DNA polymerase variants with reduced exonuclease activity and uses thereof

• US 8,921,043 B2
• Filed: 05/10/2012
• Issued: 12/30/2014
• Est. Priority Date: 05/11/2011
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Gardner, Andrew
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): LACOURCIERE, GERARD MICHAEL
• Application Number:
  US13/822,724
• Publication Number:
  US 20130196327A1
• Time in Patent Office: 964 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

Compositions and methods are described to modify Family B DNA polymerases that contain residual exonuclease activity that interferes with sequencing techniques and with detection of single nucleotide polymorphisms. The compositions are mutant proteins with reduced exonuclease activity compared with presently available “exo⁻” polymerases, and a sensitive screening assay that enables an assessment of exonuclease activity of any synthetic DNA polymerase.

Claims

1. A variant of a parent polymerase wherein the parent polymerase has at least 90% sequence homology with SEQ ID NO:

1 and/or SEQ ID NO;

2, and wherein a difference between the parent polymerase and the variant comprises an amino acid mutation in SEQ ID NO;

5 at position 5 and at least one amino acid mutation in at least one amino acid sequence selected from SEQ ID NOS;

3, 4, 6 and 8.

2. A variant according to claim 1, wherein the difference further comprises at least two amino acid mutations in SEQ ID NO:

3.

3. A variant according to claim 1, wherein the difference further comprises a ratio of exonuclease activity/polymerase activity of less than 1.5×

10<sup>−

6</sup>.

4. A variant according to claim 1, having an exonuclease activity of less than 0.01 units/mg.

5. A variant according to claim 1, wherein the parent polymerase has an amino acid sequence with at least 90% sequence homology with SEQ ID NOS:

3-7.

6. A variant according to claim 1, wherein the at least one mutation in SEQ ID NO:

5 corresponds to position 315 in SEQ ID NO;

1 where the mutated amino acid at that position is not Asp(D).

7. A variant according to claim 1, having an amino acid mutation in SEQ ID NO:

5 and a plurality of amino acid mutations in SEQ ID NO;

3.

8. A method of amplifying DNA in the absence of exonuclease activity, comprising:

combining a variant of a parent polymerase according to claim 1 with a template DNA and a primer; and

amplifying the DNA.

9. A method of sequencing a polynucleotide, comprising:

combining a variant of a parent polymerase according to claim 1 with a template polynucleotide and at least one primer to form a hybridized polynucleotide;

permitting the variant polymerase to incorporate into the template-primer hybrid, a modified nucleotide that is complementary to a nucleoside at the corresponding position on the template; and

identifying the nucleoside at the corresponding position on the template.