Recombinant Polymerases For Improved Single Molecule Sequencing

• US 20120034602A1
• Filed: 07/05/2011
• Published: 02/09/2012
• 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: Emig, Robin, Park, Insil, Jia, Lei, Bibillo, Arek, Bjornson, Keith, Hanes, Jeremiah, Lee, Harold, Clark, Sonya, Peluso, Paul, He, Molly, Christians, Fred, Vieceli, John, Kamtekar, Satwik, Miller, Erik, Cutcliffe, Colleen, Lee, Walter
• Granted Patent: US 8,999,676 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

Claims

1. A composition comprising a recombinant Φ

29-type DNA polymerase, which recombinant polymerase comprises a mutation at position E375, a mutation at position K512, and a mutation at one or more positions selected from the group consisting of L253, A484, V250, E239, Y224, Y148, E508, and T368, wherein identification of positions is relative to wild-type Φ

29 polymerase (SEQ ID NO;

1).

2. The composition of claim 1, wherein the mutation at position E375 comprises an amino acid substitution selected from the group consisting of E375Y, E375F, E375R, E375Q, E375H, E375L, E375A, E375K, E375S, E375T, E375C, E375G, and E375N.

3. The composition of claim 1, wherein the mutation at position K512 comprises an amino acid substitution selected from the group consisting of K512Y, K512F, K512I , K512M, K512C, K512E, K512G, K512H, K512N, K512Q, K512R, K512V, and K512H.

4. The composition of claim 1, wherein the mutation at position E375 comprises an E375Y substitution and the mutation at position K512 comprises a K512Y substitution.

5. The composition of claim 1, wherein the recombinant polymerase comprises one or more amino acid substitutions selected from the group consisting of L253A, L253C, L253S, A484E, A484Q, A484N, A484D, A484K, V250I , V250Q, V250L, V250M, V250C, V250F, V250N, V250R, V250T, V250Y, E239G, Y224K, Y224Q, Y224R, Y148I , Y148A, Y148K, Y148F, Y148C, Y148D, Y148E, Y148G, Y148H, Y148K, Y148L, Y148M, Y148N, Y148P, Y148Q, Y148R, Y148S, Y148T, Y148V, Y148W, E508R, and E508K.

6. The composition of claim 1, wherein the recombinant polymerase comprises a mutation at one or more positions selected from the group consisting of D510, E515, and F526, wherein identification of positions is relative to wild-type Φ

29 polymerase (SEQ ID NO;

1).

7. The composition of claim 6, wherein the recombinant polymerase comprises one or more amino acid substitutions selected from the group consisting of D510K, D510Y, D510R, D510H, D510C, E515Q, E515K, E515D, E515H, E515Y, E515C, E515M, E515N, E515P, E515R, E515S, E515T, E515V, E515A, F526L, F526Q, F526V, F526K, F526I, F526A, F526T, F526H, F526M, F526V, and F526Y.

8. The composition of claim 1, wherein the recombinant polymerase comprises a mutation at one or more positions selected from the group consisting of N62, D12, D66, and K143, E14, H61, D169, Y148, and H149, wherein identification of positions is relative to wild-type Φ

29 polymerase (SEQ ID NO;

1).

9. The composition of claim 1, wherein the recombinant polymerase comprises a combination of mutations selected from the group consisting of:

a) L253A, E375Y, A484E, and K512Y;

b) L253A, E375Y, A484E, D510K, and K512Y;

c) Y148I , Y224K, E239G, V250I , L253A, E375Y, A484E, D510K, and K512Y;

d) Y148I , Y224K, E239G, V250I , L253A, E375Y, A484E, D510K, K512Y, and E515Q;

e) E239G, L253A, E375Y, A484E, D510K, and K512Y;

f) Y224K, E239G, L253A, E375Y, A484E, D510K, and K512Y;

g) Y148I , Y224K, E239G, L253C, E375Y, A484E, D510K, and K512Y;

h) N62D, V250I , L253A, E375Y, A484E, and K512Y;

i) Y224K, E239G, L253A, E375Y, A484E, K512Y, and F526L;

j) L253A, E375Y, A484E, K512Y, and E515K;

k) E239G, L253A, E375Y, A484E, E508R, and K512Y;

l) Y148I , L253A, E375Y, A484E, and K512Y;

m) D66R, Y148I , Y224K, E239G, V250I , L253A, E375Y, A484E, D510K, and K512Y;

n) N62D, Y148I , Y224K, E239G, V250I , L253A, E375Y, A484E, D510K, and K512Y;

o) K143R, Y148I , Y224K, E239G, V250I , L253A, E375Y, A484E, D510K, and K512Y;

p) D12N, Y224K, E239G, L253A, E375Y, A484E, and K512Y;

q) Y148F, Y224K, E239G, V250I , L253A, E375Y, A484E, D510K, and K512Y;

r) Y148I, Y224K, E239G, V250I , L253A, E375Y, A484E, D510R, and K512Y;

s) Y148I, Y224K, E239G, V250I , L253A, E375Y, A484E, D510H, and K512Y; and

t) Y148I, Y224K, E239G, V250I , L253A, E375Y, A484E, E508K, D510K, and K512Y.

10. The composition of claim 9, wherein the recombinant polymerase is at least 80% identical to wild-type Φ

29 polymerase (SEQ ID NO;

1).

11. The composition of claim 10, wherein the C-terminal region of the recombinant polymerase comprises a His10 tag.

12. The composition of claim 1, wherein the recombinant polymerase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

133-193.

14. The composition of claim 1, wherein the recombinant polymerase is at least 80% identical to wild-type Φ

29 polymerase (SEQ ID NO;

1).

15. The composition of claim 1, wherein the recombinant polymerase is at least 80% identical to wild-type M2Y polymerase (SEQ ID NO:

2).

19. The composition of claim 1, wherein the recombinant polymerase comprises one of the following combinations of substitutions and/or insertions:

a) N62D, L253A, E375Y, A484E and K512Y;

b) N62D, L253A, E375Y and K512Y;

c) N62D, H149M, T368F, E375Y, D510M, K512Y and D523M;

d) N62H, E375Y, A484E, E508R and K512Y;

e) D12R, N62H, T368F, E375Y, A484E and K512Y;

f) D12R, T368F, E375Y, A484E, E508R, 511.1K, 511.2S, 512.1G and 512.2S;

g) D12R, T368F, E375Y, I378W, A484E, E508R, 511.1K, 511.2S, 512.1G and 512.2S;

h) N62D, A190E, E375Y, K422A, A484E, E508R and K512Y;

i) N62D, I93Y, T368F, T372Y, E375Y, I378W, K478Y, A484E, E508R, 511.1K, 511.2S, K512Y, 512.1G and 512.2S;

j) N62D, T368F, E375Y, P477Q, A484E and K512Y;

k) N62D, T368F, E375Y, L384M, A484E and K512Y;

l) T368F, E375Y, P477E and K512Y;

m) A176V, T368F, E375Y and K512Y;

n) T368F, E375Y, K422R and K512Y;

o) N62D, E375Y, P477Q, A484E, K512Y;

p) Y148A, E375Y, A484E and K512Y;

q) L253A, E375Y, A484E and K512Y;

r) N62D, T368F, E375Y and K512Y;

s) T368F, E375Y and K512Y;

t) N62D, T368F, E375Y, A484E and K512Y;

u) I93F, T368F, E375Y, A484E and K512Y;

or

v) L253A, E375Y and K512Y.

21. The composition of claim 1, wherein the recombinant polymerase comprises one or more exogenous features at the C-terminal and/or N-terminal region of the polymerase.

30. The composition of claim 1, comprising a phosphate-labeled nucleotide analog and a DNA template, wherein the recombinant polymerase incorporates the nucleotide analog into a copy nucleic acid in response to the DNA template.

33. The composition of claim 1, wherein the composition is present in a DNA sequencing system that comprises a zero-mode waveguide, wherein the recombinant polymerase is immobilized on a surface of the zero-mode waveguide in an active form.

34. A method of sequencing a DNA template, the method comprising:

a) providing a reaction mixture comprising;

the DNA template,

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

the recombinant polymerase of claim 1, wherein the polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction, and

one or more nucleotides and/or nucleotide analogs;

b) subjecting the reaction mixture to a polymerization reaction in which the modified recombinant 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; and

c) identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting DNA.

13. (canceled)

16-18. -18. (canceled)

20. (canceled)

22-29. -29. (canceled)

31-32. -32. (canceled)

35-39. -39. (canceled)

40. A composition comprising a recombinant Φ

29-type DNA polymerase, which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position E239, an amino acid substitution at position Y224, an amino acid substitution at position Y148, E239G, Y224K, Y224Q, Y224R, Y148I , Y148A, Y148K, Y148F, Y148C, Y148D, Y148E, Y148G, Y148H, Y148K, Y148L, Y148M, Y148N, Y148P, Y148Q, Y148R, Y148S, Y148T, Y148V, Y148W, L253A, L253C, L253S, A484E, A484Q, A484N, A484D, A484K, E375T, E375C, E375G, E375N, K512I, K512M, K512C, K512G, K512N, K512Q, K512R, K512V, D510K, D510R, D510C, V250I, V250Q, V250L, V250C, V250F, V250N, V250R, V250T, V250Y, E515Q, E515D, E515H, E515Y, E515C, E515M, E515N, E515P, E515R, E515S, E515T, E515V, E515A, E508R, E508K, F526H, F526M, and F526Y, wherein identification of positions is relative to wild-type Φ

29 polymerase (SEQ ID NO;

1).

41-43. -43. (canceled)