Polymerase variants and combination with 3'-OH non-blocking reversible terminators

Abstract

Polymerases for polynucleotide sequencing and other biotechnology applications are provided that have an improved ability to incorporate modified nucleotides, including nucleotides modified to function as reversible terminators. The present invention provides mutant polymerases with improved ability to incorporate modified nucleotides containing 3'-OH unblocked reversible terminators for use in a variety of applications, including polynucleotide sequencing, primer extension reactions and template-independent enzymatic oligonucleotide synthesis.

Claims

1. A composition comprising a 3'-OH nonblocking reversible terminator and a mutant polymerase, wherein the mutant polymerase comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 2, and a K477W mutation at a position functionally equivalent to position 477 of the Pfu polymerase, or any of the A486F mutation, A486N mutation, A486R mutation, and A486H mutation at a position functionally equivalent to position 486 of the Pfu polymerase.

2. The composition according to claim 1, wherein the mutant polymerase further comprises a mutation at a position functionally equivalent to position F494 of the Pfu polymerase.

3. The composition according to claim 2, wherein the F494 mutation is F494C, F494I, F494N, or F494T.

4. The composition according to any one of claims 1 to 3, wherein the mutant polymerase is a derivative of pyrococcus polymerase.

5. The composition according to claim 4, wherein the mutant polymerase comprises the amino acid sequence of SEQ ID NO:

2.

6. The composition according to any one of claims 1 to 5, wherein the mutant polymerase is a derivative of thermococcus polymerase.

7. A method for incorporating nucleotides into a priming strand containing nucleic acids, wherein the method is: The process involves contacting the priming chain with the nucleotide and the mutant polymerase under conditions sufficient for the integration reaction, A method wherein the mutant polymerase comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 2, and a K477W mutation at a position functionally equivalent to position 477 of the Pfu polymerase, or one of the A486F mutation, A486N mutation, A486R mutation, or A486H mutation at a position functionally equivalent to position 486 of the Pfu polymerase.

8. The method according to claim 7, wherein the nucleotide is a 3'-OH nonblocking reversible terminator.

9. A method for sequencing polynucleotides, (a) Forming a double helix comprising a template and a priming strand, wherein the template comprises a target nucleic acid to be sequenced and a primer binding site complementary to at least a portion of the priming strand. (b) Combining the priming chain with a reversible terminator nucleotide and a mutant polymerase, wherein the mutant polymerase includes an amino acid sequence that is at least 96% identical to SEQ ID NO: 2, and a K477W mutation at a position functionally equivalent to position 477 of the Pfu polymerase, or one of the A486F, A486N, A486R, or A486H mutations at a position functionally equivalent to position 486 of the Pfu polymerase. (c) Incorporating the reversible terminator into the 3' end of the priming chain by a template-dependent reaction, (d) Identifying the incorporated reversible terminator nucleotide and thereby determining the sequence of the template, Methods that include...

10. The method according to claim 9, further comprising repeating steps (c) and (d) at least 80 times.

11. A composition comprising a priming chain, a 3'-OH unmodified reversible terminator, and a mutant polymerase that is at least 96% identical to SEQ ID NO: 2, The aforementioned mutant polymerase is The Y546H mutation at a functionally equivalent position to position 546 of Pfu polymerase, L409Y, L409H, or L409F mutations at a functionally equivalent position to Pfu polymerase position 409, An A486X mutation at a position functionally equivalent to position 486 of Pfu polymerase, wherein the A486X mutation is selected from any of A486F, A486Y, A486N, A486R, and A486H, A composition containing the following:

12. The composition according to claim 11, wherein the composition does not include a template complementary to the priming chain.

13. The composition according to claim 11 or claim 12, wherein the mutant polymerase further comprises one or more mutations at functionally equivalent positions to the Pfu polymerase positions L270, E330, Q332, L333, P451, L453, L457, E476, L489, L490, N492, F494, Y497 and E581.

14. The composition according to claim 11 or claim 12, wherein the mutant polymerase comprises the amino acid sequence of SEQ ID NO:

5.

15. The composition according to any one of claims 11 to 14, wherein the mutant polymerase has an integration activity at least twice as high as that of the DNA polymerase of Sequence ID No.

11.

16. The composition according to any one of claims 11 to 15, wherein the mutant polymerase is a derivative of pyrococcus polymerase.

17. The composition according to any one of claims 11 to 15, wherein the mutant polymerase is a derivative of thermococcus polymerase.

18. A method for incorporating a single nucleotide into a priming chain by a template-independent reaction, wherein the method is The method involves combining a priming chain with a 3'-OH unmodified reversible terminator and a mutant polymerase, wherein the mutant polymerase is at least 96% identical to SEQ ID NO:

2. The Y546H mutation at a functionally equivalent position to position 546 of Pfu polymerase, One of the L409Y, L409H, and L409F mutations at a functionally equivalent position to Pfu polymerase position 409, A combination of A486X mutations at a functionally equivalent position to position 486 of Pfu polymerase, which are selected from A486F, A486Y, A486N, A486R and A486H, A method wherein the integration of the terminator is at least twice as high as that of the mutant DNA polymerase of SEQ ID NO:

11.

19. A method for synthesizing template-independent oligonucleotides, The method involves combining a priming strand, a 3'-OH unmodified reversible terminator, and a mutant DNA polymerase, wherein the mutant DNA polymerase is It consists of an amino acid sequence that is at least 96% identical to Sequence ID No. 2, Y546H mutation to histidine at a functionally equivalent position to position 546 of Pfu polymerase. L409Y, L409H, or L409F mutations at a functionally equivalent position to position 409 of Pfu polymerase, A combination of A486X mutations at a functionally equivalent position to position 486 of Pfu polymerase, which are selected from A486F, A486Y, A486N, A486R and A486H, Including, combining, A method comprising incorporating the 3'-OH unmodified reversible terminator into the priming chain.

20. The method according to claim 18 or claim 19, wherein the polymerase further comprises one or more mutations at functionally equivalent positions to the Pfu polymerase positions L270, E330, Q332, L333, P451, L453, L457, E476, L489, L490, N492, F494, Y497 and E581.

21. The method according to any one of claims 18 to 20, wherein the 3'-OH unmodified reversible terminator is a 2-nitrobenzyl modified nucleotide.

22. The method according to any one of claims 18 to 20, wherein the 3'-OH unmodified reversible terminator is a C7- or C5-hydroxymethyl-α-tert-butyl-2-nitrobenzyl modified nucleotide and its α-thio derivative.

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