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Substrate activation kit and method of detecting DNA or the like by using the same

A kit and substrate technology, applied in the field of detection of DNA or protein, can solve problems such as harmful active sites and difficulties in accurate detection of DNA

Inactive Publication Date: 2005-04-13
TOYO KOHAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Therefore, after the target DNA hybridizes to the spotted DNA, the deleterious active site also allows the DNA to self-deposit causing difficulties in the accurate detection of DNA.

Method used

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  • Substrate activation kit and method of detecting DNA or the like by using the same
  • Substrate activation kit and method of detecting DNA or the like by using the same
  • Substrate activation kit and method of detecting DNA or the like by using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Procedures for using soft diamond-coated glass slide substrates are discussed below.

[0090] (1) Preparation of soft diamond-coated glass slides

[0091] First, a 25nm thick DLC film was deposited on a 25mm wide, 75mm long, and 1mm thick glass sheet by ionized vapor deposition, using a gas mixture of 95% by volume of methane and 5% by volume of hydrogen.

[0092] Then, the surface of the glass slide is chemically modified.

[0093] Irradiate the surface of the glass substrate with a high-pressure mercury lamp in ammonia gas for 10 minutes for ammoniation, and then immerse the substrate in a solution of succinic anhydride dissolved in N-methylpyrrolidone for 60 minutes to obtain a soft glass substrate with carboxyl groups on the surface. Diamond-coated glass slides.

[0094] After derivatizing the carboxyl groups on the surface of the substrate into active esters with N-hydroxysuccinimide, as figure 1 The method shown immobilizes DNA on soft diamond-coated glass slides...

Embodiment 2

[0153] Soft diamond-coated glass slides similar to those used in Example 1 were first activated by a method similar to that used in Example 1. Spotting was performed with a solution similar to that in Example 1, except that 10% glycerol was used instead of formamide.

[0154] (4) incubation

[0155] For incubation, first place the sealed box filled with saturated aqueous sodium chloride solution in an incubator set at 4°C for about 1 hour to ensure that the temperature of the entire sealed box reaches 4°C. Then take out the airtight box from the incubator, put the substrate, and pay attention to avoid contacting the saturated sodium chloride aqueous solution (the substrate is placed on the protruding part of the bottom) when putting it in. Next, put the sealed box back into the incubator and let it stand for 1 hour. The sealed box containing the saturated aqueous sodium chloride solution was then placed in an incubator set at 65° C. for 1 hour, and then dried. After drying,...

Embodiment 3

[0161] First, a soft diamond-coated glass slide similar to that used in Example 1 was activated by a method similar to that in Example 1. Spotting was performed with a solution similar to that in Example 1, except that 10% glycerol was used instead of formamide.

[0162] (4) incubation

[0163] For incubation, first place the sealed box filled with saturated aqueous sodium chloride solution in an incubator set at 4°C for about 1 hour to ensure that the temperature of the entire sealed box reaches 4°C. Then take out the sealed box from the incubator, put the substrate into it, and avoid contacting the saturated sodium chloride aqueous solution when putting it in (the substrate is placed on the protruding part of the bottom of the sealed box). Next, put the sealed box back into the incubator and let it stand for 1 hour. Then put the sealed box filled with saturated aqueous sodium chloride solution in an incubator set at 65° C. for 1 hour, and then dry it. Add 0.5% agarose gel...

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Abstract

The purpose of the present invention is to provide a convenient and fast method for immobilizing DNA on a substrate and a method for accurately detecting DNA, and the density of DNA immobilized by the method is as high as that of the traditional method. A substrate activation kit is provided, which includes phosphate buffered saline at pH 6, solution A (aqueous) of N-hydroxysuccinimide and 1-[3-(dimethylamino)propyl]3-ethylcarbodi Solution B of imine (dioxane solution); a method for detecting DNA is provided, which is characterized in that the DNA is spotted onto the substrate activated with the above-mentioned activation kit, and then hybridized with fluorescently labeled DNA with the spotted DNA.

Description

technical field [0001] The present invention relates to a substrate activation kit used in the research of medicine, biochemistry and molecular biology, and also relates to a method for detecting DNA or protein using the kit. Background of the invention [0002] Genetic analysis is used in the fields of molecular biology and biochemistry, and more recently in medicine, for example in the diagnosis of diseases. [0003] The recent invention of a substrate that can immobilize DNA has greatly facilitated gene analysis, and the substrate is also used in the medical field to diagnose diseases. [0004] One method of immobilizing DNA on a substrate is to coat a polymer such as polylysine on the surface of a glass slide or a silicon substrate before immobilization or to synthesize the DNA onto the substrate using semiconductor techniques such as photolithography. [0005] However, in the method of immobilizing DNA using a polymer such as polylysine, there is a problem of detachmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/543G01N33/552
CPCC12Q1/6837G01N33/54353G01N33/552C12Q2565/518G01N33/53
Inventor 冈村浩丹花通文山川薰大场光芳高木研一
Owner TOYO KOHAN CO LTD
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