Modified PH129 polymerase and uses thereof
Disclosed herein, inter alia, are mutant enzymes, kits, and methods of use thereof.
1 . A polymerase comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1; comprising an amino acid substitution, wherein said amino acid substitution is A324T, A394G, A394K, A394N, A394Q, A394V D219N, D34A, D34E, D34N, D34Q, F198L, F47I, G401A, G401L, G401V, G417A, G417K, G417N, G417Q, G417R, G579A, G579S, G579T, G579Y, H485L, I119A, I119G, I119L, I119V, I179F, I378A, I378G, I378L, I378V, I93A, I93G, I93N, I93Q, K135G, K311N, K311Q, K311R, K366A, K366G, K366H, K366N, K366Q, K366R, L216A, L216F, L216G, L216P, L216W, L216Y, L381Q, L462A, L462G, L462I, L462V, L567M, P87A, P87G, P87S, P87T, P87Y, R552A, R552N, R552Q, S192A, S192C, S192G, S260N, S260Q, S487C, S578A, S578G, S578I, S578L, S578T, S578V, T203S, T231A, T231I, T231V, T434A, T434G, T434Y, V565A, V565G, V565M, W436R, Y343C, Y500A, Y500F, or Y500G.
2 . The polymerase of claim 1 , wherein said the amino acid substitution is:
D34E, F47I, P87S, I93N, I119V, I179F, S192C, F198L, T203S, D219N, T231I, K311R, A324T, Y343C, K366R, I378V, L381Q, A394V, G401V, G417R, L462I, H485L, S487C, Y500F, K539E, R552Q, V565M, L567M, S578G, or G579S.
3 . A method of incorporating a nucleotide into a nucleic acid sequence comprising combining in a reaction vessel: (i) a nucleic acid template, (ii) a nucleotide solution comprising a plurality of nucleotides, and (iii) a polymerase, wherein the polymerase is a polymerase of claim 1 .
4 . A method of amplifying a nucleic acid sequence, said method comprising:
a) hybridizing a nucleic acid template to a primer to form a primer-template hybridization complex;
b) contacting said primer-template hybridization complex with a DNA polymerase and a plurality of nucleotides, wherein the DNA polymerase is the polymerase of claim 1 ; and
c) subjecting the primer-template hybridization complex to conditions which enable the polymerase to incorporate one or more nucleotides into the primer-template hybridization complex to generate amplification products, thereby amplifying a nucleic acid sequence.
5 . A method of amplifying a template polynucleotide, the method comprising: contacting a template polynucleotide with an amplification primer, and amplifying the template polynucleotide by extending an amplification primer with a polymerase to generate an extension product comprising one or more complements of the template polynucleotide; wherein said polymerase is a polymerase of claim 1 .
6 . The method of claim 5 , comprising amplifying the template polynucleotide in a cell.
7 . The method of claim 5 , comprising amplifying the template polynucleotide in a tissue.
8 . The method of claim 5 , comprising amplifying the template polynucleotide on a solid support.
9 . The polymerase of claim 1 , further comprising a glutamic acid, aspartic acid, alanine, or glycine at an amino acid position corresponding to position 135.
10 . The polymerase of claim 1 , further comprising K135E.
11 . The polymerase of claim 1 , comprising G417A, G417K, G417N, or G417Q.
12 . The polymerase of claim 1 , comprising A394V or A394K.
13 . The polymerase of claim 1 , further comprising K536E.
14 . The polymerase of claim 1 , comprising P87S and I93N.
15 . The polymerase of claim 14 , further comprising I378V, and S578V.
16 . The polymerase of claim 1 , comprising L381Q and H485L.
17 . The polymerase of claim 1 , further comprising P87A, P87G, P87S, P87T, or P87Y.
18 . The polymerase of claim 1 , comprising F198L.
19 . The polymerase of claim 1 , comprising G417R.
20 . The polymerase of claim 1 , further comprising a glutamic acid, aspartic acid, alanine, or glycine at an amino acid position corresponding to position 135; and a glutamic acid, aspartic acid, alanine, glycine, or threonine at an amino acid position corresponding to position 536.