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Long-chain biotin label and its preparation method and use

A long-chain biotin, marker technology, used in biochemical equipment and methods, chemical instruments and methods, determination/inspection of microorganisms, etc.

Active Publication Date: 2020-10-27
伯科生物医学科技(无锡)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these organic long chains cannot fully meet current needs, especially in next-generation sequencing

Method used

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  • Long-chain biotin label and its preparation method and use
  • Long-chain biotin label and its preparation method and use
  • Long-chain biotin label and its preparation method and use

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Experimental program
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preparation example Construction

[0043] [Preparation method of long-chain biotin-labeled substance]

[0044] The second aspect of the present invention provides a method for preparing long-chain biotin-labeled substances, which includes at least the following four steps.

[0045] step 1)

[0046] The step (1) of the present invention is a step of performing DMT protection on the biotin molecule before the synthesis reaction. Specifically, it involves reacting biotin and DMT-Cl in pyridine solution to obtain intermediate product A. More specifically, the raw material biotin and DMT-Cl are dissolved in a pyridine solution in a molar amount of 1:2-1:5, and heated in a water bath at 50-90°C (for example, 80°C) for 1-8 hours, preferably 2-6 hours. hours, for example 4 hours, the crude product is obtained. Then add water and dichloromethane to the crude product, shake it well, separate the dichloromethane layer, and add anhydrous sodium sulfate to dry it. After rotary evaporation of the dichloromethane layer,...

Embodiment 1

[0077] Synthesis of Biotin Molecule:

[0078] 1. Synthesis of Molecule 1

[0079] The synthetic route of molecule 1 is as follows:

[0080]

[0081] The first step: the raw material biotin and DMT-Cl were dissolved in a pyridine solution at a molar ratio of 1:3, and heated in a water bath at 80°C for 4 hours to obtain a crude product. Add water and dichloromethane to the obtained crude product, shake it well, separate the dichloromethane layer, and add anhydrous sodium sulfate to dry it. After rotary evaporation of the dichloromethane layer, the mixture was separated on a silica gel column to obtain intermediate product A.

[0082] Step 2: Dissolve the intermediate product A in anhydrous dimethylformamide, react with 1:1 molar amount of aminohexanol for 1 hour under the catalysis of DSC, and condense to obtain the crude product. Add water and dichloromethane to the crude product, shake it well, separate the dichloromethane layer, and add anhydrous sodium sulfate to dry i...

Embodiment 2

[0095] 1. DNA synthesis of long-chain biotin molecules:

[0096] In the first step, trichloroacetic acid (TCA) reacts with the first nucleotide pre-connected on the controllable porous glass (CPG), in which the active group is in a protected state, and the 5'-hydroxyl of the nucleotide is removed Protecting group DMT to obtain free 5'-hydroxyl;

[0097] In the second step, tetrazolium mixes and activates the base phosphoramidite monomer to react with the 5'-hydroxyl group on CPG;

[0098] The third step is the capping reaction. In the condensation reaction, there may be a very small number of 5'-hydroxyl groups that do not participate in the reaction (less than 2%). After terminating with acetic anhydride and 1-methylimidazole, the reaction continues;

[0099] In the fourth step, the phosphorous acid is oxidized into a stable phosphotriester with iodine-pyridine oxidant;

[0100] The above steps are repeated until an oligonucleotide sequence (Oligo DNA) of the desired length...

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Abstract

The invention discloses a long-chain biotin marker and a preparation method and application thereof. The long-chain biotin marker comprises a biotin molecule, a spacer arm and a reaction group. The biotin molecule comprises a ring structure and a valeric acid side chain, a structure formula (I) of the spacer arm is as shown in the specification, a structural formula (II) of the reaction group is as shown in the specification, the valeric acid side chain in the biotin molecule is connected with NH at a first tail end of the spacer arm, oxygen at a second tail end of the spacer arm is combined with phosphorus in the reaction group to form an O-P bond, and accordingly connection between the spacer arm and the reaction group is realized. The long-chain biotin marker is used for gene capturingand sequencing.

Description

technical field [0001] The present invention relates to a biomarker used for gene sequencing, in particular to a long-chain biotin marker used for gene sequencing, especially for next-generation sequencing, as well as its preparation method and application. Background technique [0002] Next-generation sequencing plays an increasingly important role in molecular biology and clinical medicine. Among them, target region capture sequencing is a very cost-effective method in the application of next-generation sequencing, and has broad application space in the fields of genetic disease and tumor science research and clinical diagnosis. The principle is to design specific complementary probes for the genomic region of interest, hybridize it with genomic DNA, enrich the DNA fragments of the target genomic region, and then perform high-throughput sequencing. [0003] The key step of capture enrichment is to use biotin-modified single-stranded DNA as a capture reagent to capture the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C07F9/6561C12Q1/6869C12Q1/6806
CPCC07F9/65618C12N15/11C12Q1/6806C12Q1/6869C12Q2563/131
Inventor 郭诚常燕强卢亚明张茂华
Owner 伯科生物医学科技(无锡)有限公司
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