RECOMBINANT LIGASES AND USES THEREOF
Disclosed herein, inter alia, are ligases, kits, and methods of use thereof.
1 . A ligase comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:1 covalently attached to a polyethylene glycol moiety.
2 . The ligase of claim 1 , wherein the polyethylene glycol moiety comprises
wherein n is 2 to 24.
3 . The ligase of claim 2 , wherein n is 6, 12, 18, or 24.
4 . The ligase of claim 2 , wherein n is 24.
5 . The ligase of claim 1 , comprising a plurality of polyethylene glycol moieties.
6 . The ligase of claim 1 , wherein the polyethylene glycol moiety is covalently attached to the ligase via a bioconjugate linker.
7 . The ligase of claim 1 , further comprising one or more substitutions at amino acid position corresponding to position 7, 8, 10, 23, 24, 25, 27, 29, 30, 32, 42, 65, 67, 75, 92, 93, 94, 95, 96, 97, 98, 99, 100, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 140, 161, 164, 165, 166, 167, 171, 172, 173, 174, 175, 176, 186, 188, 190, 228, 234, 243, 244, 246, 276, 285, 286, 293, and/or 297 of SEQ ID NO:1.
8 . The ligase of claim 1 , further comprising a substitution at amino acid position corresponding to position 140, 243, 244, and/or 246.
9 . The ligase of claim 8 , wherein the substitution at amino acid position corresponding to position 140, 243, 244 is independently an arginine, lysine, histidine, glutamine, asparagine, methionine, or serine.
10 . The ligase of claim 8 , wherein the substitution at amino acid position corresponding to position 140, 243, 244 is independently a lysine, arginine, or histidine.
11 . The ligase of claim 8 , wherein the substitution at amino acid position corresponding to position 246 is an aspartic acid, glutamine, asparagine.
12 . The ligase of claim 1 , comprising an amino acid sequence that is at least 80% identical to a continuous 250 amino acid sequence within SEQ ID NO:1.
13 . The ligase of claim 1 , further comprising a lysine or arginine at amino acid position corresponding to position 140 of SEQ ID NO:1.
14 . The ligase of claim 1 , further comprising a histidine tag.
15 . The ligase of claim 1 , further comprising a lysine, arginine, glutamic acid, aspartic acid, or proline at amino acid position corresponding to position 246 of SEQ ID NO:1.
16 . A method for detecting polynucleotide sequences, comprising:
(a) hybridizing a first polynucleotide sequence to a nucleic acid molecule and hybridizing a second polynucleotide sequence to the nucleic acid molecule;
(b) ligating the first polynucleotide sequence and the second polynucleotide sequence together with a ligase to generate a ligated product, wherein the ligase comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:1; and
(c) detecting the ligated product.
17 . The method of claim 16 , wherein (a)-(c) occur in a cell or tissue.
18 . The method of claim 16 , further comprising amplifying the ligated product by extending an amplification primer hybridized to the ligated product with a polymerase to generate an extension product.
19 . The method of claim 17 , further comprising amplifying the ligated product in a cell or tissue.
20 . The method of claim 16 , wherein detecting comprises sequencing.