Polymerase compositions, methods of making and using same

• US 10,633,641 B2
• Filed: 04/23/2018
• Issued: 04/28/2020
• Est. Priority Date: 08/10/2011
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Vander Horn, Peter, Nikiforov, Theo, Luo, Guobin, Landes, Mindy, Mazur, Daniel, Tozer, Eileen, Lincecum, Tommie Lloyd
• Primary Examiner(s): Hutson, Richard G
• Application Number:
  US15/959,839
• Publication Number:
  US 20180305673A1
• Time in Patent Office: 736 Days
• Field of Search: None
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6846 Common amplification features
• 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

Abstract

The present disclosure provides compositions, methods, kits, systems and apparatus that are useful for nucleic acid polymerization. In particular, modified polymerases and biologically active fragment thereof are provided that allow for nucleic acid amplification. In one aspect, the disclosure relates to modified polymerases useful for nucleic acid sequencing, genotyping, copy number variation analysis, paired-end sequencing and other forms of genetic analysis. In some aspects, the disclosure relates to modified polymerases useful for the generation of nucleic acid libraries or nucleic acid templates for use in various downstream processes. In some aspects, the disclosure relates to the identification of homologous amino acid mutations that can be transferred across classes or families of polymerases to provide novel polymerases with altered catalytic properties. In some aspects, the disclosure provides modified polymerases having enhanced catalytic properties as compared to a reference polymerase.

Claims

1. A modified polymerase consisting of the amino acid sequence of SEQ ID NO:

15 having any one of E471K, D732R, or E745Q amino acid substitutions, wherein the numbering is relative to the amino acid sequence of SEQ ID NO;

15.

2. The modified polymerase of claim 1, wherein the modified polymerase has the D732R substitution.

3. The modified polymerase of claim 1, wherein the modified polymerase has the E745Q substitution.

4. The modified polymerase of claim 1, wherein the modified polymerase has the E471K substitution.

5. A method for performing nucleic acid amplification, comprising:

generating an amplification reaction mixture having a modified polymerase, a primer, a nucleic acid template, and one or more nucleotides, wherein the modified polymerase consists of the amino acid sequence of SEQ ID NO;

15 having any one of E471K, D732R, or E745Q amino acid substitutions, wherein the numbering is relative to the amino acid sequence of SEQ ID NO;

15; and

subjecting the amplification reaction mixture to amplifying conditions, wherein at least one of the one or more nucleotides is polymerized onto the end of the primer using the modified polymerase to generate amplification products.

6. The method of claim 5, wherein the amplifying conditions include the presence of a high ionic strength solution.

7. The method of claim 6, wherein said high ionic strength solution comprises 100 mM to 500 mM salt.

8. The method of claim 7, wherein the salt comprises NaCl and/or KCl.

9. The method of claim 5, wherein the modified polymerase has the E471K substitution.

10. The method of claim 5, wherein the modified polymerase has the E745Q substitution.

11. The method of claim 5, wherein the modified polymerase has the D732R substitution.

12. The method of claim 11, wherein the amplifying conditions include the presence of 100 mM to 200 mM salt.

13. The method of claim 5, wherein the amplifying conditions include amplification of the nucleic acid template by a polymerase chain reaction.

14. The method of claim 5, wherein the amplifying conditions include amplification of the nucleic acid template by a clonal amplification reaction.

15. The method of claim 5, wherein the amplifying conditions include amplification of the nucleic acid template by an emulsion polymerase chain reaction.

16. The method of claim 15, wherein the method further comprises subjecting the amplification products to a nucleic acid sequencing reaction.

17. The method of claim 5, wherein the amplifying conditions include amplification of the nucleic acid template onto a solid support.

18. The method of claim 17, wherein the method is performed on nucleic acid templates comprising the nucleic acid template to generate a nucleic acid library.

19. The method of claim 18, wherein the method further comprises subjecting the nucleic acid library to a nucleic acid sequencing reaction.

20. The method of claim 19, wherein the modified polymerase has the D732R substitution.