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A method for fixing targeting primers in single-molecule targeted sequencing, a single-molecule targeted sequencing kit, and applications

A fixed method and targeted sequencing technology, applied in the field of molecular biology, can solve the problem that the fixed density is only 100-200/field of view.

Active Publication Date: 2019-05-07
GENEMIND BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the primers used in the immobilization technology of the single-molecule sequencer of Helicos Company are polyT sequences modified with amino groups or DNA sequences complementary to the DNA to be tested. However, the inventors have shown through experiments that DNA modified with polyT amino groups is easy to fix On the substrate surface modified with epoxy groups, the immobilization density can reach 3000-5000 / field of view, but under the same experimental conditions, if the polyT primer modified with amino group is replaced with the primer sequence complementary to the DNA to be tested, its The fixed density is only 100~200 / field of view

Method used

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  • A method for fixing targeting primers in single-molecule targeted sequencing, a single-molecule targeted sequencing kit, and applications
  • A method for fixing targeting primers in single-molecule targeted sequencing, a single-molecule targeted sequencing kit, and applications
  • A method for fixing targeting primers in single-molecule targeted sequencing, a single-molecule targeted sequencing kit, and applications

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specific Embodiment

[0059] Material and Reagent Description:

[0060] Unless otherwise specified, the reagents used in the examples of the present invention are commercially available, and the databases used in the examples of the present invention are all public online databases.

[0061] 20x SSC: Dissolve 175.3g NaCl and 88.2g sodium citrate in 800ml water, add a few drops of 10mol / l NaOH solution to adjust the pH to 7.0, add water to 1L, aliquot and autoclave. SSC is the most standard blotting and molecular hybridization treatment solution in molecular biology, and 20xSSC is a common concentrated buffer used for denaturation and cleaning of hybridization experiments.

Embodiment 1

[0063] This embodiment provides a method for fixing targeting primers in single-molecule targeted sequencing, which specifically includes the following steps:

[0064] (1) Blow off the substrate to be fixed with a nitrogen gun for use, the surface of the substrate has an epoxy group, and the substrate slide is from an epoxy-modified slide of SCHOTT;

[0065] With 0.2M of K 2 HPO 4 Dilute the targeting primer to a concentration of 0.8nM in the fixing solution, soak the substrate in the above fixing solution containing 0.8nM targeting primer, soak for 60min;

[0066] Afterwards, the mixed solution composed of 3x SSC and 0.1% Triton, 3x SSC and K with a concentration of 150 mM and pH=8.5 were used successively. 2 HPO 4 to clean the base;

[0067] (2) Immerse the cleaned substrate into a phosphate passivation solution, the phosphate passivation solution is pH=9, and the concentration is 1M K 2 HPO 4 solution, and passivated on a shaker at room temperature at a speed of 80rpm...

Embodiment 2

[0130] The effect of shaking speed in passivation on the fluorescent spots in fixed photos and the effect of different concentrations of targeting primers on the fixation effect

[0131] Operate according to the experimental method described in above-mentioned embodiment 1, difference is:

[0132] In step 1), the targeting primer to be immobilized is T10C50-Cy3, and its concentrations are 0.4nM, 0.8nM, 1.6nM, 3.2nM respectively; in addition, in step 2), when passivating, divide into two batches and carry out parallel In the test, two groups of experiments were passivated for 15 hours under shaking conditions of 0rpm / min and 80rpm / min respectively.

[0133] Repeat three times and count the number of sequences immobilized on the substrate. The results of the fluorescence microscope are as follows: Figure 4 and Figure 5 shown. Figure 4 This is the result of fluorescence microscopy photos taken during the passivation process when the rotational speed is 0 rpm / min, including ...

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Abstract

The present invention provides a sequence immobilization method, chip, and application, the method comprising: (1) immersing a substrate in an immobilization solution of 0.4-3.2 nM containing a sequence to obtain a sequence-carrying substrate, wherein the substrate is a solid matrix having a chemically-modified surface, an end of the sequence has a 10-30 bp polyT, and the immobilization solution is a phosphate buffer; and (2) performing passivation by immersing the sequence-carrying substrate in a passivation solution to obtain a passivated sequence-carrying substrate, wherein the passivation solution is a phosphate solution. The sequence immobilization provided by the present invention has a uniform density, about 3000 points per visual field.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a method for fixing targeting primers in single-molecule targeted sequencing, a single-molecule targeted sequencing kit and applications. Background technique [0002] Single-molecule sequencing technology is known as the third-generation sequencing technology, and its notable feature is that it can directly identify DNA fragments with high fidelity. Because single-molecule sequencing technology can identify a single nucleic acid molecule, it has higher detection sensitivity. Immobilization of DNA chips is the first step in single-molecule sequencing. The quality and density of DNA immobilization on the chip determines whether subsequent single-molecule sequencing can proceed smoothly. For this reason, researchers at home and abroad are committed to modifying the surface of the chip and introducing various functional groups on the surface, such as amino groups, aldehyde groups, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6806
CPCC12N15/11C12Q1/68
Inventor 葛良进高雁纪道锐邓力蔚吴增丁蔡金森
Owner GENEMIND BIOSCIENCES CO LTD
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