Preparation method of DNA microarray chip based on gel fixed nucleic acid

A microarray chip and nucleic acid technology, applied in the field of DNA microarray chip preparation, can solve the problems of poor stability and repeatability, unstable chip quality, cumbersome process, etc., and achieve good uniformity and repeatability, high nucleic acid immobilization efficiency , the effect of improving hybridization efficiency

Inactive Publication Date: 2007-08-29
南京普东兴生物科技有限公司
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Problems solved by technology

In terms of carrier use: currently widely used carriers are hard carriers such as glass and quartz. This type of carrier can only immobilize nucleic acid on its surface, and the immobilization density of nucleic acid is too high, which often leads to a reduction in hybridization efficiency due to steric hindrance.
In terms of the chemical immobilization method used: regardless of the use of rigid supports such as glass or gel supports, the currently widely used chemical methods require a multi-step chemical reaction process to activate the support to obtain active groups such as aldehyde groups linked to nucleic acids. , the process is cumbersome, not only time-consuming and heavy workload, but also poor stability and repeatability, making the chip quality very unstable

Method used

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  • Preparation method of DNA microarray chip based on gel fixed nucleic acid

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Embodiment 1

[0011] In this example, different nucleic acids modified by acrylamide were mixed with a certain proportion of acrylamide monomer, glycerol, ammonium persulfate and water to form prepolymers, and then the prepolymers of different nucleic acids were placed in different spots on the sample plate. On the position, the nucleic acid prepolymer is spotted on different positions of the solid substrate by hand or by a spotting instrument (using position coding to make the nucleic acid molecule at each position of the solid substrate a known sample (sequence)), after spotting Place the spotting substrate in a closed humid box with tetramethylethylenediamine, under the action of vacuum, tetramethylethylenediamine volatilizes and contacts with the pre-polymer matrix on the solid substrate to cause polymerization reaction, so that Nucleic acid on each array spot is immobilized on a solid substrate through a copolymerization reaction. (See Figure 1) Oligonucleotides of different sequences ...

Embodiment 2

[0013] In this example, different PCR products modified with acrylamide at site 14417 of the low-density lipoprotein receptor gene were mixed with a certain proportion of acrylamide monomer, glycerol, ammonium persulfate and water to form a prepolymer, and then the The pre-polymers of different nucleic acids are placed on different positions of the spotting plate, and the nucleic acid pre-polymers are spotted on different positions of the solid substrate by hand or by a spotting instrument to prepare a cDNA microarray chip. The specific process is as follows: three fresh blood samples of coronary heart disease patients with known 14417 site polymorphism were prepared by acrylamide-modified front primer 5'-TAC TAT CCT TCC CAG CTC CT at the 5' end and an unmodified reverse primer 5'-TTTTCA GCA ACT TGG CAT-3'amplification. The 50ul PCR system contains 50ng genomic DNA, 1×PCR buffer, 3mM MgCl2, 250uM dNTPs, 20pmol primers of 2U Taq DNA polymerase. DNA PCR conditions were: 94°C pr...

Embodiment 3

[0015] A DNA microarray chip preparation method based on gel immobilized nucleic acid, the acrylamide modified nucleic acid, acrylamide monomer, ammonium persulfate, glycerol and water are mixed into a prepolymer, wherein the concentration of nucleic acid is 0.001-100uM Among them, the weight concentration of acrylamide monomer is between 1-30%, the weight concentration of ammonium persulfate is between 0.01-5%, the weight concentration of glycerol is 10-50%, and the optimal concentration range of nucleic acid is Between 0.1-10uM, the optimal weight range of acrylamide monomer is between 3-15%, the optimal weight range of ammonium persulfate is between 0.1-1%, specifically, the concentration of nucleic acid can be selected Between 0.001, 0.1, 8, 10, 50 or 100uM, the weight concentration of acrylamide monomer can be selected between 1%, 3%, 15%, 10%, 24% or 30%, and the weight concentration of ammonium persulfate can be selected as 0.01 %, 0.1%, 1%, 3%, 5%, the weight concentra...

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Abstract

The invention discloses a process for preparing DNA micro array chips based on gel fixed nucleic acid, which comprises mixing acrylamide modified nucleic acid, acrylamide monomer, ammonium peroxodisulfate, glycerin and water into pre-polymer, wherein the concentration of the nucleic acid is between 0.001-100uM, the monomer weight concentration of acrylamide is between 1-30%, the weight concentration of the ammonium peroxodisulfate is between 0.01-5%, the weight concentration of glycerin is between 10-50%, then applying the sample of the prepolymer onto a solid substrate, loading the sample applying substrate into an airtight case containing tetramethylethylenediamine, so as to make the tetramethylethylenediamine volatilize.

Description

technical field [0001] The invention relates to a method for preparing a DNA microarray chip, in particular to a method for preparing a DNA microarray chip based on gel immobilized nucleic acid. Background technique [0002] Gene chip is a high-tech developed rapidly in the field of life science in recent years. The so-called gene chip is a silicon chip, a glass slide, or a plastic sheet on which a large number of gene probes (oligonucleotide probes or cDNA probes) are fixed in a specific arrangement. Gene chip technology is the main means to efficiently obtain relevant biological information on a large scale. At present, the application fields of this technology mainly include gene expression profile analysis, new gene discovery, gene mutation and polymorphism analysis, genome library mapping, disease diagnosis and prediction, drug screening, gene sequencing, etc. In the early 1990s, the research and development of various biochips began mainly in the United States. In le...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 肖鹏峰陆祖宏程璐
Owner 南京普东兴生物科技有限公司
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