Generation of modified polymerases for improved accuracy in single molecule sequencing

• US 20110189659A1
• Filed: 09/30/2010
• Published: 08/04/2011
• 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: Bjornson, Keith, Lee, Harold, He, Molly, Park, Insil, Peluso, Paul, Clark, Sonya, Christians, Fred, Bibillo, Arek, Jia, Lei, Emig, Robin
• Granted Patent: US 8,420,366 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12N 11/00 Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
• CPC Class Codes: C12N 9/12 transferring phosphorus containing groups, e.g. kinases (2.7)
• 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 1/68 involving nucleic acids
• 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
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions and/or heterologous or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include reduced reaction rates at one or more steps of the polymerase kinetic cycle, increased closed polymerase/DNA complex stability, enhanced metal ion coordination, reduced exonuclease activity, decreased branching fractions, and the like. 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, are also provided. 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 DNA polymerase homologous to a Φ

29 DNA polymerase, which recombinant DNA polymerase comprises a mutation or combination of mutations relative to a wild-type Φ

29 DNA polymerase (SEQ ID NO;

1) selected from;

(a) an L253 mutation, wherein the polymerase further comprises a mutation at one or more of;

T368, E375, A484, or K512;

(b) an E375 and K512 mutation, wherein the polymerase further comprises a mutation at one or more of;

L253, T368 or A484;

(c) an I93 mutation;

(d) an S215 mutation;

(e) an E420 mutation;

(f) a P477 mutation;

(g) a D66R mutation;

(h) a K135R mutation;

(i) a K138R mutation;

(j) an L253T mutation;

(k) a Y369G mutation;

(l) a Y369L mutation;

(m) an L384M mutation;

(n) a K422A mutation;

(o) an I504R mutation;

(p) an E508K mutation;

(q) an E508R mutation;

(r) a D510K mutation;

(s) at least one mutation or combination of mutations selected from those listed in Tables 6, 9 and 10; and

(t) a T368 mutation, wherein the polymerase further comprises a mutation at one or more of;

E375 or K512.

2. The composition of claim 1, wherein the polymerase comprises a mutation that inhibits exonuclease activity of the polymerase.

3. The composition of claim 2, wherein the mutation that inhibits exonuclease activity is selected from:

an N62 mutation, a D12 mutation, a D66 mutation, and a T15 mutation.

4. The composition of claim 1, wherein the polymerase is the polymerase of (a) and comprises an E375Y and a K512Y mutation.

5. The composition of claim 1, wherein the polymerase is the polymerase of any of (c)-(r) and further comprises a mutation at one or more of:

T368, E375, A484, or K512.

6. The composition of claim 1, wherein the polymerase is the polymerase of (c) and comprises a mutation selected from I93F and I93Y.

7. The composition of claim 1, wherein the polymerase is the polymerase of (d) and comprises an S215D mutation.

8. The composition of claim 1, wherein the polymerase is the polymerase of (e) and comprises an E420M mutation.

9. The composition of claim 1, wherein the polymerase is the polymerase of (f) and comprises a mutation selected from P477E and P477Q.

10. The composition of claim 1, wherein the polymerase is the polymerase of (t) and comprises E375Y and K512Y mutations.

11. The composition of claim 1, wherein the polymerase is the polymerase of (t) and comprises E375Y, A484E and K512Y mutations.

12. The composition of claim 1, wherein the polymerase comprises one or more amino acid insertions between positions 507 and 508 as compared to SEQ ID NO:

1.

13. The composition of claim 1, wherein the polymerase comprises one or more amino acid insertions between positions 511 and 512 as compared to SEQ ID NO:

1.

14. The composition of claim 1, wherein the polymerase comprises one or more amino acid insertions between positions 512 and 513 as compared to SEQ ID NO:

1.

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

16. The composition of claim 15, wherein the one or more exogenous or heterologous features is selected from:

a polyhistidine tag, a HIS-10 tag, a HIS-6 tag, an alanine tag, an Ala10 tag, an Ala 16 tag, a biotinylation tag, a GST tag, a BiTag, an S Tag, a SNAP-tag, an HA tag, a DSB (Sso7D) tag, a lysine tag, a NanoTag, a Cmyc tag, a tag or linker comprising the amino acids glycine and serine, a tag or linker comprising the amino acids glycine, serine, alanine and histidine, a tag or linker comprising the amino acids glycine, arginine, lysine, glutamine and proline, a plurality of polyhistidine tags, a plurality of HIS-10 tags, a plurality of HIS-6 tags, a plurality of alanine tags, a plurality of Ala10 tags, a plurality of Ala16 tags, a plurality of biotinylation tags, a plurality of GST tags, a plurality of BiTags, a plurality of S Tags, a plurality of SNAP-tags, a plurality of HA tags, a plurality of DSB (Sso7D) tags, a plurality of lysine tags, a plurality of NanoTags, a plurality of Cmyc tags, a plurality of tags or linkers comprising the amino acids glycine and serine, a plurality of tags or linkers comprising the amino acids glycine, serine, alanine and histidine, a plurality of tags or linkers comprising the amino acids glycine, arginine, lysine, glutamine and proline, biotin, avidin, one or more Factor Xa sites, one or more enterokinase sites, thrombin sites, antibodies or antibody domains, antibody fragments, antigens, receptors, receptor domains, receptor fragments, ligands, dyes, acceptors, quenchers, or combinations thereof.

17. The composition of claim 15, wherein the polymerase comprises one or more exogenous or heterologous features at both the C-terminal and N-terminal regions of the polymerase.

18. The composition of claim 17, wherein the one or more exogenous or heterologous features at the C-terminal and N-terminal region are the same.

19. The composition of claim 18, wherein the one or more exogenous or heterologous features at the C-terminal and N-terminal region comprise a polyhistidine tag.

20. The composition of claim 19, wherein the polyhistidine tag comprises a His-10 tag.

21. The composition of claim 17, wherein the polymerase comprises a Btag and a polyhistidine tag.

22. The composition of claim 21, wherein the polymerase comprises a Factor Xa site.

23. The composition of claim 1, comprising a phosphate-labeled nucleotide analog.

24. The composition of claim 23, wherein the nucleotide analog comprises a fluorophore.

25. The composition of claim 23, wherein the nucleotide analog comprises 4 or more phosphate groups.

26. The composition of claim 1, 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.

27. The composition of claim 1, wherein the composition is present in a DNA sequencing system.

28. The composition of claim 27, wherein the sequencing system comprises a zero-mode waveguide.

29. The composition of claim 28, wherein the modified recombinant polymerase is immobilized on a surface of the zero-mode waveguide in an active form.

33. 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 DNA 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.

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 recombinant DNA polymerase of claim 1, 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.

30-32. -32. (canceled)

34. (canceled)

36-160. -160. (canceled)