine at position 193 to aspartic acid or glutamic acid. 
     
     
         57 . The ligase of  claim 56 , wherein the ligase has at least 80% identity to SEQ ID NO: 5. 
     
     
         58 . The ligase of  claim 56 , wherein the ligase has at least 90% identity to SEQ ID NO: 5. 
     
     
         59 . The ligase of  claim 56 , wherein the ligase has at least 95% identity to SEQ ID NO: 5. 
     
     
         60 . The ligase of  claim 56 , wherein the ligase has at least 99% identity to SEQ ID NO: 5. 
     
     
         61 . The ligase of  claim 54 , wherein the ligase is isolated, purified or recombinant. 
     
     
         62 . A nucleic acid molecule encoding the ligase of  claim 54 . 
     
     
         63 . The nucleic acid of  claim 62 , wherein the nucleic acid is isolated, purified or recombinant. 
     
     
         64 . A vector comprising the nucleic acid molecule of  claim 62 . 
     
     
         65 . The vector of  claim 64 , wherein the vector is isolated, purified or recombinant. 
     
     
         66 . A host cell comprising the vector of  claim 64 . 
     
     
         67 . The host cell of  claim 66 , wherein the host cell is isolated, purified or recombinant. 
     
     
         68 . A method of making a ligase comprising:
 a. culturing the isolated recombinant host cell of  claim 66  or  67  under conditions such that the ligase is expressed; and   b. recovering said ligase.   
     
     
         69 . The ligase of  claim 56 , wherein the amino acid mutation consists of changing the glycine at position 193 to aspartic acid. 
     
     
         70 . The ligase of  claim 56 , wherein the amino acid mutation consists of changing the glycine at position 193 to glutamic acid. 
     
     
         71 . The ligase of  claim 54 , wherein the ligase is attached to a solid support. 
     
     
         72 . The ligase of  claim 54 , wherein the ligase comprises a N-terminal nucleotidyltransferase (NTase) domain, the NTase domain including at least one motif selected from the group consisting of motif I, motif Ia, motif III, motif IIIa, motif IV, motif V, and motif VI. 
     
     
         73 . The ligase of  claim 54 , wherein the activity of the ligase comprises at least one of the following:
 (1) provide a nucleophilic attack on ATP or NAD+ resulting in release of PPi or NMN and formation of a covalent ligase-adenylate intermediate;   (2) transfer adenylate to a 5′-end of a 5′-phosphate-terminated DNA strand to form DNA-adenylate; or   (3) form a covalent bond joining a polynucleotide termini and liberation of AMP.   
     
     
         74 . The ligase of  claim 54 , wherein the ligase can mediate at least one of the following bond transformations: phosphoanhydride to phosphoramidate, phosphoramidate to phosphoanhydride, or phosphoanhydride to phosphodiester.