DNA Polymerases and Related Methods
Provided herein is an engineered DNA polymerases containing an amino acid sequence selected from: an amino acid sequence that is at least 80% identical to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:24; and an amino acid sequence that is at least 92% identical to an amino acid sequence selected from: SEQ ID NO:2 and SEQ ID NO:3. Also provided are methods employing the described DNA polymerases.
1 . A DNA polymerase comprising an amino acid sequence selected from:
an amino acid sequence that is at least 80% identical to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:24; and
an amino acid sequence that is at least 92% identical to an amino acid sequence selected from: SEQ ID NO:2 and SEQ ID NO:3.
2 . The DNA polymerase of claim 1 , comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:24.
3 . The DNA polymerase of claim 1 , comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from: SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:24.
4 . The DNA polymerase of claim 1 , comprising an amino acid sequence that is identical to an amino acid sequence selected from: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 SEQ ID NO:6, and SEQ ID NO:24.
5 . A DNA polymerase, wherein the DNA polymerase is a fusion protein comprising an amino acid sequence of claim 1 and an exogenous amino acid sequence.
6 . The DNA polymerase of claim 5 , wherein the exogenous amino acid sequence comprises a DNA binding domain.
7 . The DNA polymerase of claim 5 , wherein the exogenous amino acid sequence comprises a purification tag.
8 . A method, comprising:
incubating a reaction mixture comprising:
(i) a DNA polymerase of claim 1 ;
(ii) a target nucleic acid;
(iii) dNTPs; and
(iv) one or more primers,
under conditions suitable for polynucleotide extension of the target nucleic acid to produce a copy of the target nucleic acid.
9 . The method of claim 8 , wherein the conditions are isothermal.
10 . The method of claim 9 , wherein the one or more primers comprise Loop-Mediated Isothermal Amplification (LAMP) primers.
11 . A composition comprising: (i) an aqueous solution of or (ii) a freeze-dried/lyophilized form of the DNA polymerase of claim 1 .
12 . The composition of claim 11 , wherein the DNA polymerase is in a solution that comprises glycerol.
13 . The composition of claim 11 , further comprising a solid support.
14 . The composition of claim 11 , further comprising one or more components selected from a buffering agent, a salt, a primer, and an aptamer.
15 . A kit comprising:
a DNA polymerase of claim 1 ; and
a reaction buffer.
16 . The kit of claim 15 , further comprising dNTPs.
17 . The kit of claim 15 , wherein the DNA polymerase is in a form selected from: dried form, lyophilized form, solution form.
18 . The kit of claim 15 , wherein the reaction buffer comprises a buffering agent and a salt.
19 . The kit of claim 15 , wherein the DNA polymerase is provided in a separate tube from reaction buffer.
20 . The kit of claim 15 , wherein the reaction mixture is suitable for receiving and extending a target nucleic acid in the presence of the DNA polymerase and one or more primers.
21 . The kit of claim 15 , further comprising one or more additional enzymes, optionally selected from a reverse transcriptase and a uracil DNA glycosidase (UDG).