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Mutant pores

a technology of msp and pores, applied in the field of msp mutant forms, can solve the problems of slow and expensive technology, and achieve the effect of reducing the net negative charg

Active Publication Date: 2017-12-14
OXFORD NANOPORE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a modification to a protein structure that reduces the negative charge of certain amino acids. This modification may improve the function of the protein.

Problems solved by technology

Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of polynucleotide and require a high quantity of specialist fluorescent chemicals for signal detection.

Method used

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Examples

Experimental program
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Effect test

example 1

[0345]This Example describes the use of a helicase—T4 Dda—E94C / C109A / C136A / A360C (SEQ ID NO: 24 with mutations E94C / C109A / C136A / A360C) to control the movement of DNA construct X (shown in FIG. 1) through a number of different MspA nanopores. All of the nanopores tested exhibited changes in current as the DNA translocated through the nanopore.

Materials and Methods

[0346]Prior to setting up the experiment, DNA construct X or Y (see FIGS. 1 and 2 respectively for diagram and sequences used in constructs X and Y, final concentration added to the nanopore system 0.1 nM) was pre-incubated at room temperature for five minutes with T4 Dda—E94C / C109A / C136A / A360C (final concentration added to the nanopore system 10 nM, SEQ ID NO: 24 with mutations E94C / A360C which was provided in buffer (253 mM KCl, 50 mM potassium phosphate, pH 8.0, 2 mM EDTA)). After five minutes, TMAD (100 μM final concentration added to the nanopore system) was added to the pre-mix and the mixture incubated for a further 5...

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Abstract

The invention relates to mutant forms of Msp. The invention also relates to polynucleotide characterisation using Msp.

Description

FIELD OF THE INVENTION[0001]The invention relates to mutant forms of Msp. The invention also relates to polynucleotide characterisation using Msp.BACKGROUND OF THE INVENTION[0002]There is currently a need for rapid and cheap polynucleotide (e.g. DNA or RNA) sequencing and identification technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of polynucleotide and require a high quantity of specialist fluorescent chemicals for signal detection.[0003]Transmembrane pores (nanopores) have great potential as direct, electrical biosensors for polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as a potential DNA sequencing technology.[0004]When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time. Nan...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07K14/35
CPCC12Q1/6869C07K14/35C12Q2565/607C12Q2565/631C12Q2522/101C12Q2521/513C12Q2563/157
Inventor CLARKE, JAMES ANTHONYHERON, ANDREW JOHNJAYASINGHE, LAKMALWALLACE, ELIZABETH JAYNE
Owner OXFORD NANOPORE TECH LTD