Polymerase compositions and methods of making and using same

Patent metadata

Field	Value
Publication number	US-9976178-B2
Application number	US-201514970818-A
Country	US
Kind code	B2
Filing date	Dec 16, 2015
Priority date	Dec 16, 2014
Publication date	May 22, 2018
Grant date	May 22, 2018

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 fragments thereof, such as modified Taq polymerases, are provided that allow for improved nucleic acid amplification. In some aspects, the disclosure provides modified polymerases having improved thermostability, accuracy, processivity and/or read length as compared to a reference Taq polymerase. In some aspects, the disclosure relates to modified polymerases or biologically active fragments thereof, useful for amplification methods, and in practically illustrative embodiments, emulsion PCR.

First claim

What is claimed is:

1. A method for amplifying a nucleic acid, comprising contacting the nucleic acid with a modified polymerase, under suitable conditions for amplifying the nucleic acid, and amplifying the nucleic acid, wherein the modified polymerase consists of the amino acid sequence of SEQ ID NO: 1 having a single amino acid substitution selected from the group consisting of E397V, G418C, E745T, and E805I, wherein the modified polymerase exhibits polymerase activity and exhibits an improvement in thermostability relative to the reference polymerase of SEQ ID NO: 34.

2. A method according to claim 1 , wherein the modified polymerase has a G418C amino acid substitution.

3. The method of claim 1 , wherein the suitable conditions comprise suitable conditions for performing a polymerase chain reaction.

4. The method of claim 1 , wherein the suitable conditions comprise suitable conditions for performing an emulsion polymerase chain reaction.

5. The method of claim 1 , wherein the amplifying is clonally amplifying the nucleic acid in solution or on a solid support.

6. The method of claim 1 , further comprising determining the nucleic acid sequence of at least a portion of the nucleic acid.

7. The method of claim 1 , wherein the modified polymerase has an E805I amino acid substitution.

8. The method of claim 1 , wherein the modified polymerase has an E745T amino acid substitution.

9. A method for amplifying a nucleic acid, comprising contacting the nucleic acid with a modified polymerase, under suitable conditions for amplifying the nucleic acid, and amplifying the nucleic acid, wherein the modified polymerase consists of the amino acid sequence of SEQ ID NO: 1 having an E397V amino acid substitution, wherein the modified polymerase exhibits polymerase activity and exhibits an improvement in thermostability relative to the reference polymerase of SEQ ID NO: 34.

10. The method of claim 9 , wherein the suitable conditions comprise suitable conditions for performing a polymerase chain reaction.

11. The method of claim 9 , wherein the suitable conditions comprise suitable conditions for performing an emulsion polymerase chain reaction.

12. The method of claim 9 , wherein the amplifying is clonally amplifying the nucleic acid in solution or on a solid support.

13. The method of claim 9 , further comprising determining the nucleic acid sequence of at least a portion of the nucleic acid.

14. A method for amplifying a nucleic acid, comprising contacting the nucleic acid with a modified polymerase, under suitable conditions for amplifying the nucleic acid, and amplifying the nucleic acid, wherein the modified polymerase consists of the amino acid sequence of SEQ ID NO: 1 having E397V and E805I amino acid substitutions, wherein the modified polymerase exhibits polymerase activity and exhibits an improvement in thermostability relative to the reference polymerase of SEQ ID NO: 34.

15. The method of claim 14 , wherein the suitable conditions comprise suitable conditions for performing a polymerase chain reaction.

16. The method of claim 14 , wherein the suitable conditions comprise suitable conditions for performing an emulsion polymerase chain reaction.

17. The method of claim 14 , wherein the amplifying is clonally amplifying the nucleic acid in solution or on a solid support.

18. The method of claim 14 , further comprising determining the nucleic acid sequence of at least a portion of the nucleic acid.

Assignees

• Life Technologies Corp

Inventors

• Mazur Daniel
• Tozer Eileen
• Chen Sihong
• Vander Horn Peter
• Lincecum Tommie

Classifications

• C12N9/1252

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q2521/101

  DNA polymerase · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title