POLYMERASES ENGINEERED TO REDUCE NUCLEOTIDE-INDEPENDENT DNA BINDING
Provided are engineered DNA polymerases exhibiting modified functionality, and polynucleotides encoding same. Modified features include: (1) reduced catalytic activity in the presence of magnesium ions and/or (2) reduced affinity for primed template nucleic acid molecules in the absence of cognate nucleotide, and an ability to discriminate between cognate and non-cognate nucleotides under low salt conditions. Sequencing By Binding™ procedures employing the engineered polymerases have certain advantages. The engineered polymerases can have other uses as well.
1 . A method of detecting a next correct nucleotide of a primed template nucleic acid, comprising the steps of:
(a) contacting the primed template nucleic acid with a first reaction mixture that comprises a crippled DNA polymerase and a test nucleotide,
wherein the test nucleotide is the next correct nucleotide of the primed template nucleic acid,
wherein a ternary complex is formed, the ternary complex comprising the primed template nucleic acid, the crippled DNA polymerase and the test nucleotide, and
wherein the crippled DNA polymerase comprises a variant of the sequence of SEQ ID NO:3, said variant being at least 80% identical to SEQ ID NO:3 and comprising an amino acid substitution mutation at one or more of positions K250, Q281, D355, Q425, and D532; and
(b) detecting the ternary complex without chemical incorporation of the test nucleotide into the primer of the primed template nucleic acid, thereby detecting the next correct nucleotide.
2 . The method of claim 1 , wherein the primer of the primed template nucleic acid comprises a reversible terminator nucleotide at its 3′-end.
3 . The method of claim 1 , further comprising (c) determining from the results of step (b) the identity of the next template nucleotide of the primed template nucleic acid.
4 . The method of claim 1 , wherein the variant is at least 90% identical to SEQ ID NO:3.
5 . The method of claim 1 ,
wherein the substitution mutation at position K250 comprises a mutation to a polar amino acid,
wherein the substitution mutation at position Q281 comprises a mutation to an acidic amino acid,
wherein the substitution mutation at position D355 comprises a mutation to a different acidic amino acid,
wherein the substitution mutation at position Q425 comprises a mutation to a different polar amino acid, and
wherein the substitution mutation at position D532 comprises a mutation to a different acidic amino acid.
6 . The method of claim 5 ,
wherein the substitution mutation at position K250 comprises a mutation to Cys,
wherein the substitution mutation at position Q281 comprises a mutation to Glu,
wherein the substitution mutation at position D355 comprises a mutation to Glu,
wherein the substitution mutation at position Q425 comprises a mutation to Cys, and
wherein the substitution mutation at position D532 comprises a mutation to Glu.
7 . The method of claim 1 , wherein the test nucleotide comprises an exogenous fluorescent label.
8 . The method of claim 1 , wherein the at least one amino acid substitution mutation is a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E).
9 . The method of claim 1 , wherein the at least one amino acid substitution mutation is a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).
10 . The method of claim 1 , wherein the at least one amino acid substitution mutation is a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E), a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).
11 . The method of claim 1 , wherein the at least one amino acid substitution mutation is a substitution mutation at position D355 that replaces Asp (D) with Glu (E), and a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E).
12 . The method of claim 1 , wherein the at least one amino acid substitution mutation is a substitution mutation at position D355 that replaces Asp (D) with Glu (E), a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).
13 . The method of claim 1 , wherein the at least one amino acid substitution mutation is a substitution mutation at position D355 that replaces Asp (D) with Glu (E), a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E), a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).
14 . The method of claim 1 , wherein the crippled polymerase comprises an exogenous label.
15 . A method of detecting a next correct nucleotide of a primed template nucleic acid, comprising the steps of:
(a) contacting the primed template nucleic acid with a first reaction mixture that comprises a crippled DNA polymerase and a test nucleotide,
wherein the test nucleotide is the next correct nucleotide of the primed template nucleic acid,
wherein a ternary complex is formed, the ternary complex comprising the primed template nucleic acid, the crippled DNA polymerase and the test nucleotide, and
wherein the crippled DNA polymerase comprises a variant of the sequence of SEQ ID NO:2, said variant being at least 80% identical to SEQ ID NO:2 and comprising an amino acid substitution mutation at one or more of positions Q290, K259 and Q434; and
(b) detecting the ternary complex without chemical incorporation of the test nucleotide into the primer of the primed template nucleic acid, thereby detecting the next correct nucleotide.
16 . The method of claim 15 , wherein the substitution mutation comprises Glu (E) at position 290.
17 . The method of claim 15 , wherein the substitution mutation comprises Cys (C) at position 259, and Cys (C) at position 434.
18 . The method of claim 15 , wherein the substitution mutation comprises Glu (E) at position 290, Cys (C) at position 259, and Cys (C) at position 434.
19 . A method of detecting a next correct nucleotide of a primed template nucleic acid, comprising the steps of:
(a) contacting the primed template nucleic acid with a first reaction mixture that comprises a crippled DNA polymerase and a test nucleotide,
wherein the test nucleotide is the next correct nucleotide of the primed template nucleic acid,
wherein a ternary complex is formed, the ternary complex comprising the primed template nucleic acid, the crippled DNA polymerase and the test nucleotide, and
wherein the crippled DNA polymerase comprises a variant of the sequence of SEQ ID NO:1, said variant being at least 80% identical to SEQ ID NO:1 and comprising an amino acid substitution mutation at one or more of positions Q307, K276 and Q451; and
(b) detecting the ternary complex without chemical incorporation of the test nucleotide into the primer of the primed template nucleic acid, thereby detecting the next correct nucleotide.
20 . The method of claim 19 , wherein the substitution mutation comprises Glu (E) at position 307.
21 . The method of claim 19 , wherein the substitution mutation comprises Cys (C) at position 276, and Cys (C) at position 451.
22 . The method of claim 19 , wherein the substitution mutation comprises Glu (E) at position 307, Cys (C) at position 276, and Cys (C) at position 451.