DNA polymerase variants with reduced exonuclease activity and uses thereof

• US 8,921,044 B2
• Filed: 04/08/2014
• 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:
  US14/247,760
• Publication Number:
  US 20140296082A1
• Time in Patent Office: 266 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase

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

(i) the parent polymerase has at least 90% sequence homology with SEQ ID NO:

1 and/or SEQ ID NO;

2,

(ii) 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; and

(iii) wherein the variant is characterized by one or more properties selected from;

reducing average phasing by 1%-51% compared to the parent polymerase;

increasing average sequencing read quality by 7%-75% compared to parent enzyme;

increasing average sequencing read length by 1%-200% and increasing average full length sequencing reads by 25%-200%.

2. A variant according to claim 1 wherein the variant is characterized by one or more properties selected from:

reducing average phasing by at least 1%-10% compared to the parent polymerase;

increasing average sequencing read quality by at least 10%-50% compared to parent enzyme;

increasing average sequencing read length by at least 5%-20% and increasing average full length sequencing reads by at least 25%-100%.

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

combining 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, with a template DNA and a primer; and

amplifying the DNA.

4. A method of sequencing a polynucleotide, comprising:

(a) combining 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 with a template polynucleotide and at least one primer to form a hybridized polynucleotide;

(b) 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;

(c) identifying the nucleoside at the corresponding position on the template; and

(d) optionally repeating (a) through (c).

5. A method according to claim 4 wherein the variant is characterized by of one or more properties selected from:

reducing average phasing by 1%-51% compared to the parent polymerase;

increasing average sequencing read quality by 7%-75% compared to parent enzyme;

increasing average sequencing read length by 1%-200% and increasing average full length sequencing reads by 25%-200%.

6. A method according to claim 4, wherein the variant is characterized by one or more properties selected from:

reducing average phasing by at least 1%-10% compared to the parent polymerase;

increasing average sequencing read quality by at least 10%-50% compared to parent enzyme;

increasing average sequencing read length by at least 5%-20% and increasing average full length sequencing reads by at least 25%-100%.

7. A method according to claim 4 wherein (d) further comprises at least one of:

reducing average phasing by 1%-51% compared to the parent polymerase;

increasing average sequencing read quality by 7%-75% compared to parent enzyme;

increasing average sequencing read length by 1%-200% or increasing average full length sequencing reads by 25%-200%.

8. A method according to claim 4 wherein (d) further comprises at least one of:

reducing average phasing by at least 1%-10% compared to the parent polymerase;

increasing average sequencing read quality by at least 10%-50% compared to parent enzyme;

increasing average sequencing read length by at least 5%-20% and increasing average full length sequencing reads by at least 25%-100%.