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Microarray for detection of mutations in beta-globin genes and detection method thereof

A polymorphism detection and sequence technology, applied in biochemical equipment and methods, specific-purpose bioreactors/fermenters, measurement devices, etc., can solve the problems of complex experimental procedures, expensive kits, and rising costs. Achieving excellent usefulness

Inactive Publication Date: 2014-12-10
MITSUBISHI CHEM CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the kit is very expensive, expensive equipment is required, and the experimental process is also complicated, so the cost increases when it is used for the analysis of a large number of analytes

Method used

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  • Microarray for detection of mutations in beta-globin genes and detection method thereof
  • Microarray for detection of mutations in beta-globin genes and detection method thereof
  • Microarray for detection of mutations in beta-globin genes and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0232] studied variation at 25 positions of the beta-globin gene at one time detection using DNA microarrays. The positions of the mutations to be detected are shown in Table 1 below.

[0233] [Table 1] Mutation position of β globulin

[0234]

[0235] Among them, probes for detecting variations c.52A>T, c.84_85insC, c.364G>C, and c.380T>G are difficult to design due to the characteristics of the surrounding base sequences, so they are studied first.

[0236] 1. Fabrication of through-hole DNA microarrays

[0237] DNA microarrays were fabricated as described below.

[0238]

[0239] Oligonucleotides described in SEQ ID NO: 1 to 18 were synthesized as probes.

[0240] The synthesized oligonucleotides were oligonucleotides with an aminohexyl group introduced at the 5' end of the oligonucleotides. After synthesis, the oligonucleotide was reacted with methacrylic anhydride, purified by HPLC, and fractionated to obtain 5'-terminal vinylated oligonucleotides having the base...

Embodiment 2

[0308] In order to detect mutations at 25 positions of the β globin gene at one time using a DNA microarray, probes equipped with SEQ ID NOs: 3, 4, 7, 8, 11, 12, 17, 18 and SEQ ID NOs: 25 to 66 were produced. array of needles.

[0309] The mutation positions to be detected were the same as in Table 1 of Example 1, and a DNA microarray was prepared in the same manner as in Example 1.

[0310]

[0311] Using the mutated plasmid DNA with sequence numbers 1 to 25 described in Table 1 as a template, two sets of primer pairs with sequence numbers 21 to 24 were used to carry out PCR reaction. Ampdirect Plus kit (Shimadzu Corporation) was used for the PCR reaction.

[0312]

[0313]

[0314] The PCR reaction was performed in the Max mode using a GeneAmp9700 thermal cycler. The temperature conditions are as follows.

[0315]

[0316] 95°C for 10 minutes

[0317] (94°C for 30 seconds, 68°C for 30 seconds, 72°C for 30 seconds)×35 cycles

[0318] 4°C reaction ends

[0319] ...

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Abstract

An objective of the present invention is to provide a microarray for detection of mutations in beta-globin genes with which a large number of mutations (subject for detection) can be simply and quickly detected. The present invention provides a probe group for detection of mutations in beta-globin genes including genes represented by SEQ ID NOs: 3, 4, 7, 8, 11, 12, 17, 18 and 25 to 66, a microarray in which the probe group is immobilized, a method for detection of mutations in beta-globin genes using the microarray, and a kit for detection of mutations in beta-globin genes using the microarray and primers.

Description

technical field [0001] The present invention relates to a probe set for detecting variation of β globin gene, a microarray having the probe set, and a method for detecting variation of β globin gene using the same. Background technique [0002] The human genome consists of approximately 3 billion genetic codes (bases), and it has gradually become known that there are many differences in genetic codes (base sequences) between individuals. Currently, among these base sequence differences, single nucleotide polymorphisms (SNPs) are particularly attracting attention. [0003] Single nucleotide polymorphism (SNP) means that only one nucleotide differs in the nucleotide sequence of DNA, and it is the smallest unit corresponding to human personality such as whether alcohol can be drunk or whether drugs are easy to work. Among the 3 billion base pairs in the human genome, there are approximately 3 million (1 in 500 to 1,000 base pairs) to 10 million single nucleotide polymorphisms ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12M1/00C12N15/09G16B25/20
CPCC12Q2600/156C12Q1/6876C12Q1/6883G16B25/20G16B25/00G16B30/00C12Q2600/16G01N21/6428G01N2021/6439
Inventor 外川直之
Owner MITSUBISHI CHEM CORP
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