Method of detecting methylated DNA in sample

A technology for detecting samples and methylation, which is used in biochemical equipment and methods, determination/inspection of microorganisms, etc., to achieve the effect of simple detection

Active Publication Date: 2012-09-05
SYSMEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this regard, when the CpG island is not methylated, transcription of the gene becomes possible due to the binding of transcription factors to the promoter

Method used

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  • Method of detecting methylated DNA in sample
  • Method of detecting methylated DNA in sample
  • Method of detecting methylated DNA in sample

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Experimental program
Comparison scheme
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Embodiment approach

[0037] Preferred embodiments of the present invention will be described below with reference to the drawings.

[0038] The "CpG site" in the present specification refers to a site where cytosine (C) and guanine (G) are adjacent to each other in this order from the 5' to the 3' direction in the base sequence of DNA. In addition, the character "p" of CpG represents a phosphodiester bond between cytosine and guanine.

[0039] The "methylated CpG site" in the present specification refers to a CpG site modified by methylation at the 5-position of cytosine. That is, at the methylated CpG site, 5-methylcytosine (methylated cytosine) and guanine are adjacent in this order from the 5' to the 3' direction.

[0040] "Methylated DNA" in this specification refers to DNA containing at least one 5-methylcytosine.

[0041] In the method of detecting methylated DNA in a sample of the present invention (hereinafter also referred to as "detection method"), first, a sample that may contain meth...

Embodiment 1

[0085] As the methylated DNA to be detected, 6MeCG oligonucleotide, which is an oligonucleotide containing six 5-methylcytosines, was used. The base sequence of the 6MeCG oligonucleotide is shown below.

[0086]

[0087] 5'-CGAGGTCGACGGTATTGATm5cGAGTATm5cGATAGTm5cGATATm5cGATATm5cGATATm5cGATATACAACGTCGTGACTGG-3' (SEQ ID NO: 1)

[0088] ("m5c" in the base sequence indicates 5-methylcytosine.)

[0089] (1) Sample preparation

[0090] An aqueous solution of 10 pM 6MeCG oligonucleotide was prepared as a 6MeCG oligonucleotide solution. The 6MeCG oligonucleotide solution was denatured by heating at 95° C. for 10 minutes, and then left to stand on ice for 1 minute. 2 µl was taken from the denatured 6MeCG oligonucleotide solution as an input sample. The remaining solution was used as a sample provided in the detection method of the present invention.

[0091] (2) Contact between sample and anti-methylated DNA antibody

[0092] The above-mentioned sample was divided into two, an...

Embodiment 2

[0113] (1) Sample preparation

[0114] A solution of 6MeCG oligonucleotide was prepared as in Example 1, from which the input sample and the sample provided for the detection method of the present invention were obtained.

[0115] (2) Contact with anti-methylated DNA antibody

[0116] From the above samples, as in Example 1, a test sample and a control sample were obtained.

[0117] (3) Decomposition of DNA by deoxyribonuclease

[0118] 4 μl of exonuclease I (20 U / μl: NEB Co., Ltd.), one type of deoxyribonuclease, was added to each of the above test samples and control samples, and reacted at 37° C. for 1 hour. After the reaction, exonuclease I was inactivated by heating each sample at 80°C for 20 minutes.

[0119] (4) Detection of methylated DNA

[0120] In order to check whether the 6MeCG oligonucleotide remained in the sample, quantitative PCR was performed as in Example 1.

[0121] The obtained reaction solution was subjected to electrophoresis using 3% agarose gel, a...

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Abstract

In order to simply obtain a methylated DNA sample, a method including bringing a sample which may include methylated DNA into contact with a protein capable of binding to methylated DNA to bind the methylated DNA in the sample to the protein, bringing the sample obtained into contact with at least one deoxyribonuclease to degrade DNA in the sample; and detecting methylated DNA which is not degraded by the deoxyribonuclease by the binding of the sample obtained to the protein is used. In the method, at least one type of deoxyribonucleases is a deoxyribonuclease different from a methylation-sensitive restriction enzyme capable of degradating a single stranded DNA.

Description

【Technical field】 [0001] The present invention relates to a method for detecting methylated DNA from a DNA-containing sample. 【Background technique】 [0002] In the chromosomal DNA of higher eukaryotes, it is known that the 5th position of C (cytosine) among the bases constituting DNA is methylated. Such DNA methylation functions as a control structure for gene expression. For example, when a region rich in CpG sequences (also referred to as "CpG island" or "CG island") present in the promoter region of a certain gene is methylated, the transcription of the gene is repressed. This phenomenon is also known as "gene silencing". In contrast, when the CpG island is not methylated, since the transcription factor can bind to the promoter, transcription of the gene becomes possible. [0003] As mentioned above, methylation of DNA is one of the control structures of gene expression. That is, DNA methylation is involved in various aspects such as early embryogenesis, expression o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6804C12Q1/6827C12Q2521/331C12Q2522/101
Inventor 酒井绫子梶田昌裕
Owner SYSMEX CORP
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