Modified DNA polymerases for improved amplification

• US 9,315,787 B2
• Filed: 01/13/2012
• Issued: 04/19/2016
• Est. Priority Date: 01/14/2011
• Status: Active Grant

• Current Assignee: Kapa Biosystems (Roche Holding AG)
• Original Assignee: Kapa Biosystems, Inc. (Roche Holding AG)
• Inventors: McEwan, Paul, Foskett, John, Bourn, William, Schafer, Wolfgang, Van Der Walt, Eric
• Primary Examiner(s): Hutson, Richard
• Application Number:
  US13/979,509
• Publication Number:
  US 20140030765A1
• Time in Patent Office: 1,558 Days
• Field of Search: None
• US Class Current: 1/1
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The present invention provides improved DNA polymerases that may be better suited for applications in recombinant DNA technologies, in particular technologies involving plant-derived samples. Among other things, the present invention provides modified DNA polymerases derived from directed evolution experiments designed to select mutations that confer advantageous phenotypes under conditions used in industrial or research applications.

Claims

1. A DNA polymerase whose amino acid sequence is at least 95% identical to that of SEQ ID NO:

38, but differs from SEQ ID NO;

38 at a position corresponding to F749 of SEQ ID NO;

38 and furthermore differs from SEQ ID NO;

38 by at least two additional amino acids at positions corresponding to at least two of A61, K346, 5357, E507 or I707 of SEQ ID NO;

38.

2. The DNA polymerase of claim 1, wherein the amino acid at a position corresponding to F749 of SEQ ID NO:

38, is selected from the group consisting of L, I, V, T, Y, and M.

3. The DNA polymerase of claim 1, wherein the amino acid at a position corresponding to 749 of SEQ ID NO:

38 is L, and the at least two additional amino acids correspond to amino acids in SEQ ID NO;

38, but are substitutions relative to SEQ ID NO;

38 and are selected from the group consisting of;

A61T, K346E, S357C, E507K, I707M and combinations thereof.

4. The DNA polymerase of claim 3, wherein the DNA polymerase has increased resistance to a plant-derived PCR inhibitor as compared to that of SEQ ID NO:

38.

12. The DNA polymerase of claim 1, whose amino acid sequence is at least 96% identical to that of SEQ ID NO:

38.

13. The DNA polymerase of claim 1, whose amino acid sequence is at least 97% identical to that of SEQ ID NO:

38.

14. The DNA polymerase of claim 1, whose amino acid sequence is at least 98% identical to that of SEQ ID NO:

38.

15. The DNA polymerase of claim 3, wherein the DNA polymerase has increased resistance to salt relative to that of SEQ ID NO:

38.

5. A formulation of DNA polymerases comprising:

(i) a DNA polymerase whose amino acid sequence is at least 95% identical to that of SEQ ID NO;

38, but differs from SEQ ID NO;

38 by an amino acid at a position corresponding to F749 of SEQ ID NO;

38; and

furthermore differs from SEQ ID NO;

38 by at least two additional amino acids at positions corresponding to at least two of A61, K346, S357, E507 or I707 of SEQ ID NO;

38; and

(ii) at least one DNA polymerase exhibiting 3′

-exonuclease activity.

6. The formulation of claim 5, wherein the DNA polymerase and the at least one DNA polymerase exhibiting 3′

-exonuclease activity are present in a ratio of about 1;

1 to about 1;

2000 relative units of enzyme.

7. A method comprising a step of amplifying nucleic acids in a biological sample using:

(i) a DNA polymerase whose amino acid sequence is at least 95% identical to that of SEQ ID NO;

38, but differs from SEQ ID NO;

38 by an amino acid at a position corresponding to F749 of SEQ ID NO;

38; and

furthermore differs from SEQ ID NO;

38 by at least two additional amino acids at positions corresponding to at least two of A61, K346, S357, E507, or I707 of SEQ ID NO;

38; and

(ii) at least one additional DNA polymerase, wherein the least one additional DNA polymerase has 3′

-exonuclease activity.

8. The method of claim 7, wherein the biological sample includes purified DNA or a crude DNA extract.

9. The method of claim 7, wherein the biological sample is a plant sample.

10. The method of claim 9, wherein the plant sample is selected from the group consisting of:

leaf tissue, seed tissue, plant tissue, organ tissue, stored plant sample, crude plant DNA extract, and combinations thereof.

11. The method of claim 7, wherein the DNA polymerase and the at least one additional DNA polymerase are present in a ratio of about 1:

1 to about 1;

2000 relative units of enzyme.

16. A DNA polymerase whose amino acid sequence is that of SEQ ID NO:

47.

17. A DNA polymerase whose amino acid sequence is that of SEQ ID NO:

48.

18. A DNA polymerase whose amino acid sequence is at least 98% identical to that of SEQ ID NO:

47, including in that it has I at a position corresponding to F749 of SEQ ID NO;

38, and in that it has at least two other amino acid substitutions at positions relative to wild type DNA polymerase of SEQ ID NO;

38, which positions are selected from the group consisting of 61, 346, 357, 507, 707, and combinations thereof.

19. A DNA polymerase whose amino acid sequence is at least 98% identical to that of SEQ ID NO:

48, including in that it has L at a position corresponding to F749 of SEQ ID NO;

38 and in that it has at least two other amino acid substitutions at positions relative to wild type DNA polymerase of SEQ ID NO;

38, which positions are selected from the group consisting of 61, 346, 357, 507, 707 and combinations thereof.