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A kind of dna methylation detection kit and its detection method

A detection method and methylation technology, applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of low detection throughput, low accuracy, incomplete methylation information, etc., and achieve simple and effective implementation. , the effect of high detection specificity

Inactive Publication Date: 2014-10-29
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Several methylation detection methods currently exist, such as methylation-specific polymerase chain reaction, methylation-sensitive single nucleotide primer extension, DNA sequencing, etc., have some limitations: methylation information is incomplete, Low detection throughput and low accuracy

Method used

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  • A kind of dna methylation detection kit and its detection method
  • A kind of dna methylation detection kit and its detection method
  • A kind of dna methylation detection kit and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Make a DNA methylation detection kit according to the following formula

[0025] (1) Reaction solution A: 0.3mg / ml graphene oxide solution.

[0026] (2) Reaction solution B: 0.5 mM EDCI.

[0027] (3) Reaction solution C: 1.0 mM sulfo-NHS.

Embodiment 2

[0029] The detection process is as figure 1 .

[0030] 1. Preparation of DNA methylation samples: extract genomic DNA from tissue or blood, treat genomic DNA with bisulfite, convert unmethylated cytosine (C) into uracil (U), methyl The modified cytosine is unchanged. The specific steps are as follows: (1) Put an appropriate amount of gastric cancer tissue into a centrifuge tube, add 50 μl 10% SDS and 500 μl TE, and cut it into pieces with scissors; (2) Centrifuge at 12,000 rpm for 5 minutes; (3) Add 2.5 μl RNase enzyme after removing the supernatant and 500 μl TE, in a water bath at 37°C for 60 minutes; (4) Add 9 μl proteinase K to a centrifuge tube, and bathe in a water bath at 50°C overnight; (5) Add an equal volume of saturated phenol, mix well for 10 minutes, and centrifuge at 11,000 rpm for 5 minutes to separate the phases of the mixture. Place the supernatant in another centrifuge tube and repeat this step 4 times; (6) add 2 times the volume of pre-cooled ethanol to th...

Embodiment 3

[0035]1. Preparation of DNA methylation samples: (1) Take an appropriate amount of rectal cancer tissue into a centrifuge tube, add 50 μl of 10% SDS and 500 μl of TE, and cut it into pieces with scissors; (2) Centrifuge at 12,000 rpm for 5 minutes; (3) Remove the supernatant Add 2.5 μl RNase enzyme and 500 μl TE, and bathe in water at 37°C for 60 minutes; (4) Add 9 μl proteinase K in a centrifuge tube, bathe in water at 50°C overnight; (5) Add an equal volume of saturated phenol, mix well for 10 minutes, and centrifuge at 11,000 rpm for 5 minutes The mixture was separated into phases, and the supernatant was placed in another centrifuge tube, and this step was repeated 4 times; (6) Add 2 times the volume of pre-cooled ethanol to the centrifuge tube, mix well and place it at -20°C for 30 min; (7 ) centrifuge at 12000rpm for 20min, remove the solution rapidly; (8) wash the precipitate with 70% ethanol, remove the solution; (9) add 100μl sterilized water to dissolve after drying a...

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Abstract

The invention discloses a DNA (deoxyribonucleic acid) methylation detection kit and detection method. The kit is characterized in that a glass slide with graphene oxide is prepared, methylation detection probes are designed according to a specific gene sequence, a single-chain DNA marked with AuNP or other substances capable of quenching detection fluorescence of the graphene oxide is synthesized, and the single-chain DNA is physically adsorbed onto the glass slide with the graphene oxide dot matrix. Based on the characteristic that the graphene oxide adsorbs the single-chain DNA and a double-chain DNA departs from the graphene oxide, the invention realizes accurate and fast DNA methylation detection with the advantages of high flux and low cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a DNA methylation detection kit and a method for detecting DNA methylation using the kit. Background technique [0002] Several methylation detection methods currently exist, such as methylation-specific polymerase chain reaction, methylation-sensitive single nucleotide primer extension, DNA sequencing, etc., have some limitations: methylation information is incomplete, The detection throughput is low and the accuracy is low. [0003] In 2004, two scientists from the University of Manchester, Andre Geim and Konstantin Novoselov, prepared for the first time a new type of two-dimensional carbon nanomaterial - graphene. And won the Nobel Prize in Physics in 2010, which has attracted the attention of researchers all over the world. [0004] Graphene oxide is the oxide of graphene, and its preparation is simple, and cost is also relatively low, and single-stranded DNA has stronger adsorpt...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 高力周阳陈克平张春霞陈亮连超群
Owner JIANGSU UNIV
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