Amplified nucleic acids and immobilized products thereof

A technology for amplifying nucleic acid and nucleic acid, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve problems such as difficult mass production and supply of nucleic acids

Inactive Publication Date: 2004-05-26
TAKARA HOLDINGS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] As described above, according to the conventional nucleic acid amplification method, it is difficult to mass-produce and supply nucleic acid for substrates with immobilized nucleic acid

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0124] The following examples illustrate the present invention in more detail, but should not be construed as limiting the scope of the present invention.

[0125] Reference Example 1

[0126] (1) Preparation of genomic DNA from Pyrococcus furiosus

[0127] It will contain 1% tryptone (Difco Laboratories), 0.5% yeast extract (DifcoLaboratories), 1% soluble starch (Nacalai Tesque), 3.5% Jamarin S Solid (Jamarin Laboratory), 0.5% Jamarin S Liquid (Jamarin Laboratory), 0.003% MgSO 4 , 0.001% NaCl, 0.0001% FeSO 4 ·7H 2 O, 0.0001% CoSO 4 , 0.0001%CaCl 2 ·7H 2 O, 0.0001% ZnSO 4 , 0.1ppm CuSO 4 ·5H 2 O, 0.1ppmKAl(SO 4 ) 2 , 0.1ppm H 3 BO 4 , 0.1ppm Na 2 MoO 4 ·2H 2 O and 0.25ppmNiCl 2 ·6H 2 The 2L medium of O was placed in a 2L culture flask, sterilized at 120°C for 20 minutes, and nitrogen gas was introduced to remove dissolved oxygen. Then the medium was inoculated with Pyrococcus furiosus (from Deutsche Sammlung von Mikroorganismen (German Collection of Microorganisms)) Purchase; DSM3...

Embodiment 2

[0141] Reference Example 2: Cloning of RNase HII gene from Pyrococcus horikoshii

[0142] (1) Preparation of genomic DNA from Pyrococcus horikoshii

[0143] It will contain 1% tryptone (Difco Laboratories), 0.5% yeast extract (DifcoLaboratories), 1% soluble starch (Nacalai Tesque), 3.5% Jamarin S Solid (Jamarin Laboratory), 0.5% Jamarin S Liquid (Jamarin Laboratory), 0.003% MgSO 4 , 0.001% NaCl, 0.0001% FeSO 4 ·7H 2 O, 0.0001% CoSO 4 , 0.0001%CaCl 2 ·7H 2 O, 0.0001% ZnSO 4 , 0.1ppm CuSO 4 ·5H 2 O, 0.1ppmKAl(SO 4 ) 2 , 0.1ppm H 3 BO 4 , 0.1ppm Na 2 MoO 4 ·2H 2 O and 0.25ppmNiCl 2 ·6H 2 The 2L medium of O was placed in a 2L culture flask, sterilized at 120℃ for 20 minutes, and nitrogen gas was introduced to remove dissolved oxygen. Then Pyrococcus horikoshiiOT3 (purchased from the Institute of Physical and Chemical Research (RIKEN)) was inoculated into the medium ; JCM9974), cultured at 95°C for 16 hours without shaking. After cultivation, the cells were collected by centrifugation....

Embodiment 3

[0162] Reference Example 3: Measurement of Thermostable RNase H Activity

[0163] 1 mg of poly(rA) or poly(dT) (both from Amersham Pharmacia Biotech) was dissolved in 1 ml of 40 mM tris-HCl (pH 7.7) containing 1 mM EDTA to prepare poly(rA) and poly(dT) solutions.

[0164] Then add poly(rA) solution (to a final concentration of 20μg / ml) and poly(dT) solution (to a final concentration of 30μg / ml) to contain 4mM MgCl 2 , 1 mM DTT, 0.003% BSA and 4% glycerol in 40 mM tris-HCl (pH 7.7). The mixture was allowed to react at 37°C for 10 minutes, and then cooled to 4°C to prepare a poly(rA)-poly(dT) solution.

[0165] Add 1 μl of enzyme solution to 100 μl of the poly(rA)-poly(dT) solution. The mixture was allowed to react at 40°C for 10 minutes. 10μl 0.5M EDTA was added to stop the reaction. Then measure the absorbance at 260 nm. As a control, 10 μl of 0.5 M EDTA was added to the reaction mixture, the resulting mixture was allowed to react at 40° C. for 10 minutes, and then the absorbance ...

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Abstract

A nucleic acid, which is provided in a large amount through a nucleic acid amplification reaction with the use of chimeric oligonucleotide primers, is constructed in a state of containing a modified deoxyribonucleotide for immobilizing the nucleic acid to a solid phase and then immobilized to a solid phase at a high efficiency, thereby giving an immobilized nucleic acid product with excellent qualities.

Description

Technical field [0001] The invention relates to a high-sensitivity DNA chip that can be used in the field of gene analysis. Background technique [0002] A matrix (such as a DNA chip) on which nucleic acid is immobilized has been developed as a tool for rapid promotion of gene function analysis projects. It has been widely used to analyze all yeast genes and the expression of genes in cultured cells and tissues. Known methods for preparing DNA chips (or DNA arrays) include, for example, a method in which single-stranded DNA is synthesized in a predetermined region on the solid phase of the chip, and a method in which pre-prepared single-stranded or double-stranded DNA is spotted on the solid phase of the chip. The method in the predetermined area of ​​the phase. Examples of DNA chips prepared according to the previous method are the high-density oligonucleotide arrays described in U.S. Patent Nos. 5,445,934, 5,744,305, and 5,700,637. Examples of DNA chips prepared in accordance ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6837C12Q2565/518C12Q2531/119C12Q1/68
Inventor 峰野纯一武田理六嶋正知上森隆司外园成和向井博之山下周作佐川裕章浅田起代藏加藤郁之进
Owner TAKARA HOLDINGS
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