Polymerase compositions and kits, and methods of using and making the same

Patent metadata

Field	Value
Publication number	US-11866740-B2
Application number	US-202117302621-A
Country	US
Kind code	B2
Filing date	May 7, 2021
Priority date	Oct 1, 2015
Publication date	Jan 9, 2024
Grant date	Jan 9, 2024

Abstract

The present disclosure provides compositions, methods, kits, systems and apparatus that are useful for nucleic acid polymerization. In particular, recombinant polymerases and biologically active fragments thereof are provided that allow for nucleic acid amplification. In some aspects, the disclosure provides recombinant polymerases that yield lower systematic error rates and/or improved accuracy, when used in sequencing by synthesis reactions as compared to a control polymerase. In one aspect, the disclosure relates to recombinant polymerases useful for nucleic acid sequencing, genotyping, copy number variation analysis, paired-end sequencing and other forms of genetic analysis. In another aspect, the recombinant polymerases are useful for the amplification of nucleic acid templates during PCR, emPCR, isothermal amplification, recombinase polymerase amplification, rolling circle amplification, strand displacement amplification and proximity ligation amplification. In some aspects, the disclosure relates to recombinant polymerases useful for the generation of nucleic acid libraries and/or nucleic acid templates.

First claim

What is claimed is:

1. A method for obtaining sequence information from a nucleic acid template, comprising:
(a) providing a reaction mixture comprising a template nucleic acid, one or more nucleotides, a sequencing primer and a recombinant polymerase;
(b) incorporating one or more nucleotides onto the end of the sequencing primer generating an extended primer product; and
(c) detecting the extended primer product by detecting an ion released in generation of the extended primer product;
wherein the recombinant polymerase comprises a polypeptide sequence selected from SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 121, wherein the method comprises template-dependent nucleic acid amplification, and wherein the method comprises amplifying one or more nucleic acids using a solid support system thereby clonally amplifying the nucleic acids on the solid support.

2. The method of claim 1 , wherein the ion is detected using an ion-sensitive field effect transistor (ISFET).

3. The method of claim 1 , wherein the ion released in generation of the extended primer product is a hydrogen ion.

4. The method of claim 1 , wherein the polypeptide sequence is SEQ ID NO: 117.

5. The method of claim 1 , wherein the polypeptide sequence is SEQ ID NO: 119.

6. The method of claim 1 , wherein the polypeptide sequence is SEQ ID NO: 121.

7. A method for performing a polymerization reaction, comprising:
a) contacting a recombinant polymerase with a nucleic acid template in the presence of one or more nucleotides; and
(b) polymerizing at least one of the one or more nucleotides using the recombinant polymerase;
wherein the recombinant polymerase comprises a polypeptide sequence selected from SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 121, wherein the method comprises template-dependent nucleic acid amplification, and wherein the method comprises amplifying one or more nucleic acids using a solid support system thereby clonally amplifying the nucleic acids on the solid support.

8. The method of claim 7 , wherein the polypeptide sequence is SEQ ID NO: 117.

9. The method of claim 7 , wherein the polypeptide sequence is SEQ ID NO: 119.

10. The method of claim 7 , wherein the polypeptide sequence is SEQ ID NO: 121.

11. The method of claim 7 , wherein amplification comprises polymerization selected from emulsion PCR, bridge PCR, PCR, qPCR, RT-PCR, isothermal amplification, recombinase polymerase amplification, rolling circle amplification, strand displacement amplification and proximity ligation amplification.

12. The method of claim 7 , wherein the method comprises isothermal amplification or emulsion PCR amplification of one or more nucleic acids using a solid support system thereby clonally amplifying the nucleic acids on the solid support, wherein the polypeptide sequence is SEQ ID NO: 117.

13. The method of claim 7 , wherein the method comprises isothermal amplification or emulsion PCR amplification of one or more nucleic acids using a solid support system thereby clonally amplifying the nucleic acids on the solid support, wherein the polypeptide sequence is SEQ ID NO: 119.

14. The method of claim 7 , wherein the method comprises isothermal amplification or emulsion PCR amplification of one or more nucleic acids using a solid support system thereby clonally amplifying the nucleic acids on the solid support, wherein the polypeptide sequence is SEQ ID NO: 121.

Assignees

• Life Technologies Corp

Inventors

• Mazur Daniel
• Vander Horn Peter B
• Tozer Eileen
• Chen Sihong
• Luo Guobin
• Shirley Joshua
• HEINEMANN Kevin

Classifications

• C12N9/1252 Primary

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

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Y207/07007

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

• C12Q2521/101

  DNA polymerase · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title