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Molecular conformation change-based sequence measuring method

A molecular conformation and molecular motor technology, applied in the field of sequencing based on molecular conformation changes, can solve problems such as low signal-to-noise ratio and short read length

Inactive Publication Date: 2015-11-25
CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods have their own advantages, but are limited by low signal-to-noise ratios and short read lengths

Method used

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  • Molecular conformation change-based sequence measuring method
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  • Molecular conformation change-based sequence measuring method

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Embodiment 1

[0025] The preparation of embodiment 1 nanopore:

[0026] Press as figure 1 The nanopore preparation process is carried out:

[0027] 1) Provide a silicon substrate, grow a monoatomic layer film 2 with a thickness of 0.2 nm above the silicon substrate 1, and then grow electrically insulating film layers 3 and 4 with a film thickness of 30 nm on the upper and lower sides;

[0028] 2) A photolithographic etching process is used to etch a trapezoidal opening 5 in the electrical insulating film layer 4 and the silicon substrate 1, and a hole 6 with a diameter of 5 nm is etched in the electrical insulating film layer 3 by electron beam lithography. The breakdown method etches a nanopore 7 with a diameter of 3 nm in the monoatomic thin film layer 2;

[0029] 3) Forming biological streptomycin on the surface of well 6 by biotin modification method.

Embodiment 2

[0030] Example 2 Detection of sequences by conformational changes of molecular motors

[0031] Such as figure 2 Schematic diagram of the sequence detected by the conformational change of the molecular motor shown, figure 2 Among them, 1 represents the graphene film layer, 3 represents the silicon nitride film layer, 6 represents the hole etched in the silicon nitride film layer 3, 7 represents the nanopore etched in the graphene film layer, and 8 represents the hole used for fixing The chemical layer of the molecular motor, 9 represents the two stages in the circuit, 10 represents the voltage source that provides the potential, and 11 represents the ammeter used to detect the change in current;

[0032] a is the molecular motor phi29 DNA polymerase, b is the double-stranded part of the DNA molecular template, and c is the single-stranded part of the DNA molecular template. Molecular motor a forms a synthesis with DNA and moves one base at a time. The synthesis is at an exte...

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Abstract

The invention relates to molecular conformation change-based sequence measuring method which can be used for detecting sequences of DNA, RNR, polypeptides or proteins. The method for detecting the sequence of the DNA comprises the following steps: connecting a graphene nanopore device into a circuit; fixing a molecular motor above or in each graphene nanopore; forming a compound body by the molecular motor and DNA template molecules; adding a solution containing single basic groups; when each basic group is compounded into a DNA molecule template by the molecular motor, generating the change of conformation of the molecular motor to influence ionic current passing through each nanopore, wherein the change of the conformation of the molecular motor, caused by each basic group, is different; and analyzing the sequence of the DNA by detecting electrical signals generating when DNA penetrates through the graphene nanopore.

Description

technical field [0001] The invention belongs to the field of sequence testing, and relates to a sequencing method based on molecular conformation changes, in particular to detecting the influence of molecular conformation changes through nanopores on ion currents passing through nanopores, and then determining each base and analyzing the sequence. Background technique [0002] At present, people use ionic current, tunneling current, fluorescence and other methods to distinguish single bases and realize the measurement of sequences. These methods have their own advantages, but are limited by low signal-to-noise ratios and short read lengths. To overcome the above limitations, we employed a sequencing technique that utilizes graphene nanopores to detect molecular conformational changes. By further utilizing the advantages of semiconductor process integration, devices can be miniaturized, integrated, and scaled, further reducing the production cost of final products, and reali...

Claims

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Application Information

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IPC IPC(8): G01N27/00
Inventor 王德强冯艳晓于军杜春雷
Owner CHONGQING INST OF GREEN & INTELLIGENT TECH CHINESE ACADEMY OF SCI
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