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Method

A polynucleotide and substrate technology, applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., can solve expensive, slow and other problems

Active Publication Date: 2019-01-15
OXFORD NANOPORE TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Existing techniques are slow and expensive, mainly because they rely on amplification techniques to generate large quantities of polynucleotides and require large quantities of specialized fluorescent chemicals for signal detection

Method used

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Embodiment 1

[0536] MuA binds to transposons in a tetramer that is very stable; it remains tightly bound after strand transfer of the transposon. If MuA is not removed from the DNA, this can be characterized using nanopore system inhibition. MuA can be removed by heating to 75°C. However, this relies on the use of a thermal cycler or water bath and may damage other components in the solution. Here, we describe an alternative technique for the removal of MuA using a helicase without the need for a heated reaction. Hel308Mbu-E284C / S615C-STrEP (SEQ ID NO: 10, with mutations E284C / S615C, with a streptavidin tag attached to its C-terminus) is a progressive helicase that binds to single-stranded DNA and 3' to 5' direction movement. When the transposon has a 3' overhang on the bottom strand, Hel308Mbu-E284C / S615C-STrEP (SEQ ID NO: 10, with the mutation E284C / S615C attached to its C-terminus with a streptavidin tag) can bind And while moving along the DNA, the MuA complex is forced to separate...

Embodiment 2

[0560] This example describes the removal of MuA transposase using a number of different translocases.

[0561] Materials and methods

[0562] adapter annealing

[0563] MuA adapters consisting of SEQ ID NO: 117 and 121 were annealed to 10 μΜ in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl at 2°C / min from 95°C to 22°C. This adapter contains a minimal MuA recognition sequence with a pre-formed 5' bottom strand flap, and a 12nt 5' tail on the top strand and a 10nt 3' tail on the bottom strand.

[0564] transposome formation

[0565] Form transposome complexes, but add 1 μl MuA adapter, 4.5 μl nuclease-free water, 2 μl of 5x transposon buffer (125 mM Tris pH 8, 550 mM NaCl, 2.5 mM EDTA, 50% glycerol, 0.25% Triton-X100 ) and 2.5ul concentrated MuA transposase (Thermofisher). The mixture was then incubated at 30°C for 1.5 hours.

[0566] Transposition

[0567] Then a transposition reaction containing 10 μl of 5x transposase buffer (125 mM Tris pH8, 550 mM NaCl, 50 mM MgCl2), 5 μl of...

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Abstract

The invention relates to a method for modifying a template double stranded polynucleotide, especially for characterisation using nanopore sequencing. The method produces from the template a pluralityof modified double stranded polynucleotides. These modified polynucleotides can then be characterised.

Description

technical field [0001] The present invention relates to methods of modifying template double-stranded polynucleotides, particularly for characterization using nanopore sequencing. Background technique [0002] There are many commercial situations that require the preparation of nucleic acid libraries. This is usually accomplished using transposases. Depending on the transposase used to prepare the library, it may be desirable that transposition events can be repaired in vitro prior to use of the library, for example in sequencing. [0003] There is currently a need for rapid and inexpensive nucleic acid (eg, DNA or RNA) sequencing and identification technologies across a wide range of applications. Existing techniques are slow and expensive, mainly because they rely on amplification techniques to generate large quantities of polynucleotides and require large quantities of specialized fluorescent chemicals for signal detection. [0004] Transmembrane pores (nanopores) hold...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2521/513C12Q2525/155C12Q2525/191C12Q2535/122C12Q2565/631C12Q2521/10C12Q2522/101C12Q1/6869
Inventor 戴维·杰克逊·斯托达特詹姆斯·怀特
Owner OXFORD NANOPORE TECH LTD