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Method for detecting DNA methylation

A methylation and demethylation technology, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of expensive equipment, low methylation level, limited sensitivity, etc., and reach the detection DNA concentration Low, sensitive detection, high sensitivity effect

Active Publication Date: 2008-07-30
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether it is based on methyl-sensitive single-base extension technology (Ms-SNuPE) or methyl-specific PCR technology (MSP), harmful radioactive isotopes are used in detection to improve sensitivity, or enzyme digestion reactions and gene sequencing are used. , High-pressure liquid chromatography (HPLC) and electrophoresis analysis, these detection methods require cumbersome separation and purification procedures, increase the detection time, and use more electrophoretic analysis with limited sensitivity, which is not conducive to the detection of low methylation levels , limiting the application of Ms-SNuPE and MSP technologies in clinical cancer diagnosis
Real-time quantitative PCR technology that uses high-sensitivity fluorescent detection methods requires the use of double-labeled fluorescent probe DNA, which is expensive. Due to the limitation of length and specificity, fluorescent probes are not easy to design, and not every DNA sequence can be designed. suitable dual-labeled probes, thus limiting the broad application of this technique in clinical cancer diagnosis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1, the application of PFP1 in the detection of plasmid 2 methylation

[0063] PFP1

[0064] Formula (II)

[0065] 1. Synthesis of PFP1

[0066] The structure of PFP1 is shown in formula (II), and the synthesis method is as follows:

[0067] Add 50mL of toluene, 5.2g of neopentyl glycol and 0.5g of p-toluenesulfonic acid to a 100mL single-necked bottle, reflux for 24 hours, evaporate the solvent and separate on a silica gel column (eluent: dichloromethane) to obtain 4.8g of 1,4- Neopentyldiboronate. 1 H NMR (400 MHz, CDCl 3 ): δ (ppm) 7.78 (4H, s), 3.77 (8H, s), 1.02 (12H, s).

[0068] Add 5.4mL toluene and 3.6mL 2M potassium carbonate to a 25mL two-necked flask, and after bubbling nitrogen gas for 30 minutes, add 325 mg of 2,7-dibromo-9,9-bis(6-bromohexyl)fluorene, 151 mg 1,4-Neopentyldiborate and 20 mg tetrakis(triphenylphosphine)palladium, stirred and reacted at 95°C under nitrogen for 24 hours, added chloro...

Embodiment 2

[0087] Example 2, Application of PFP1 in Plasmid 1 Methylation Detection

[0088] 1. Synthesis of PFP1

[0089] Same as Step 1 of Example 1.

[0090] Two, sodium bisulfite treatment

[0091] 500 ng of plasmid 1 was treated according to the literature to deaminate the unmethylated cytosine in the DNA to be tested into uracil, embed it in agarose, and store it at 4°C.

[0092] 3. PCR amplification

[0093] Same as Step 3 of Example 1.

[0094] 4. Single base extension reaction

[0095] Same as Step 4 of Example 1.

[0096] 5. Measurement of Fluorescence Spectroscopy

[0097] Same as Step 5 of Example 1.

[0098] Figure 4 shows the fluorescence spectra obtained by single-base extension with primer 3 and primer 4 respectively. When primer 4 is used, there is a significant energy transfer, but when primer 3 is used, only background signals appear, indicating that the target CpG site is demethylated. of.

Embodiment 3

[0099] Example 3, the application of PFP1 in the methylation detection of the mixture of plasmid 1 and plasmid 2

[0100] 1. Synthesis of PFP1

[0101] Same as Step 1 of Example 1.

[0102] Two, sodium bisulfite treatment

[0103] Plasmid 1 and plasmid 2 were mixed, and the mass ratio of plasmid 1 and plasmid 2 was 1:1. 500ng of the mixture was treated according to the literature to deaminate the unmethylated cytosine in the DNA to be tested into uracil, embed it in agarose, and store it at 4°C.

[0104] 3. PCR amplification

[0105] Same as Step 3 of Example 1.

[0106] 4. Single base extension reaction

[0107] Same as Step 4 of Example 1.

[0108] 5. Measurement of Fluorescence Spectroscopy

[0109] Same as Step 5 of Example 1.

[0110] Figure 5 shows the fluorescence spectra obtained by single-base extension with primers 3 and 4, respectively. Significant energy transfer occurred when primers 3 and 4 were used, indicating that the target CpG site was partially methyl...

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Abstract

The invention discloses a method of detecting the methylation of DNA. With high sensitivity and fast processing, the method provided by the invention can detect DAN with lower concentration and can conveniently detect low-level methylation of DNA without steps of separation and purification. Compared with the clinical histological method which has high demand onto samples, the invention only needs a little mount of DNA to sensitively detect the methylation level of tumor suppressor gene, thereby having significance in clinical diagnoses of early cancers.

Description

technical field [0001] The invention relates to a method for detecting DNA methylation. Background technique [0002] The methylation of mammalian DNA only occurs on the C of CpG dinucleotides, and the promoters of gene regulatory regions are rich in CpG sequences, and the methylation level of these CpG sequences is closely related to gene expression. [0003] In recent years, with the rapid development of polymerase chain reaction (PCR), the relationship between the methylation of tumor suppressor genes and cancer has been continuously and deeply studied. Studies have shown that the abnormal methylation of CpG islands in the promoter region of tumor suppressor genes leads to the inactivation of tumor suppressor genes, which is an important mechanism of cancer, and the methylation level increases with the development of cancer. The high methylation level of some genes may be a marker for the formation of tumors and even cancer cells. How to understand the methylation statu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王树冯福德
Owner INST OF CHEM CHINESE ACAD OF SCI
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