Polymerase compositions, methods of making and using same

Patent metadata

Field	Value
Publication number	US-10240134-B2
Application number	US-201715481858-A
Country	US
Kind code	B2
Filing date	Apr 7, 2017
Priority date	Sep 30, 2013
Publication date	Mar 26, 2019
Grant date	Mar 26, 2019

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 are provided that allow for nucleic acid amplification. In some aspects, the disclosure provides modified polymerases having lower systematic error as compared to a reference polymerase. 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. 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 properties.

First claim

What is claimed is:

1. A composition comprising an isolated recombinant polypeptide comprising a polymerase polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 34 and maintaining substitutions H46R, E446Q, H572R, N487R, H281M, D264A, H273N, and E493R, wherein the isolated recombinant polypeptide has DNA polymerase activity.

2. The composition of claim 1 , wherein the isolated recombinant polypeptide has the amino acid sequence of SEQ ID NO: 34.

3. The composition of claim 1 , wherein the polymerase polypeptide has the amino acid sequence of SEQ ID NO: 34.

4. The composition of claim 1 , wherein the polymerase polypeptide has an amino acid sequence having at least 95% identity to SEQ ID NO: 34.

5. The composition of claim 1 , wherein the polymerase polypeptide has an amino acid sequence having at least 98% identity to SEQ ID NO: 34.

6. The composition of claim 1 , wherein the polymerase polypeptide has an amino acid sequence having at least 99% identity to SEQ ID NO: 34.

7. The composition of claim 1 , wherein the isolated recombinant polypeptide exhibits a decreased systematic error in a sequencing system that uses an ion-based DNA sequencing reaction as compared to LR2 which is a DNA polymerase having the amino acid sequence of SEQ ID NO: 2 further comprising mutations D264K and E493Q.

8. The composition of claim 1 , wherein the polymerase activity is measured as raw read accuracy, systematic error, strand bias, average read length, or total sequencing throughput.

9. A method for performing DNA polymerization comprising:
(a) providing a reaction mixture comprising the composition of claim 1 , and a nucleic acid hybridized to a primer; and
(b) contacting the nucleic acid with at least one type of nucleotide, wherein the contacting includes incorporating at least one nucleotide onto the primer thereby generating an extended primer product.

10. The method of claim 9 , wherein the polymerase polypeptide in the composition of claim 1 further includes one or more amino acid substitutions selected from the group consisting of: N485K, A263K, H528S, H528F, D423K, and D480R.

11. The method of claim 9 , wherein the method further includes detecting the presence of the extended primer product in the reaction mixture, thereby determining whether nucleotide incorporation has occurred.

12. The method of claim 11 , wherein the contacting and the detecting are repeated more than once, thereby detecting a plurality of nucleotide incorporations.

13. The method of claim 12 , wherein the method further includes identifying at least one of the plurality of nucleotide incorporations.

14. The method of claim 9 , wherein the polymerization method includes PCR, qPCR, bridge PCR, RT-PCR, ligation mediated PCR, isothermal amplification, or emulsion PCR.

15. A method for obtaining DNA sequence information from a nucleic acid template, comprising:
(a) providing a reaction mixture comprising the composition of claim 1 , and a template nucleic acid hybridized to a sequencing primer;
(b) contacting the template nucleic acid with at least one type of nucleotide, wherein the contacting includes incorporating one or more nucleotides from the at least one type of nucleotide onto the 3′ end of the sequencing primer and generating an extended primer product;
(c) detecting the presence of the extended primer product in the reaction mixture, thereby determining whether nucleotide incorporation has occurred; and
(d) identifying at least one of the one or more nucleotides incorporated from the at least one type of nucleotide.

16. The method of claim 15 , wherein the polymerase polypeptide in the composition of claim 1 further includes one or more amino acid substitutions selected from the group consisting of N485K, A263K, H528S, H528F, D423K, and D480R.

17. The method of claim 16 , wherein the contacting, detecting, and identifying steps are repeated more than once, thereby identifying a plurality of sequential nucleotide incorporations.

Assignees

• Life Technologies Corp

Inventors

• Vander Horn Peter
• Mazur Daniel
• Nikiforov Theo
• Landes Mindy
• Tozer Eileen

Classifications

• 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

• C12Q1/6869

  Methods for sequencing · CPC title