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Preparation method of long DNA (deoxyribonucleic acid) sequence silicon dioxide microspheres

A technology of DNA sequence and silica, which is applied in the field of preparation of long DNA sequence silica microbeads, can solve the problems of many nucleic acid biological information, missing important information, large amount of data, etc., and achieve comprehensive biological information and activation efficiency High, high connection efficiency effect

Pending Publication Date: 2022-04-29
BIOMARKER TECH
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Problems solved by technology

[0004] Due to the limitation of the mechanism of action, the traditional sequencing method may lead to the omission of important information. However, nucleic acid contains a lot of biological information and a large amount of data. Therefore, the oligonucleotide chain with long sequence coding is modified on the substrate, and then used for DNA sequencing. The collection of information is of great significance

Method used

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  • Preparation method of long DNA (deoxyribonucleic acid) sequence silicon dioxide microspheres
  • Preparation method of long DNA (deoxyribonucleic acid) sequence silicon dioxide microspheres
  • Preparation method of long DNA (deoxyribonucleic acid) sequence silicon dioxide microspheres

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preparation example Construction

[0031] The invention provides a method for synthesizing long-sequence coded silica microbeads. This method adopts carboxylation reaction to connect carboxylated silica and the sequence with amino group, then adds the complementary sequence of subsequent barcode sequence, and uses primer annealing and polymerase polymerization reaction to obtain silica with long sequence coding Microbeads, so that the obtained microbeads are further applied to DNA chip decoding.

[0032] The present invention adopts following technical scheme:

[0033] 1. Design primers: design coding primer sequences.

[0034] 2. Activation of carboxylated silica microbeads: use EDC and NHS to activate silica microbeads with carboxyl groups to obtain silica microbeads with good dispersion and exposed carboxyl groups on the surface.

[0035] 3. Ligation reaction: Carboxylated silica microbeads and oligonucleotide chains with amino-terminal modification are connected through carboxylation reaction to obtain si...

Embodiment 1

[0042] The synthetic method of the silica microbead of embodiment 1 long sequence code

[0043] The synthesis method of the long-sequence-coded silica microbeads provided in this example comprises the following steps:

[0044] 1. Activated carboxyl silica

[0045] Accurately weigh 1.09mg EDC and 0.65mg NHS, prepare 0.1M MES at the same time, dissolve the weighed EDC and NHS with 100ul MES, and obtain a mixture of EDC and NHS; ), washed twice with the prepared MES solution, and then the above-mentioned EDC and NHS mixture was added to the microbeads, and the final reaction volume was 100 μL; then the microbeads were placed at room temperature, and the metal bath was shaken (2000rpm) to react for 30min.

[0046] 2. Connect barcode 1 amino-modified oligonucleotide chains

[0047]After the microbeads were reacted, they were evenly divided into 5 tubes, and then 2.5 μL of amino-modified oligonucleotides (dissolved in 0.1M MES, with a final concentration of 50 μM) were added to ea...

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Abstract

The invention discloses a preparation method of long DNA (deoxyribonucleic acid) sequence silicon dioxide microspheres. According to the method, silicon dioxide is taken as a carrier, a surface carboxyl functional group is activated by adopting a chemical reagent, and covalent linkage with an amino-modified oligonucleotide sequence can be realized. And meanwhile, a subsequent sequence can be introduced in a manner of combining complementary sequence annealing with polymerization reaction (sequence extension). By utilizing the method, the silicon dioxide microspheres coded by various long sequences can be obtained. Compared with a traditional method, the method has the advantages that more oligonucleotide sequences are modified on the silicon dioxide microbeads, and the contained biological information is more comprehensive.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing long DNA sequence silica microbeads. Background technique [0002] Nucleic acid, including deoxyribonucleic acid and ribonucleic acid, is one of the most basic substances of life and plays a vital role in various life activities such as growth, development, mutation, inflammation, and cancer. Nucleic acid molecules are closely related to and play an important role in the occurrence and development of various diseases that affect human health. Therefore, the development of accurate and effective methods for sensitive and accurate detection is of great significance for in-depth exploration of nucleic acid functional regulation, drug screening, early detection, clinical diagnosis and treatment, and prognosis evaluation of related diseases. [0003] Silica is a typical inorganic powder material with large specific surface area and good chemical stability. Monodis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C01B33/12
CPCC12Q1/6869C01B33/12C12Q2563/185C12Q2535/122C12Q2525/173C12Q2533/101C12Q2563/149
Inventor 郑洪坤刘敏张梦龙李佳
Owner BIOMARKER TECH
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