Generation of modified polymerases for improved accuracy in single molecule sequencing

• US 20100112645A1
• Filed: 03/30/2009
• Published: 05/06/2010
• Est. Priority Date: 03/31/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Park, Insil, Peluso, Paul, Clark, Sonya, Christians, Fred, Bibillo, Arek, Bjornson, Keith, Jia, Lei, Emig, Robin, Lee, Harold, He, Molly
• Granted Patent: US 8,257,954 B2
• Time in Patent Office: Days
• US Class Current: 435/91.500
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase

Abstract

Provided are compositions comprising modified recombinant polymerases that exhibit branching fractions that are less than the branching fractions of the polymerases from which they were derived, or branching fractions that are less than about 25% for a phosphate-labeled nucleotide analog. Also provided are compositions comprising modified recombinant polymerases that exhibit closed polymerase/DNA complexes with increased stability relative to the parental polymerases. Also provided are compositions comprising modified recombinant polymerases that exhibit decreased rate constants relative to the parental polymerases. Provided are methods for generating polymerases with the aforementioned phenotypes. Provided are methods of using such polymerases to make a DNA or to sequence a DNA template.

Claims

1. A composition comprising a modified recombinant Φ

29-type DNA polymerase, which modified recombinant polymerase comprises one or more mutations relative to a parental polymerase at one or more positions selected from the group consisting of;

a) positions that form a binding site for a metal ion that interacts with an epsilon and/or digamma phosphate of a bound nucleotide analog having five or more phosphate groups;

b) positions 372-397 and 507-514;

c) positions that form a binding site for a terminal fluorophore on a phosphate-labeled nucleotide analog;

d) positions at an intramolecular interface in a closed conformation of a ternary complex comprising the polymerase, a DNA, and a nucleotide or nucleotide analog;

e) positions that form a binding site for a polyphosphate group of a bound nucleotide or nucleotide analog;

f) positions that interact with the base of a bound nucleotide or nucleotide analog; and

g) positions that interact with a bound DNA;

wherein the one or more mutations comprise at least one mutation other than a 514Y, 514W, 514F, 5141, 514K, 259S, 370V, 370K, 372D, 372E, 372R, 372K, 372N, 372L, 387A, 387D, 478D, 478E, 478R, 480K, 480M, 480R, 371Q, 379E, 379T, 486D, 486A, 188A, 188S, 254F, 254V, 254A, 390F, or 390A substitution;

wherein numbering of positions is relative to wild-type Φ

29 polymerase.

3. The composition of claim 1, wherein the one or more mutations comprise at least one amino acid substitution at at least one residue selected from the group consisting of positions 484, 249, 179, 198, 211, 255, 259, 360, 363, 365, 370, 372, 378, 381, 383, 387, 389, 393, 433, 478, 480, 514, 251, 371, 379, 380, 383, 458, 486, 101, 188, 189, 303, 313, 395, 414, 497, 500, 531, 532, 534, 558, 570, 572, 574, 64, 305, 392, 402, 422, 496, 529, 538, 555, 575, 254, and 390.

4. The composition of claim 1, wherein the modified recombinant polymerase comprises at least one amino acid substitution or combination of substitutions selected from the group consisting of:

E375Y, K512Y, and T368F;

A484E;

A484Y;

N387L;

T372Q;

T372Y;

T372Y and K478Y;

K478Y;

1370W;

F198W;

L381A;

T368F;

A484E, E375Y, K512Y, and T368F;

A484Y, E375Y, K512Y, and T368F;

N387L, E375Y, K512Y, and T368F;

T372Q, E375Y, K512Y, and T368F;

T372L, E375Y, K512Y, and T368F;

T372Y, K478Y, E375Y, K512Y, and T368F;

1370W, E375Y, K512Y, and T368F;

F198W, E375Y, K512Y, and T368F; and

L381A, E375Y, K512Y, and T368F;

A484K;

A484R;

Q380K;

N251Q;

N251K;

I378W; and

S395K.

5. The composition of claim 1, wherein the one or more mutations comprise an insertion of at least one amino acid within residues 372-397 and/or 507-514.

2. (canceled)

6. A composition comprising a modified recombinant DNA polymerase, which modified recombinant polymerase comprises one or more mutations relative to a parental polymerase, and which modified recombinant polymerase exhibits a first rate constant for a first step in its catalytic cycle that is less than a first rate constant for the first step exhibited by the parental polymerase.

7. The composition of claim 6, wherein the first rate constant exhibited by the modified recombinant polymerase is less than 0.25 times the first rate constant exhibited by the parental polymerase.

8. The composition of claim 6, wherein the first step is not rate limiting in the catalytic cycle of the parental polymerase.

9. The composition of claim 6, wherein the modified recombinant polymerase exhibits a second rate constant for a second step in its catalytic cycle, where the second rate constant is between 0.1 and 10 times the first rate constant.

10. The composition of claim 9, wherein the second step is rate limiting in the catalytic cycle of the parental polymerase.

11. The composition of claim 9, wherein the second step involves incorporation of a bound nucleotide or nucleotide analog.

12. The composition of claim 11, wherein the second step involves incorporation of a nucleotide analog having from 3-7 phosphate groups.

13. The composition of claim 6, wherein the first step involves release of a polyphosphate product.

14. The composition of claim 13, wherein the first rate constant exhibited by the modified recombinant polymerase is less than about 100/second.

18. The composition of claim 9, wherein the second step involves incorporation of a bound nucleotide or nucleotide analog, wherein the first step involves release of a polyphosphate product, and wherein the second rate constant exhibited by the modified recombinant polymerase is between 0.2 and 1 times the first rate constant exhibited by the modified recombinant polymerase.

19. The composition of claim 9, wherein the second rate constant exhibited by the modified recombinant polymerase is smaller than the second rate constant exhibited by the parental polymerase for the second step.

20. The composition of claim 6, wherein the modified recombinant polymerase is a modified recombinant Φ

29-type DNA polymerase.

21. The composition of claim 20, wherein the modified recombinant polymerase is a modified recombinant Φ

29, B103, GA-1, PZA, Φ

15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.

22. The composition of claim 20, wherein the modified recombinant polymerase comprises at least one amino acid substitution or combination of substitutions selected from the group consisting of:

an amino acid substitution at position 484;

an amino acid substitution at position 198;

an amino acid substitution at position 381;

A484E;

A484Y;

N387L;

T372Q;

T372Y;

T372Y and K478Y;

K478Y;

1370W;

F198W;

L381A;

T368F;

A484E, E375Y, K512Y, and T368F;

A484Y, E375Y, K512Y, and T368F;

N387L, E375Y, K512Y, and T368F;

T372Q, E375Y, K512Y, and T368F;

T372L, E375Y, K512Y, and T368F;

T372Y, K478Y, E375Y, K512Y, and T368F;

1370W, E375Y, K512Y, and T368F;

F198W, E375Y, K512Y, and T368F;

L381A, E375Y, K512Y, and T368F;

E375Y, K512Y, and T368F;

A484K;

A484R;

Q380K;

N251Q;

N251K;

I378W; and

S395K;

, wherein numbering of positions is relative to wild-type Φ

29 polymerase.

23. The composition of claim 6, wherein the modified recombinant polymerase comprises a mutation that inhibits exonuclease activity of the polymerase.

25. The composition of claim 6, wherein the parental polymerase is a wild-type or an exonuclease deficient polymerase.

26. The composition of claim 6, comprising a phosphate-labeled nucleotide analog.

27. The composition of claim 26, wherein the nucleotide analog comprises a fluorophore.

28. The composition of claim 26, wherein the nucleotide analog has from 3-7 phosphate groups.

29. The composition of claim 6, comprising a phosphate-labeled nucleotide analog and a DNA template, wherein the modified recombinant polymerase incorporates the nucleotide analog into a copy nucleic acid in response to the DNA template.

30. The composition of claim 6, wherein the composition is present in a DNA sequencing system.

31. The composition of claim 30, wherein the sequencing system comprises a zero mode waveguide.

32. The composition of claim 6, wherein the modified recombinant polymerase is immobilized on a surface.

35. A method of making a DNA, the method comprising:

a) providing a reaction mixture comprising;

a template,

a replication initiating moiety that complexes with or is integral to the template,

the modified recombinant DNA polymerase of claim 6, which polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, and

one or more nucleotides and/or nucleotide analogs; and

b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA.

121. The composition of claim 6, wherein the first step involves translocation.

15-17. -17. (canceled)

24. (canceled)

33-34. -34. (canceled)

36-37. -37. (canceled)

38. A composition comprising a modified recombinant DNA polymerase, which modified recombinant polymerase exhibits a rate constant for release of a polyphosphate product that is less than about 50/second.

39-51. -51. (canceled)

52. A composition comprising a modified recombinant Φ

29-type DNA polymerase, which modified recombinant polymerase exhibits a branching fraction that is a) less than about 25% for a phosphate-labeled nucleotide analog and b) less than a branching fraction for the analog exhibited by a parental polymerase;

wherein, when the modified recombinant polymerase is a modified recombinant Φ

29-type DNA polymerase, the modified recombinant polymerase comprises at least one mutation relative to the parental polymerase other than a T368D, T368E, or T368G substitution, wherein numbering of positions is relative to wild-type Φ

29 polymerase.

59. The composition of claim 52, wherein the modified recombinant polymerase is a modified recombinant Φ

29-type DNA polymerase;

wherein the modified recombinant polymerase comprises at least one amino acid substitution or combination of substitutions selected from the group consisting of;

an amino acid substitution at position 153;

an amino acid substitution at position 191;

an amino acid substitution at position 388;

an amino acid substitution at position 422;

an amino acid substitution at position 128;

an amino acid substitution at position 253;

an amino acid substitution at position 504;

an amino acid substitution at position 143;

an amino acid substitution at position 183;

an amino acid substitution at position 236;

an amino acid substitution at position 363;

P153L;

G191A;

T368F;

T368P;

T368S;

T368V;

T368N;

T368A;

T373N;

T373V;

T373C;

1378V;

I378F;

K379S;

K379A;

S388A;

S388T;

K422R;

F128M;

F128V;

1504V;

K143D;

K512R;

Q183S;

R236N;

L253A;

F363Y;

L253A, F363Y, and_L480M;

T368F, K379S, E375Y, and K512Y;

T368F and K379S;

T368G and K379S;

T368F and T373A;

E375Y, K512Y and K379S;

E375Y, K512Y and T368F;

T368F and V514K;

T368F and K379T;

S388A and P153L;

E375Y, K512Y and T368G;

T368G and T373A;

E375W and T368G;

I378K and K379S;

T368F and I378K;

T368G and I378K;

T368G and V514K;

E375W and K379T;

T373A and K379S;

E375W and T373A;

E375Y, K512Y and T373A;

E375W and I378K;

E375Y, K512Y and I378K;

T373A and V514K;

T373A and I378K;

E375Y, K512Y and K379T;

I378K and V514K;

E375W and V514K;

T368G and K379T; and

E375Y, K512Y and V514K;

wherein numbering of positions is relative to wild-type Φ

29 polymerase.

73. A method of making a DNA, the method comprising:

a) providing a reaction mixture comprising;

a template,

a replication initiating moiety that complexes with or is integral to the template,

the modified recombinant DNA polymerase of claim 52, which polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, and

one or more nucleotides and/or nucleotide analogs; and

b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA.

53-58. -58. (canceled)

60-72. -72. (canceled)

74-75. -75. (canceled)

76. A composition comprising a modified recombinant Φ

29-type DNA polymerase, which modified recombinant polymerase comprises at least one amino acid substitution or combination of substitutions selected from the group consisting of;

an amino acid substitution at position 153;

an amino acid substitution at position 191;

an amino acid substitution at position 388;

an amino acid substitution at position 422;

an amino acid substitution at position 128;

an amino acid substitution at position 253;

an amino acid substitution at position 504;

an amino acid substitution at position 143;

an amino acid substitution at position 183;

an amino acid substitution at position 236;

an amino acid substitution at position 363;

P153L;

G191A;

T368F;

T368P;

T368S;

T368V;

T368N;

T368A;

T373N;

T373V;

T373C;

I378V;

I378F;

K379S;

K379A;

S388A;

S388T;

K422R;

F128M;

F128V;

1504V;

K143D;

K512R;

Q183S;

R236N;

L253A;

F363Y;

L253A, F363Y, and L480M;

T368F, K379S, E375Y, and K512Y;

T368F and K379S;

T368G and K379S;

T368F and T373A;

E375Y, K512Y and K379S;

E375Y, K512Y and T368F;

T368F and V514K;

T368F and K379T;

S388A and P153L;

E375Y, K512Y and T368G;

T368G and T373A;

E375W and T368G;

I378K and K379S;

T368F and I378K;

T368G and I378K;

T368G and V514K;

E375W and K379T;

T373A and K379S;

E375W and T373A;

E375Y, K512Y and T373A;

E375W and I378K;

E375Y, K512Y and I378K;

T373A and V514K;

T373A and I378K;

E375Y, K512Y and K379T;

I378K and V514K;

E375W and V514K;

T368G and K379T;

E375Y, K512Y and V514K;

A484E, E375Y, K512Y, and T368F;

A484E;

A484K;

A484R;

T3720;

Q380K;

N251Q;

N251K;

I378W; and

S395K;

wherein numbering of positions is relative to wild-type Φ

2 polymerase.

77-83. -83. (canceled)

84. A composition comprising a modified recombinant DNA polymerase, which modified recombinant polymerase comprises at least one mutation relative to a parental polymerase that increases or is predicted to increase stability of a closed complex comprising the modified recombinant DNA polymerase and a DNA.

85-97. -97. (canceled)

98. A composition comprising a modified recombinant Φ

29-type DNA polymerase, which modified recombinant polymerase comprises one or more amino acid substitutions relative to a parental polymerase at one or more positions selected from the group consisting of;

position 68-position 76, position 92, position 405-position 413, and position 560-position 564, wherein numbering of positions is relative to wild-type Φ

29 DNA polymerase, and which modified recombinant polymerase exhibits increased stability of a closed complex comprising the recombinant DNA polymerase and a DNA as compared to the parental polymerase.

100. The composition of claim 98, wherein the modified recombinant polymerase is a modified recombinant Φ

29 polymerase, and wherein the modified recombinant polymerase comprises at least one amino acid substitution or combination of substitutions selected from the group consisting of;

T92F, T92I, G410D, T92I and G410D, N72A, N721, N72F, and N72S.

113. A method of making a DNA, the method comprising:

a) providing a reaction mixture comprising;

a template,

a replication initiating moiety that complexes with or is integral to the template,

the modified recombinant DNA polymerase of claim 98, which polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, and

one or more nucleotides and/or nucleotide analogs; and

b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA.

99. (canceled)

101-112. -112. (canceled)

114-120. -120. (canceled)