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Method for detecting karyon DNA (deoxyribonucleic acid) and mitochondrion DNA methylation levels of corn through high-performance liquid chromatography

A high-performance liquid chromatography and cell nucleus technology, which is applied to the detection of corn nuclear DNA and mitochondrial DNA methylation levels, can solve the problems of cumbersome experiments and the inability of three enzymes to act at the same time

Inactive Publication Date: 2013-02-27
SICHUAN AGRI UNIV
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Problems solved by technology

Nuclease P1 can only act on the single strand of DNA; the optimum reaction temperature of nuclease P1 is higher than that of the other two enzymes, but the optimum pH value is lower than that of the other two enzymes, which means that the three enzymes cannot be simultaneously function, and the pH value needs to be adjusted artificially, which makes the experiment extremely cumbersome

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  • Method for detecting karyon DNA (deoxyribonucleic acid) and mitochondrion DNA methylation levels of corn through high-performance liquid chromatography
  • Method for detecting karyon DNA (deoxyribonucleic acid) and mitochondrion DNA methylation levels of corn through high-performance liquid chromatography
  • Method for detecting karyon DNA (deoxyribonucleic acid) and mitochondrion DNA methylation levels of corn through high-performance liquid chromatography

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Embodiment Construction

[0012] The present invention will be described in detail below with reference to specific embodiments.

[0013] DNA samples are hydrolyzed into bases / nucleosides / nucleotides by acid or enzyme. In this experiment, Benzonase, phosphodiesterase and alkaline phosphatase are used to prepare a hydrolysis mixture, which can be prepared for 100 parts of 1μg DNA hydrolysis mixture The method is as follows: 250U Benzonase, 300mU Phosphodiesterase I, and 200U Alkaline Phosphatase are added into 5mL Tris-HCl hydrolysis buffer with pH 7.9.

[0014] Add 50 μL of the hydrolysis mixture to 1 μg of 20 ng / μL DNA, incubate at 37 °C for 6 hours, and refrigerate at -20 °C for measurement. Start the computer, pump A, pump B, SPD detector, column thermostat, etc. of the high performance liquid chromatograph, first rinse the column with 100% methanol for 1 hour, then transition with water: methanol (95:5, v / v) for 30 minutes, and finally Rinse with buffered mobile phase for 1 h, and equilibrate the ...

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Abstract

The invention discloses a method for detecting the karyon DNA (deoxyribonucleic acid) and mitochondrion DNA methylation levels of a corn through high-performance liquid chromatography. The preparation method of a hydrolysis mixed solution for treating 100 parts of DNA of 1 mu g comprises the following step: adding 250 U of Benzonase, 300 muU of phosphodiesterase I and 200 U of alkaline phosphatase into 5 mL of Tris-HCl hydrolysis buffer solution having a pH value of 7.9, wherein the pH values of mobile phases are as follows: the pH value of mitochondrion DNA is regulated to 3.42 with phosphoric acid, and the pH value of karyon DNA is regulated to 3.0 with phosphoric acid; the chromatographic conditions are as follows: a C18 filler chromatographic column is used, the flow rate is 0.8 mL / min, the detector wavelength is 287 nm, the column temperature is 35 DEG C, and the sample size is 20 muL; and methanol, 5mM sodium pentanesulfonate and triethylamine of which the ratio is 10:90:0.2 (V / V) are used as the mobile phases, and the pH values are respectively regulated to 3.42 and 3.0 with phosphoric acid. The method is simple and easy to implement, and all the components can be completely separated within 20 minutes.

Description

technical field [0001] The present invention relates, in particular, to a method for detecting methylation levels of maize nuclear DNA and mitochondrial DNA by using high performance liquid chromatography. Background technique [0002] With the gradual deepening of DNA methylation research, the detection of DNA methylation levels has developed into a rich methodology. The commonly used analysis method for DNA methylation is the MSAP method, which is widely used in the assessment of cytosine methylation in genomes such as rice, cotton and Arabidopsis. However, the isoschizomers (such as HpaII and MspI) used in the MSAP method cannot cleave all methylated sites and have limited recognition range. In contrast, the HPLC method can conveniently, quickly and accurately measure the methylation level of the entire genome, and it can perform large-scale analysis and determination of the DNA methylation level of the entire genome at a macro level, which is precisely the recognition s...

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Application Information

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IPC IPC(8): G01N30/88
Inventor 曹墨菊荣廷昭张晨张艳花汪静卢艳丽刘和洋汪瀚宇王继玥曾文兵汪生庆刘坚高世斌周树峰胡尔良
Owner SICHUAN AGRI UNIV
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