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DNA (Deoxyribonucleic Acid) methylation detection kit and application method thereof

A detection kit and methylation technology, applied in the field of biological analysis, can solve the problems of time-consuming and low sensitivity, and achieve the effect of simple operation and high sensitivity

Inactive Publication Date: 2018-05-11
SHANDONG NORMAL UNIV
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  • Description
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Problems solved by technology

Ligation-based polymerase chain reaction (PCR) can simultaneously detect multiple sites of 5-methylated cytosines including non-cytosine / guanine dinucleotide (CpG) sites, but the use of denaturing polyacrylamide gels Analyzing reaction products by electrophoresis is low sensitivity and time-consuming

Method used

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  • DNA (Deoxyribonucleic Acid) methylation detection kit and application method thereof
  • DNA (Deoxyribonucleic Acid) methylation detection kit and application method thereof
  • DNA (Deoxyribonucleic Acid) methylation detection kit and application method thereof

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Embodiment 1

[0047] Embodiment 1 A kind of DNA methylation detection kit

[0048] The kit includes: sodium bisulfite, hydroquinone, desalting column, 0.3 mole per liter of sodium hydroxide, ammonium acetate, ethanol, trisaminomethane hydrochloride, potassium chloride, magnesium chloride, nicotinamide adenine Dinucleotide, polyethylene glycol octylphenyl ether, probe X, probe Y, probe X', probe Y', exonuclease I, exonuclease III, 10×NEBufferI, sulfuric acid Ammonium, Cy5-labeled reporter probe, biotin-labeled capture probe, thermostable ligase, and 605 quantum dots.

[0049] The nucleotide sequences of the probe X, probe Y, probe X' and probe Y' are shown in SEQ ID NO.1-4; the probe Y is modified with a phosphate group at the 5' end, and the 3' The end is modified by phosphorothioation, and the probe Y' is modified with a phosphate group at the 5' end.

[0050] The Cy5-labeled reporter probe is: 5'-TGA CTC TGT GGA GTC CTG CC-3', as shown in SEQ ID NO.5, wherein the fourth base T base at t...

Embodiment 2

[0051] The using method of DNA methylation detection kit described in embodiment 2

[0052] The method for using the DNA methylation detection kit is divided into the following steps:

[0053] (1) DNA bisulfite treatment, after denaturing the DNA to be tested at 42°C and 0.35 moles per liter of sodium hydroxide for 30 minutes, carry out sodium bisulfite reaction: add the denatured DNA to be tested into fresh The prepared 3.2 moles per liter of sodium bisulfite and 0.5 millimoles per liter of hydroquinone were reacted at 50°C for 16 to 18 hours; after the reaction, DNA was recovered using a desalting column, and 0.3 moles per liter of sodium hydroxide was used to React at 37°C for 15 minutes to complete the modification, then neutralize with ammonium acetate, ethanol precipitate and dry, and store the DNA solution in ultrapure water at 20°C for later use;

[0054] (2) Three-step circular ligase chain reaction, the reaction system is: 20 millimoles per liter of trisaminomethane...

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Abstract

The invention belongs to the technical field of biological analysis and in particular relates to a DNA (Deoxyribonucleic Acid) methylation detection kit and an application method thereof. The kit is prepared from sodium bisulfite, hydroquinone, a desalting column, 0.3mol / L sodium hydroxide, ammonium acetate, ethanol, triaminomethane hydrochloride, potassium chloride, magnesium chloride, nicotinamide adenine dinucleotide, polyethylene glycol octylphenol ether, a probe X, a probe Y, a probe X', a probe Y', exonuclease I, exonuclease III, 10*NEBuffer I, ammonium sulfate, a Cy5-labeled reporter probe, a biotin-labeled capturing probe, thermal stabilization ligase and a 605 quantum point. The kit provided by the invention has the advantages of high sensitivity, strong high specificity and simple operation and DNA methylation detection can be carried out on cytosine / guanine dinucleotide (CpG) and non-cytosine / guanine dinucleotide (CpG) sites.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a DNA methylation detection kit and a use method thereof. Background technique [0002] DNA methylation is a highly characterized epigenetic modification in the human genome, which often occurs at the carbon five-position cytosine residue of the cytosine / guanine dinucleotide (CpG) island, and is catalyzed by a DNA methyltransferase. methyl group to carbon 5 of cytosine. Each cytosine / guanine dinucleotide (CpG) island may consist of dozens to hundreds of cytosine / guanine dinucleotides (CpG) constituting the main gene promoter region. Methylation of cytosine / guanine dinucleotide (CpG) islands in promoter regions may result in deregulated regulation of multiple functions in human cells, including DNA replication, DNA repair, gene transcription, X chromosome inactivation, genomic imprinting and cell differentiation. Abnormal DNA methylation patterns are clo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/137C12Q2563/107C12Q2523/125
Inventor 张春阳王黎娟王子月
Owner SHANDONG NORMAL UNIV
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