阅读说明：本技术 一种dna亚硫酸氢盐转化及纯化的方法 (Method for converting and purifying DNA bisulfite ) 是由 易吉 解青青 李泽卿 于 2020-05-29 设计创作，主要内容包括：本发明公开了一种DNA亚硫酸氢盐转化及纯化的方法,采用热变性法95℃处理DNA样本3-5分钟后,立即冰置以防止复性,再通过变温热循环进行转化,然后采用磁珠法对转化的DNA进行纯化。本发明方法操作简单方便,耗时短,并能够得到高转化率、高质量的DNA,提高了转化后的qPCR及其他分析技术的灵敏度,有利于甲基化研究的全自动化及标准化操作。(The invention discloses a method for converting and purifying DNA bisulfite, which comprises the steps of treating a DNA sample for 3-5 minutes at 95 ℃ by a thermal denaturation method, immediately freezing to prevent renaturation, converting by a temperature-variable thermal cycle, and purifying the converted DNA by a magnetic bead method. The method is simple and convenient to operate, consumes short time, can obtain DNA with high conversion rate and high quality, improves the sensitivity of qPCR and other analysis technologies after conversion, and is favorable for full-automatic and standardized operation of methylation research.)

1. A method for bisulfite conversion and purification of DNA, comprising the steps of:

s1, adding 20-25 mu L of DNA to be processed into a centrifuge tube;

s2, incubating at 95 ℃ for 3min-5min, immediately taking out the centrifuge tube, placing on ice, adding 100-150 μ L of the transformation solution, reversing or shaking for uniform mixing, and centrifuging for a short time;

s3, placing the centrifuge tube in an instrument capable of setting a temperature change program to perform variable temperature thermal cycle reaction:

s4, adding 240-350 mu L (2X) magnetic bead solution into the reaction product, and incubating at room temperature for 5-10 min;

s5, keeping the centrifugal tube on a magnetic frame, and carefully removing supernatant after the solution in the centrifugal tube is clarified;

s6, keeping the centrifugal tube on a magnetic rack, adding 500-800 μ L of freshly prepared washing liquid, incubating at room temperature for 30-60S, and carefully removing the supernatant;

s7, adding 50-60 μ L TE buffer solution, gently blowing and mixing with a pipette gun, adding 5-6 μ L3M sodium hydroxide solution, reversing or shaking for mixing, and incubating at 42 ℃ for 15-20 min;

s8, adding a 5M ammonium acetate solution with the volume 0.25 time that of the solution in the centrifuge tube in the step S7, reversing or shaking for uniform mixing, adding a magnetic bead solution with the volume 2.5 times that of the solution in the centrifuge tube in the step S7, slightly blowing and uniformly mixing by using a pipette gun, and incubating for 10min at room temperature;

s9, keeping the centrifugal tube on a magnetic frame, and carefully removing supernatant after the solution in the centrifugal tube is clarified;

s10, keeping the centrifugal tube on a magnetic rack, adding 500-800 μ L of freshly prepared washing liquid, incubating at room temperature for 30-60S, and carefully removing the supernatant;

s11, repeating the step S10 once;

s12, keeping the centrifugal tube on a magnetic frame, and opening the cover to dry for 5-10 min at room temperature;

s13, taking out the centrifugal tube from the magnetic frame, adding 52.5-62.5 μ L ddH₂O, lightly blowing and uniformly mixing by using a pipette gun, keeping the centrifugal tube on a magnetic frame, and carefully sucking 50-60 mu L of supernatant into a new centrifugal tube after the solution in the centrifugal tube is clarified;

the conversion solution in step S2 was an aqueous solution containing 4.32M-4.61M sodium bisulfite, 0.18M-0.27M sodium hydroxide, 6.4mM-9.6mM hydroquinone, 0.06M-0.17M water soluble vitamin C, 1.0mM-2.0mM tetraethylammonium chloride, and 0.21M-0.42M guanidine hydrochloride.

2. The method for bisulfite conversion and purification of DNA according to claim 1, characterized in that: the conversion solution was an aqueous solution containing 4.61M sodium bisulfite, 0.24M sodium hydroxide, 6.4mM hydroquinone, 84.0mM water-soluble vitamin C, 1.0mM tetraethylammonium chloride, and 0.30M guanidine hydrochloride.

3. The method for bisulfite conversion and purification of DNA according to claim 1, characterized in that: in step S3, the apparatus capable of setting the temperature variation program is a PCR apparatus.

4. The method for bisulfite conversion and purification of DNA according to claim 1, wherein in step S3, the temperature-variable thermal cycling reaction comprises the steps of:

s3-1, incubating at 95 ℃ for 30S;

s3-2, incubating for 20min at 58 ℃;

s3-3, incubating at 95 ℃ for 10S;

s3-4, incubating for 20min at 58 ℃;

s3-5, repeating the steps S3-3 and S3-4, 4-6 times;

s3-6, and keeping at 4 ℃.

5. The method for bisulfite conversion and purification of DNA according to claim 1, characterized in that: the magnetic bead solution is an aqueous solution containing 1mM EDTA, 10mM Tris-HCl, 2.5M sodium chloride, 19% (v/v) PEG-8000, 0.6% (v/v) Tween20 and 2% (v/v) magnetic beads.

6. The method for bisulfite conversion and purification of DNA according to claim 1, characterized in that: the washing solution is 70-80% (v/v) ethanol solution.

7. The method for bisulfite conversion and purification of DNA according to claim 1, characterized in that: the TE buffer contained 10mM Tri-HCl and 1mM EDTA, pH 7.5-8.5.

Technical Field

The invention relates to the field of nucleic acid conversion and purification, in particular to a method for converting and purifying DNA bisulfite.

Background

DNA methylation is a form of chemical modification of DNA that alters genetic expression without altering the DNA sequence. DNA methylation refers to the transfer of an active methyl group to a specific base in a DNA strand by DNA methyltransferase using S-adenosylmethionine as a methyl donor. DNA methylation typically occurs at CpG sites. Cytosine is converted to 5-methylcytosine by DNA methyltransferase. Approximately 80% -90% of the CpG sites in a human gene have been methylated, but in certain regions, such as CpG islands rich in cytosine and guanine, are unmethylated. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression. DNA methylation can shut down the activity of certain genes, and demethylation induces reactivation and expression of genes. A great deal of research in recent years shows that DNA abnormal methylation is closely related to the occurrence, development and canceration of tumors. The intensive study of DNA methylation levels is a necessary trend and will likely become a routine technical indicator in medicine for the treatment of certain diseases.

There are many ways to study DNA methylation, and one technique that researchers often use is bisulfite conversion, among many. DNA will convert unmethylated cytosines to uracil during bisulfite treatment, while methylated cytosines remain unchanged. Subsequent analysis by sequencing, quantitative PCR or chip analysis to compare the sequence of the treated and untreated DNA will allow determination of which sites in the DNA sequence are methylated.

The basic steps of the current bisulfite conversion are: high-temperature cracking of DNA, variable-temperature conversion of bisulfite, desulfonation, purification and recovery. However, the bisulfite conversion process often suffers from a number of problems: (1) a long-time conversion and temperature change process is needed, and sodium hydroxide solution is needed for removing sulfonic acid groups; (2) the transformation efficiency is low, sometimes the transformation is not complete, sometimes the transformation is excessive; (3) the recovery rate is not high, a large amount of DNA can be lost, and sometimes even reaches 50%; (4) due to the harsh bisulfite treatment conditions, severe degradation and fragmentation of the DNA may occur, thereby reducing the sensitivity of PCR and subsequent analytical techniques. Therefore, based on the rapid development of epigenetics at present, bisulfite conversion and purification methods are in need of improvement.

Disclosure of Invention

In view of the above, the present invention provides a method for converting and purifying DNA bisulfite, which optimizes and improves the existing method to make the DNA conversion rate reach 100%, and adopts a magnetic bead method to purify the converted DNA, so as to obtain DNA with high conversion efficiency, high quality and high purity, and is favorable for full automation and standardized operation of methylation research, and is used for subsequent molecular biology research, especially clinical molecular diagnosis.

The technical scheme of the invention is realized as follows:

the invention provides a method for converting and purifying DNA bisulfite, which comprises the following steps:

s1, adding 20-25 mu L of DNA to be processed into a centrifuge tube;

s2, incubating at 95 ℃ for 3min-5min, taking out the centrifuge tube, placing on ice, adding 100-150 μ L of the transformation solution, reversing or shaking for mixing uniformly, and centrifuging for a short time;

s3, placing the centrifuge tube in an instrument capable of setting a temperature change program to perform variable temperature thermal cycle reaction:

s4, adding 240-350 mu L (2X) magnetic bead solution into the reaction product, and incubating at room temperature for 5-10 min;

s5, keeping the centrifugal tube on a magnetic frame, and carefully removing supernatant after the solution in the centrifugal tube is clarified;

s6, keeping the centrifugal tube on a magnetic rack, adding 500-800 μ L of freshly prepared washing liquid, incubating at room temperature for 30-60S, and carefully removing the supernatant;

s7, adding 50-60 μ L TE buffer solution, gently blowing and mixing with a pipette gun, adding 5-6 μ L3M sodium hydroxide solution, reversing or shaking for mixing, and incubating at 42 ℃ for 15-20 min;

s8, adding a 5M ammonium acetate solution with the volume 0.25 time that of the solution in the centrifuge tube in the step S7, reversing or shaking for uniform mixing, adding a magnetic bead solution with the volume 2.5 times that of the solution in the centrifuge tube in the step S7, slightly blowing and uniformly mixing by using a pipette gun, and incubating for 10min at room temperature;

s9, keeping the centrifugal tube on a magnetic frame, and carefully removing supernatant after the solution in the centrifugal tube is clarified;

s10, keeping the centrifugal tube on a magnetic rack, adding 500-800 μ L of freshly prepared washing liquid, incubating at room temperature for 30-60S, and carefully removing the supernatant;

s11, repeating the step S10 once;

s12, keeping the centrifugal tube on a magnetic frame, and opening the cover to dry for 5-10 min at room temperature;

s13, taking out the centrifugal tube from the magnetic frame, adding 52.5-62.5 μ L ddH₂O, lightly blowing and uniformly mixing by using a pipette gun, keeping the centrifugal tube on a magnetic frame, and carefully sucking 50-60 mu L of supernatant into a new centrifugal tube after the solution in the centrifugal tube is clarified;

the conversion solution in step S2 was an aqueous solution containing 4.32M-4.61M sodium bisulfite, 0.18M-0.27M sodium hydroxide, 6.4mM-9.6mM hydroquinone, 0.06M-0.17M water soluble vitamin C, 1.0mM-2.0mM tetraethylammonium chloride, and 0.21M-0.42M guanidine hydrochloride.

Based on the above technical solution, preferably, the conversion solution is an aqueous solution containing 4.61M sodium bisulfite, 0.24M sodium hydroxide, 6.4mM hydroquinone, 84.0mM water-soluble vitamin C, 1.0mM tetraethylammonium chloride and 0.30M guanidine hydrochloride.

In addition to the above technical solution, preferably, in step S3, the apparatus capable of setting the temperature variation program is a PCR apparatus.

On the basis of the above technical solution, preferably, in step S3, the temperature-variable thermal cycle reaction includes the following steps:

s3-1, incubating at 95 ℃ for 30S;

s3-2, incubating for 20min at 58 ℃;

s3-3, incubating at 95 ℃ for 10S;

s3-4, incubating for 20min at 58 ℃;

s3-5, repeating the steps S3-3 and S3-4, 4-6 times;

s3-6, and keeping at 4 ℃.

Based on the above technical scheme, preferably, the magnetic bead solution is an aqueous solution containing 1mM EDTA, 10mM Tris-HCl, 2.5M sodium chloride, 19% (v/v) PEG-8000, 0.6% (v/v) Tween20 and 2% (v/v) magnetic beads.

On the basis of the technical scheme, preferably, the washing solution is 70-80% (v/v) ethanol solution.

Based on the above technical scheme, preferably, the TE buffer solution contains 10mM Tri-HCl and 1mM EDTA, and has a pH value of 7.5-8.5.

Compared with the prior art, the method for converting and purifying the DNA bisulfite has the following beneficial effects:

(1) the conversion solution adopted in the conversion process of the method can ensure that the DNA conversion is more effective and complete, and hydroquinone and water-soluble vitamin C in the conversion solution form a protective solution so that a DNA sample stably exists in the experimental reaction; tetraethylammonium chloride is used as a catalyst, and can promote DNA denaturation and conversion reaction; guanidine hydrochloride is used as a protein denaturant to remove protein in a sample, and DNA is combined to improve the yield of the DNA after conversion; wherein, the sodium hydroxide plays a role in adjusting the pH of the solution, so that the DNA is in the optimal pH value of the solution and can stably exist;

(2) the method has the advantages that the conversion is carried out through the temperature change effect, so that the DNA conversion is more sufficient, the DNA is extracted and purified by combining the magnetic bead method, and the DNA is obtained according to the detection result data;

(3) DNA conversion and purification are carried out by using the kit, about 3.5 hours is needed for processing one sample, and 4.5 hours is needed for processing a batch of samples (16 samples); the invention adjusts the transformation time and the purification method of the transformed DNA, takes 3 hours for processing a sample, only needs 3.5 hours for processing a batch of samples (16 samples), and has more advantages when processing a large batch of samples in comparison.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a real-time fluorescent PCR amplification curve for methylation index detection after human genomic DNA has been transformed and purified by the method of the present invention and the method of the commercial kit.

FIG. 2 is a real-time fluorescent PCR amplification curve for unmethylated index detection after conversion and purification of human genomic DNA by the method and commercial kit of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.