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Patterning microarray flowing tank, manufacturing method, detection method and application of flowing tank

A flow cell and patterning technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as high cost, low throughput of sequencer, and no practical value of sequencing information

Pending Publication Date: 2019-07-30
南京拓远生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Third-generation sequencers (Pac Bio and Oxford Nanopore) have low throughput and high cost
The Passion distribution is limited to only 30% of the clusters being monoclonal, and the sequencing information generated by other clusters higher than 60% has no practical value

Method used

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  • Patterning microarray flowing tank, manufacturing method, detection method and application of flowing tank
  • Patterning microarray flowing tank, manufacturing method, detection method and application of flowing tank
  • Patterning microarray flowing tank, manufacturing method, detection method and application of flowing tank

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Surface activation of glass slides.

[0106] Take a 25 mm x 75 mm x 0.5 mm glass slide, sonicate it in 0.5 wt% SDS aqueous solution for 20 minutes, and then wash it thoroughly with deionized water. Then ultrasonic treatment in 29% ammonia water, 30% hydrogen peroxide (H2O2), deionized water, 1:1:5 (volume ratio) solution for 20 minutes and then thoroughly cleaned with deionized water. Then ultrasonically treat for 20 minutes in a solution of 38% HCl and 30% hydrogen peroxide (H2O2) and deionized water with a volume ratio of 1:1:6, and then rinse thoroughly with deionized water. The treated slides were placed in deionized water and kept tightly covered. When in use, they were taken out and dried with argon gas, and then baked at 110°C for 5 minutes. The water contact angle of this pre-treated slide is less than 10 degrees. The prepared substrate can be used to form a layer of NH2 groups, and then immobilize a layer of photodegradable chains on the surface of the substr...

Embodiment 2

[0108] 3-Aminopropyltrimethoxysilane was used to form NH2 groups on the slide surface.

[0109] Add 30 milliliters of absolute ethanol, 500 microliters of 3-aminopropyltrimethoxysilane and 20 microliters of triethylamine into a polypropylene chip tube with a screw cap, and put 5 slides pretreated in Example 1 , shake for 3 hours. Take out the glass slide, wash it with a sufficient amount of 95% ethanol, blow it dry with argon, and place it in an oven at 110°C for annealing treatment for 5 minutes. Prepared slides are immediately used in the next step to covalently immobilize a layer of photolyzable linkages on the substrate surface.

Embodiment 3

[0111] 1-(bromomethyl)-2-nitrobenzene is covalently immobilized on the surface of the glass slide (Example 2) with NH2, so that there is no reactive group on the surface of the glass slide.

[0112] Weigh 2.16 mg of 1-(bromomethyl)-2-nitrobenzene and add it to 2 ml of absolute ethanol (FW214.96, 0.01 mmol, 5 mM), add 2 microliters of DIPEA (diisopropylethylamine, FW129. 24, ρ=0.782, 0.012mmol, 6mM) and added dropwise onto the surface of the glass slide (Example 2) with NH2 groups, left to stand for 3 hours, rinsed with ethanol, and dried with argon. The covalent immobilization reaction process of embodiment 3 is as follows:

[0113]

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PUM

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Abstract

The invention relates to a patterning microarray flowing tank, a manufacturing method, a detection method and application of the flowing tank and relates to the technical field of biology. A layer ofreactive functional groups formed on the surface of a chain-protection transparent solid substrate through activation are chained through photolysis, then a patterning photolysis chain is chained withmolecules to form a reactive microarray, a layer of reactive functional polymers are fixed to the reactive microarray through covalence for forming a three-dimensional reactive functional polymer microarray, and finally a reactive DNA primer and the reactive functional polymers on the microarray are used for a reaction to form a three-dimensional DNA primer microarray. By adopting the surface ofthe patterning microarray flowing tank, the proportion of a monoclonal cluster is further increased, more effective sequencing information is generated, the three-dimensional surface polymers are usedfor forming the three-dimensional surface structure of the DNA primer, the high-density DNA primer structure can be formed, through the surface, the high-signal-to-noise-ratio sequencing informationcan be generated for DNA sequencing, and the manufacturing process is effective, simple and clean.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a patterned microarray flow cell, a manufacturing method, a detection method and applications thereof. Background technique [0002] The core of high-throughput DNA sequencing is the flow cell of the sequencer. The surface of the initial flow cell was not patterned. According to the Poisson distribution theory, the maximum number of monoclonal clusters is about 30%. Moreover, the position of monoclonal clusters is disordered, which is difficult for data collection and processing. Using a patterned flow cell can increase the proportion of monoclonal clusters to over 60%, which greatly improves the sequencing throughput, and the position of the monoclonal clusters is relatively fixed. [0003] DNA sequencing has a history of nearly half a century. In the early 1970s, Chinese biochemist Wu Rui used DNA polymerase catalysis to detect radiolabeled oligonucleotide DNA sequencing technolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2535/122C12Q2531/113
Inventor 向国兵张明航吕华施琦
Owner 南京拓远生物科技有限公司
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