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Sample treatment solution and reagent kit for preparing sample for detecting DNA methylation

A treatment solution and methylation technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of complex pre-treatment, time-consuming standard method, etc., and achieve simple and easy pre-treatment, simplified extraction process, The effect of fewer steps

Inactive Publication Date: 2008-09-03
SYSMEX CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Thus, the standard method is very time-consuming (approximately 22 hours in total)
The problem is that the pretreatment (preparation of DNA methylation detection sample) of the biological sample before the PCR reaction is very complicated, and it takes about 20 hours only for the pretreatment (1-3 above)

Method used

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  • Sample treatment solution and reagent kit for preparing sample for detecting DNA methylation
  • Sample treatment solution and reagent kit for preparing sample for detecting DNA methylation
  • Sample treatment solution and reagent kit for preparing sample for detecting DNA methylation

Examples

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preparation example Construction

[0046] The method for preparing a sample for detection of DNA methylation (hereinafter sometimes referred to as "sample for detection of DNA methylation") according to this embodiment includes: mixing a sample treatment liquid containing protease and an aqueous solvent with a biological sample containing cells The step (mixing step) and the step of adding a mutagen to the resulting mixture to convert unmethylated cytosine contained in DNA in the mixture into other bases (mutation step).

[0047] In this embodiment, the so-called aqueous solvent refers to general water, such as distilled water, ion-exchanged water and pure water. The aqueous solvent may contain an organic solution as long as it does not adversely affect the enzymatic reaction of protease.

[0048]The protease in the present embodiment is not particularly limited as long as it decomposes protein. For example, it can be thermolysin, pyroprotease, papain, trypsin, pronase, α-chymotrypsin, subtilisin, pepsin, plas...

example

[0101]

[0102] 300 µl of human serum was added to 1 ng of genomic DNA derived from cultured human breast cancer cell MDA231 to prepare a biological sample. As quality control, a biological sample containing only 300 μl of human serum, which does not contain genomic DNA derived from MDA231, was used.

[0103]

[0104] Sample treatment solutions A to F were prepared as follows. 320μl distilled water was used for quality control.

[0105] Sample treatment solution A:

[0106] Mix 300 μl of urea aqueous solution with a final concentration of 4M and 20 μl of 20 mg / ml proteinase K aqueous solution to obtain 320 μl of sample treatment solution A.

[0107] Sample treatment solution B:

[0108] Mix 300 μl of guanidine hydrochloride aqueous solution with a final concentration of 4M and 20 μl of 20 mg / ml proteinase K aqueous solution to obtain 320 μl of sample treatment solution B.

[0109] Sample treatment solution C:

[0110] Mix 300 μl of SDS aqueous solution with a final co...

example 1

[0155] Figure 2 shows the detection results of methylated DNA when using the sample treatment solution A, the sample treatment solution B and the quality control. As shown in Figure 2, methylation can be detected in both the ERα gene and the E-Cad gene using the sample treatment solution A and the sample treatment solution B containing proteinase K as a protease and urea or guanidine hydrochloride as a chaotropic ion DNA, but distilled water used as a quality control could not detect methylated DNA. Regarding the biological sample not containing DMA231-derived genomic DNA, methylated DNA was not detected in both the sample treatment solution A and the sample treatment solution B.

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Abstract

The present invention provides a sample treatment solution for preparing a sample for DNA methylation which can achieve stable detection results in detection of DNA methylation and be easily pretreated, comprising aqueous solution and protease, and a kit, a sample preparation method and DNA methylation detecting method thereof.

Description

Technical field: [0001] The invention relates to a sample processing solution and a kit for preparing a sample for detecting DNA methylation. That is, the present invention relates to a sample preparation method and a DNA methylation detection method using the sample treatment solution and the kit. Background technique: [0002] In the chromosomal DNA of higher eukaryotes, among the bases constituting the DNA, the 5-terminus of C (cytosine) may be methylated. Unlike prokaryotes, DNA methylation in higher eukaryotes functions as a mechanism for suppressing the expression of genetic information. For example, when the CpG region (CpG island, CG island) containing a large number of certain genes is methylated, the transcription of the gene is inhibited. On the contrary, when the CpG island is not methylated, the transcribed gene can bind to the promoter region, and the gene is in a transcribable state. In this way, DNA methylation is one of the regulatory mechanisms of gene e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q1/6827C12Q2527/125C12Q2523/125C12Q2521/537
Inventor 梶田昌裕酒井绫子山本宪明石原英干
Owner SYSMEX CORP
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