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Nucleotide derivatives and DNA microarray

A technology of derivatives and nucleotides, applied in the field of nucleotide derivatives and DNA microarrays, can solve the problems of excessive labor, time and cost

Inactive Publication Date: 2008-03-05
斋藤烈 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, methods such as PCR amplification using labeled dNTPs are used when preparing the target nucleotide sequence, but this requires excessive labor, time, and cost
In addition, when detecting SNP, etc., the method of using the melting temperature of the hybridization probe and the target nucleotide sequence as an index is generally used, but in this case, it is necessary to adjust the hybridization stringency conditions for each single target nucleotide The sequence is strictly set, and even if the conditions are set in this way, there are disadvantages that measurement errors caused by mishybridization and the like cannot be avoided

Method used

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  • Nucleotide derivatives and DNA microarray
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Synthesis of Nucleotide Derivatives (PyU(5), PyC(5), PyA(7))

[0115] According to Figures 6 and 7, nucleotide derivatives (PyU(5), PyC(5), PyA(7)) were synthesized as follows. Wherein, the serial numbers of the compounds correspond to those in Figures 6 and 7.

[0116] Scheme i (synthesis of compound 2)

[0117] Propargylamine (1, Wako Pure Chemical Industries, Ltd.) and 1-pyrenylcarboxylic acid (2, Aldrich (Aldrich)) (1:1), in the presence of condensing agent PyBOP (1 equivalent, NOVA Biochem), in N, N - in dimethylformamide, stirred at room temperature for 2.5 hours, extracted, and purified by column chromatography to obtain the product 2 (91%).

[0118] Scheme ii (compound 4: synthesis of nucleoside derivatives of PyU(5))

[0119] 3 (obtained by stirring 5-iodo-2'-deoxyuridine (Sigma) in 4,4'-dimethoxytrityl chloride (Tokyo Chemical) and pyridine) and 2 (1:1 ) in the presence of (tetraphenylphosphine) palladium (0.15 equivalents, Wako Pure Chemicals), copper iod...

Embodiment 2

[0141] Synthesis of Nucleotide Derivatives (PyG(8))

[0142] According to Fig. 8, a nucleotide derivative (PyG(8)) was synthesized as follows. Wherein, the serial numbers of the compounds correspond to the serial numbers in FIG. 8 .

[0143] Scheme 1 (synthesis of compound 2)

[0144] Using 1-bromopyrene as a starting material, compound 1 was obtained by Sonogashira coupling reaction with trimethylsilyl acetate glyceride. Next, the protective trimethylsilyl group was removed from sodium methoxide in methanol to obtain compound 2 (pyrene unit) (yield 70%).

[0145] Scheme 2 (compound 6: synthesis of nucleoside derivatives of PyG(8))

[0146] Compound 3 was obtained by adding N-bromosuccinimide to 2'-deoxyguanosine and reacting in water (yield 60%). Next, use tert-butyldimethylsilyl chloride and imidazole to protect the 3' and 5' hydroxyl groups of the sugar of compound 3 by tert-butyldimethylsilyl, and then carry out Sonogashira coupling reaction with compound 2 to obtain ...

Embodiment 3

[0150] Synthesis of oligodeoxyribonucleotides

[0151] Using the nucleotide derivatives (PyU(5), PyC(5), PyA(7)) made in Example 1 and the nucleotide derivatives (PyG(8)) made in Example 2, Synthesis of oligodeoxyribonucleotides containing nucleotide derivatives. Oligodeoxyribonucleotides were synthesized by a 392 DNA / RNA synthesizer from Applied Biosystems in accordance with the usual phosphoramidite method. Extraction from the solid phase carrier and deprotection are carried out by incubating in 25% ammonia water for a certain temperature and for a certain time, and then refining by high performance liquid chromatography.

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Abstract

A novel nucleotide derivative showing a change in the fluorescent signal intensity depending on the corresponding base species of the counterpart chain of hybridization. And occurring as a member of a single-strand nucleotide sequence, where the corresponding base of the counterpart chain of the hybridization of the single-strand sequence is as followed:(1) which is a thymine / uracil derivative showing the most intense light emission in the case where the corresponding base is adenine; (2) which is a cytosine derivative showing the most intense light emission in the case where the corresponding base is guanine; (3) which is an adenine derivative showing the most intense light emission in the case where the corresponding base is cytosine; or (4) which is a guanine derivative showing the most intense light emission in the case where the corresponding base is cytosine or thymine / uracil.

Description

technical field [0001] The present invention relates to a nucleotide derivative for identifying a specific base type in a nucleotide sequence, and a DNA microarray having capture probes containing the nucleotide derivative. Background technique [0002] With the advent of the post-genome era, there is a demand for a new technology capable of accurately and efficiently detecting the type of base in a nucleotide sequence at low cost. For example, SNP (Single Nucleotide Polymorphism: Single Nucleotide Polymorphism) is the polymorphism with the highest frequency existing in the ratio of about 0.1% (about 1000 base single nucleotides) in the human genome, and has been increasingly It is clear that its presence or absence affects various diseases (such as the SNP of p53 gene related to lung cancer: non-patent document 1), so that the technology of correctly judging the presence or absence of SNP for the purpose of diagnosis and genetic therapy (SNP type) is becoming more and more...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H19/04C07H19/10C07H19/20C07H21/00C12Q1/68C12N15/00
Inventor 斋藤烈冈本晃充吉田安子
Owner 斋藤烈
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