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DNA immobilization method based on nucleic acid immobilization by gel and use thereof

An immobilization method and nucleic acid technology, applied in the field of target nucleic acid detection, can solve problems such as the reduction of immobilization efficiency, and achieve the effects of eliminating possibility, high immobilization efficiency and high loading capacity.

Inactive Publication Date: 2009-07-15
HANGZHOU ENSHI GENE TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to improve the immobilization efficiency with the support, it is necessary to pre-heat denature the DNA, and the immobilization efficiency is significantly reduced in the case of no denaturation

Method used

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  • DNA immobilization method based on nucleic acid immobilization by gel and use thereof
  • DNA immobilization method based on nucleic acid immobilization by gel and use thereof
  • DNA immobilization method based on nucleic acid immobilization by gel and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Detection of Efficiency of DNA Immobilization by Hydrogel Copolymerization

[0040] (1) Preparation of end-modified PCR amplification products

[0041] Using the plasmid DNA containing the Staphylococcus aureus enterotoxin E gene (pUCm-T-SEE, see GenBank accession number M21319) as a template, a fragment of about 850 bp of the gene was amplified by PCR. The base sequences of the two primers used for this PCR are shown in SEQ ID NO.1 and SEQ ID NO.2; during primer synthesis, (CH2) was introduced at the 5' end of the upstream and downstream primers 6 -NH 2Modification; after the PCR reaction is completed, the PCR product is recovered, quantitatively analyzed by GeneQuant, and stored at -20°C for later use;

[0042] (2) Take 5% polymethacrylamide gel and 20% polymethacrylamide gel for use; configure the gel according to the following formula

[0043] 5% polymethacrylamide gel 20% polymethacrylamide gel

[0044] h 2 O 3.167mL —

[0045] 10×TBE(pH1...

Embodiment 2

[0054] Thermal Stability Experiment of Fixation Method

[0055] (1) Preparation of end-modified PCR amplification products

[0056] Using the plasmid DNA (pUCm-T-SEE, GenBank accession number M21319 of which see) containing the Staphylococcus aureus enterotoxin E gene as a template, a fragment of about 850 bp of the gene was amplified by PCR; two primers for this PCR See SEQ ID NO.1 and SEQ ID NO.2 for the base sequence; in the primer synthesis process, (CH2) was introduced at the 5' end of the upstream and downstream primers 6 -NH 2 Modification; after the PCR reaction is completed, the PCR product is recovered, quantitatively analyzed by GeneQuant, and stored at -20°C for later use;

[0057] (2) Immobilization of PCR products

[0058] Take the previously amplified and purified PCR product (with NH at the end 2 Modification), take 1 μL and dilute to 10ng-1μg / μL with double-distilled water; take the diluted PCR product, heat and denature it at 100°C for 5 minutes, and imme...

Embodiment 3

[0070] Immobilization of pUCm-T-SEE plasmid fragment

[0071] (1) Fragmentation and terminal modification of pUCm-T-SEE

[0072] Extract the pUCm-T-SEE plasmid according to the conventional method, take 100 μL of the purified pUCm-T-SEE plasmid, add 50 μL of formic acid, mix well and place it in a water bath at 30°C for a reaction time of 20 minutes; add NaOH milk immediately after the reaction Adjust the pH of the turbid solution to 7.0; according to the kit instructions, use the DNA gel recovery kit to recover the plasmid DNA after formic acid treatment, recover the DNA on the column with 100 μL of double distilled water, and the liquid in the centrifuge tube is the recovered plasmid DNA ; Add 25 μL of 2.5mol.L to the depurinated plasmid solution -1 ethylenediamine hydrochloride solution, after mixing, add NaOH emulsion to adjust the pH to 7.4, and react in a water bath at 37°C for 4 hours; after 4 hours, add 25 μL of freshly prepared 0.1mol L -1 Sodium borohydride solutio...

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Abstract

The invention relates to a method of immobilizing DNA on gel, in particular to a DNA immobilization method based on immobilization of nucleic acid on the gel and the application thereof. The immobilization method of a DNA sample comprises three steps of raw material selection, mixing and polyreaction. In the step of raw material selection, the selected raw materials and dosage are as follows: 10 percent of 10*TBE(pH13), 10-70 percent of 40 percent methacrylamide storage liquid, 1muL-5ml diluted degeneration amino terminal modified nucleic acid, 1-10 percent of N, N, N, N-tetramethyl ethylenediamine, 1-10 percent of 10 percent ammonium persulfate, and water. In the step of mixing, configured gel raw material is added with amino terminal modified nucleic acid to be mixed. The nucleic acid amount in the mixture is 10-1,000ng / 3muL. In the step of polyreaction, 5muL mixture is put in a PCR reaction tube at 37 DEG C for 30min. After the polyreaction is finished, the mixture is molded into gel shape, and the amino terminal modified nucleic acid is immobilized by the polyreaction in the gel. The method has the advantages of high bearing capacity, immobilization of the terminal modified nucleic acid molecule by stable covalent bond formed between the molecule and a solid phase carrier and capacity of enduring dramatic conditions of the PCR reaction, etc.

Description

technical field [0001] The invention relates to a method for immobilizing DNA on a gel, which can be used for preparing DNA microarrays, repeated utilization of immobilized DNA, and the like. The invention also relates to a material on which DNA is immobilized according to said method and a method for detecting a target nucleic acid using said material. Background technique [0002] The integrity of our genetic material is constantly challenged due to constant chemical exposure (environmental substances, food, etc.). Genomic DNA is not static in the process of disease and throughout a person's life, and it is increasingly necessary to study the dynamic changes at the genome level. [0003] Due to technical bottlenecks, systematic comparative studies on the dynamic changes at the genome level during disease processes are still not possible. However, the need to preserve individual genome DNA for current or future research obviously has extremely important practical signific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 厉朝龙贾丹梅
Owner HANGZHOU ENSHI GENE TECH DEV
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