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Mutant DNA polymerase(s) with improved strand displacement ability

A polymerase, strand displacement technology, applied in recombinant DNA technology, enzymes, transferases, etc., can solve problems such as low efficiency of strand displacement DNA synthesis

Pending Publication Date: 2021-04-23
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many thermostable DNA polymerases exhibit rapid and sustained primer extension DNA synthesis, but strand displacement DNA synthesis is inefficient

Method used

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  • Mutant DNA polymerase(s) with improved strand displacement ability
  • Mutant DNA polymerase(s) with improved strand displacement ability
  • Mutant DNA polymerase(s) with improved strand displacement ability

Examples

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example

[0275] The following examples are provided to illustrate but not limit the claimed invention.

example 1

[0276] Example 1: Library Generation

[0277] Briefly, the steps in this screening process include library generation, expression and partial purification of mutant enzymes, screening of enzymes for desired properties, DNA sequencing, clone purification, and further characterization of selected candidate mutants. Each of these steps is further described below.

[0278] Clone library generation: Nucleic acid encoding the G46E mutation of C21 DNA polymerase using GeneMorph II for error-prone PCR TM Random mutagenesis kit (Agilent Technologies) was used to undergo error-prone (mutagenic) PCR. Using In-Fusion TM Cloning System (Takara Bio USA, Inc) to clone PCR fragments to create mutagenesis libraries. Cloned inserts were transformed into chemically competent LK4 cells. The library is then screened for increased strand displacement activity of the expressed mutant polymerase.

[0279] Assay design: Assay design in image 3 shown in . Four complementary oligonucleotides Oli...

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Abstract

Disclosed are DNA polymerases having increased 5'-3' strand displacement activity and substantially reduced 5'-3' exonuclease and endonuclease activity relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

Description

[0001] priority information [0002] This patent application claims priority to U.S. Provisional Patent Application No. 62 / 730,908, filed September 13, 2018, the contents of which are hereby incorporated by reference in their entirety for all purposes. The sequence listing written in the file SequenceListing_31934-US.txt of the 150,900-byte machine format IBM-PC, MS-Windows operating system created on August 29, 2018 is incorporated herein by reference in its entirety for all purposes. technical field [0003] The present invention provides DNA polymerases with improved activity, including increased 5'-3' strand displacement activity and significantly reduced 5'-3' exonuclease / endonuclease activity, and such polymerases in Use in a variety of applications including nucleic acid polynucleotide extension and amplification. Background technique [0004] DNA polymerase is responsible for the replication and maintenance of the genome, a role that is critical for the accurate tra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12Q1/6869C12N15/09
CPCC12N9/1252C12Q1/6844C12Y207/07007
Inventor F·库尔巴诺夫
Owner F HOFFMANN LA ROCHE & CO AG