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DNA sulfite conversion and purification method

A sulfite and centrifuge tube technology, applied in the field of transformation DNA purification, can solve the problems of difficult automatic operation, reduced extraction efficiency, cumbersome operation, etc., and achieves the effects of saving experimental operation time, improving extraction purity, and simple equipment and equipment.

Active Publication Date: 2016-04-06
杭州千基生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the main problem of sulfite conversion is that it is necessary to first denature the nucleic acid to be converted with sodium hydroxide, and then perform a long-term conversion and variable temperature process, and a sodium hydroxide solution is required for desulfonation.
In this process, it will lead to severe degradation and fragmentation of DNA
In addition to the problem of DNA degradation, there is no good solution to the DNA purification method after sulfite conversion. The current commercial kits basically use the spin column method for product purification, and require carrier RNA. At the same time, a multi-step washing process is required. Too many results in high DNA loss and reduced extraction efficiency, and this method requires a centrifuge, which is difficult to achieve automatic operation and has low purification efficiency, cumbersome operation, and low yield

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The comparison of different conversion temperatures of embodiment 1

[0033] Acquisition of methylated human genome: Use QIAGEN commercial blood human genome extraction kit to extract human genomic DNA, and then use NEB company's SssI methyltransferase for methylation treatment. The specific operation is carried out according to the manufacturer's instructions. Methylation After the treatment, the methylated DNA was purified using Tiangen’s PCR product purification kit, and the purified DNA was stored in a -20°C refrigerator for later use.

[0034] Sulphite conversion of the present invention and purification specific implementation steps:

[0035] 1. Add 20ul of DNA to be treated (methylated DNA or unmethylated DNA) in a 0.2ml centrifuge tube, and set up 3 copies.

[0036] 2. Add 85ul transformation solution to each portion, then add 35ul protection solution, and mix well.

[0037] 3. Place the three centrifuge tubes in a constant temperature metal bath at 70°C, 80°C...

Embodiment 2

[0054] Embodiment 2 Different purification reagents purify DNA effect contrast

[0055] Acquisition of methylated human genome: same as in Example 1.

[0056] Concrete implementation steps of sulfite conversion of the present invention:

[0057] 1. Add 60ul of DNA to be treated (methylated DNA or unmethylated DNA) into a 1.5ml centrifuge tube.

[0058] 2. Add 85ul transformation solution, then add 15ul protection solution, and mix well.

[0059] 3. Place the centrifuge tube in a constant temperature metal bath at 80°C for 60 minutes;

[0060] 4. Cool to room temperature;

[0061] The conversion solution is an aqueous solution containing 1.5M sodium bisulfite, 1.0M magnesium bisulfite, and 10mM diglyme, pH 5.5.

[0062] The protection solution is an aqueous solution containing 50mM hydroquinone, 100mM water-soluble vitamin C, and 10mM sodium bisulfite, with a pH of 5.0.

[0063] Concrete implementation steps of purification of the present invention:

[0064] 1. Add 600ul ...

Embodiment 3

[0086] Embodiment 3 The inventive method compares with Qiagen commercialization kit method

[0087] Acquisition of methylated human genome: same as in Example 1.

[0088] Sulphite conversion of the present invention and purification specific implementation steps:

[0089] 1. Add 20ul of DNA to be treated (methylated DNA or unmethylated DNA) into a 1.5ml centrifuge tube.

[0090] 2. Add 85ul transformation solution, then add 35ul protection solution, and mix well.

[0091] 3. Place the centrifuge tube in a constant temperature metal bath at 80°C for 60 minutes;

[0092] 4. Cool to room temperature;

[0093] 5. Add 300ul binding solution and 20ul magnetic bead solution to the 1.5ml centrifuge tube containing transformed DNA, mix well, and place at room temperature for 10min;

[0094] 6. Place the centrifuge tube on the magnetic stand for 2 minutes. After the magnetic beads are separated, discard the supernatant;

[0095] 7. Add 1ml of washing solution, mix the magnetic bead...

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Abstract

The invention discloses a DNA sulfite conversion and purification method. The method does need early sodium hydroxide denaturating treatment conducted on nucleic acid, adopts varying-temperature conversion conditions different from a commercialized reagent on the market and can perform a conversion experiment under the constant temperature condition. A magnetic bead method is adopted to perform nucleic acid purification after nucleic acid methylation conversion, a high-salt low pH value is utilized to perform nucleic acid separation and purification, then a low-salt high pH value is utilized to perform elution, the step of performing desulfonating by adopting sodium hydroxide on the market at present is omitted in the purification process, and meanwhile high-conversion-rate, high-quality and high-purity DNA can be obtained only by adopting a one-step washing step. The DNA sulfite conversion and purification method has the advantages of being quick, simple and convenient to operate and improving the conversion efficiency, the extraction efficiency and the extraction purity.

Description

technical field [0001] The invention belongs to the field of nucleic acid methylation conversion and purification, and relates to a method of converting DNA using sulfite under constant temperature conditions, denaturants and DNA protection reagents, and using purification reagents to purify the converted DNA, and is applied to various molecules biological research. Background technique [0002] Methylation refers to the process of catalytic transfer of methyl groups from reactive methyl compounds such as S-adenosylmethionine to other compounds. The most common methylation modifications are DNA methylation and histone methylation, and the present invention involves DNA methylation. [0003] DNA methylation refers to the transfer of active methyl groups to specific bases in the DNA chain under the catalysis of DNA methyltransferase (DNMT) using S-adenosylmethionine as the methyl donor. chemical modification process. DNA methylation generally occurs at the CpG site (cytosin...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/10C12N15/1013C12Q2523/125
Inventor 尹华立郑银娜裘惠良
Owner 杭州千基生物科技有限公司
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