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DNA methylation detection kit and application thereof

A detection kit and methylation technology, applied in the field of molecular biology, can solve problems such as low recovery rate and loss of genomic DNA fragments, achieve high recovery efficiency, good integrity, and save a lot of time

Inactive Publication Date: 2018-06-08
生工生物工程(上海)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the methylation detection kits on the market use silica gel membrane adsorption columns as the adsorption medium for modified DNA, but the recovery experiment of silica gel membrane adsorption columns proves that the recovery rate of DNA fragments less than 200bp is very low, resulting in genomic DNA partial fragment loss of

Method used

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  • DNA methylation detection kit and application thereof
  • DNA methylation detection kit and application thereof
  • DNA methylation detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Preparation and preparation of the kit:

[0052] CT conversion solution: 2.5mol / L Na 2 S 2 O 5 , NaOH adjusts the pH of the solution to 5.0

[0053] Antioxidant: 10mM C 6 H 4 (OH) 2 (Quinol)

[0054] Binding solution: 4mol / L Gu-HCl (guanidine hydrochloride), 1mol / L KAC, adjust the pH of the solution to 4.5

[0055] Washing solution: 70% ethanol

[0056] Desulfonation solution: 100mM NaOH, 70% ethanol

[0057] Eluent: 10mM Tris, 1mM EDTA, pH8.0

[0058] Magnetic bead suspension: carboxyl magnetic beads with a particle size of 200nm

[0059] The CT conversion solution, antioxidant, binding solution, desulfonation solution and eluent solvent are all double distilled water.

[0060] After the above reagents are configured separately, the kit is assembled.

[0061] The following detailed steps are applicable to the operation of the kit of the present invention:

[0062] Step 1. Add 0.2-2μg human gDNA to the PCR tube. If the volume of the DNA sample is less than 20ul, add ddH 2 O to 20μl.

[...

example

[0081] There is no significant difference in the effect of using the kit within the above range. For parallel comparison, the following examples uniformly adopt the following steps:

[0082] Step 1. Add 0.5μg human gDNA to the PCR tube and add ddH 2 O to 20μl.

[0083] Step 2. Add 208 μl of CT conversion solution and 12 μl of antioxidant to the above tube, mix thoroughly and gently, and react according to the following procedure: 98°C for 10 minutes; 98°C for 30s and 60°C for 150 minutes.

[0084] Step 3. After the reaction is completed, transfer the above reaction solution to a 1.5 ml EP tube.

[0085] Step 4. Add 240 μl binding solution and 30 μl magnetic bead suspension to the above reaction solution, shake and mix thoroughly, combine for 3 minutes at room temperature, and mix occasionally.

[0086] Step 5. Place the above EP tube on the magnetic separation rack for magnetic separation, aspirate the supernatant, save the magnetic beads, and take out the centrifuge tube from the magn...

Embodiment 1-1

[0098] In this example, the kit of the present invention is used to detect whether a segment of the promoter region of the human phosphatase and tensin homolog (PTEN) gene is methylated. PTEN is a tumor suppressor gene that can negatively regulate the growth of tumor cells. Human genomic DNA is extracted from cultured gastric cancer cells using the Magnetic Bead Method Animal Tissue Genomic Extraction Kit (B518721).

[0099] In order to confirm the reliability of the test results of the kit of the present invention, a commercially available similar product was used as a control kit to simultaneously process 500 ng of human cell genomic DNA.

[0100] experiment procedure:

[0101] BSP experiment:

[0102] Primer design: Take the sequence of the promoter region of the PTEN gene as the target sequence, use ABI's Methyl primerExpress software to analyze and find the CPG island in the promoter region, and then design a pair of BSP primers. The primer sequence is shown below.

[0103] BSP-F...

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Abstract

The invention relates to the field of molecular biology and discloses a DNA methylation detection kit and an application thereof. The DNA methylation detection kit comprises a CT conversion solution,an antioxidant, a binding solution, a washing solution, a desulfonation solution, an eluent and a magnetic bead suspension. According to the DNA methylation detection kit, DNA fragments are recoveredfrom a modified DNA solution with a magnetic separation technology, the kit has high recovery efficiency, the completeness of the recovered fragments is good, the kit can be matched with an automaticnucleic acid extractor to realize high-throughput methylation detection, a plenty of time is saved, and the detection efficiency is improved.

Description

Technical field: [0001] The invention relates to the field of molecular biology, in particular to a high-sensitivity DNA methylation detection kit and its application. Background technique: [0002] Epigenetics means that the DNA sequence has not changed but the gene expression has undergone heritable changes. Epigenetics is caused by non-genetic mechanisms, including regulatory mechanisms such as DNA methylation, histone covalent modification, chromatin remodeling, gene silencing, and RNA editing. Among them, DNA methylation is an important part of epigenetic research. one. [0003] DNA methylation refers to the use of S-adenosyl-methionine (SAM) to provide a methyl group under the catalysis of DNA Methyltransferase (DNMT). In CpG dinucleotide The covalent modification process of adding a methyl group to the fifth carbon atom of the cytosine pyrimidine ring. DNA methylation does not change the primary structure of DNA, but can cause changes in chromatin structure, DNA conformat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/10
CPCC12Q1/6806C12Q2523/125C12Q2563/143C12Q2563/149
Inventor 张云静李威张中娜
Owner 生工生物工程(上海)股份有限公司
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