Polymerase compositions, methods of making and using same

Patent metadata

Field	Value
Publication number	US-9976128-B2
Application number	US-201514754203-A
Country	US
Kind code	B2
Filing date	Jun 29, 2015
Priority date	Aug 10, 2011
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 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.

First claim

The invention claimed is:

1. A modified Bst DNA polymerase consisting of the amino acid sequence of SEQ ID NO: 2 having a E493Q amino acid substitution and any one of D264K, D264Q or D264S amino acid substitutions, wherein the numbering is relative to the amino acid sequence of SEQ ID NO: 2, and wherein the modified Bst DNA polymerase exhibits improved accuracy in a sequencing by synthesis reaction compared to the DNA polymerase of SEQ ID NO: 2 and/or exhibits an increased dissociation time constant compared to the DNA polymerase of SEQ ID NO: 2.

2. The modified Bst DNA polymerase of claim 1 , wherein the modified Bst DNA polymerase has the D264K amino acid substitution.

3. The modified Bst DNA polymerase of claim 1 , wherein the modified Bst DNA polymerase has the D264S amino acid substitution.

4. The modified Bst DNA polymerase of claim 1 , wherein the modified Bst DNA polymerase has the D264Q amino acid substitution.

5. A method for performing a nucleotide polymerization reaction, comprising:
contacting the modified Bst DNA polymerase of claim 1 with a nucleic acid template in the presence of one or more nucleotides,
polymerizing at least one of the one or more nucleotides using the modified Bst DNA polymerase, and
detecting a signal indicating the polymerization of the at least one of the one or more nucleotides by the modified Bst DNA polymerase using a sensor.

6. The method of claim 5 , wherein the modified Bst DNA polymerase exhibits improved accuracy in a sequencing by synthesis reaction compared to SEQ ID NO:2.

7. The method of claim 5 , wherein the sensor is an ISFET.

8. The method of claim 5 , wherein the sensor detects a hydrogen ion.

9. The method of claim 5 , wherein the method further comprises determining the identity of the one or more nucleotides polymerized by the modified polymerase.

10. The method of claim 5 , wherein the polymerizing occurs in the presence of 200 mM to 300 mM salt.

11. A method for performing nucleic acid amplification, comprising:
generating an amplification reaction mixture comprising the modified Bst DNA polymerase of claim 1 , a primer, a nucleic acid template, and one or more nucleotides,
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.

12. The method of claim 11 , wherein the amplifying conditions comprise 125 mM to 400 mM salt.

13. The method of claim 12 , wherein the amplifying conditions comprise 200 mM to 300 mM salt.

14. The method of claim 12 , wherein the salt comprises NaCl and/or KCl.

15. The method of claim 11 , wherein the modified Bst DNA polymerase exhibits improved accuracy in a sequencing by synthesis reaction compared to SEQ ID NO:2.

Assignees

• Life Technologies Corp

Inventors

• Vander Horn Peter
• Nikiforov Theo
• Luo Guobin
• Landes Mindy
• Mazur Daniel
• Tozer Eileen
• Lincecum Tommie

Classifications

• C12Y207/07007

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

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12N9/1252 Primary

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

• C12Q1/6846 Primary

  Common amplification features · CPC title