CN111593092A - Method for converting and purifying DNA bisulfite - Google Patents
Method for converting and purifying DNA bisulfite Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 39
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 title claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 51
- 239000011324 bead Substances 0.000 claims abstract description 27
- 239000000243 solution Substances 0.000 claims description 93
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 48
- 238000002156 mixing Methods 0.000 claims description 25
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims description 22
- 239000006228 supernatant Substances 0.000 claims description 20
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 18
- 238000000746 purification Methods 0.000 claims description 18
- 230000009466 transformation Effects 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000007664 blowing Methods 0.000 claims description 12
- YMBCJWGVCUEGHA-UHFFFAOYSA-M tetraethylammonium chloride Chemical compound [Cl-].CC[N+](CC)(CC)CC YMBCJWGVCUEGHA-UHFFFAOYSA-M 0.000 claims description 11
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- 229930003268 Vitamin C Natural products 0.000 claims description 9
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 9
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 9
- 235000019154 vitamin C Nutrition 0.000 claims description 9
- 239000011718 vitamin C Substances 0.000 claims description 9
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- 239000005695 Ammonium acetate Substances 0.000 claims description 8
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- 239000007788 liquid Substances 0.000 claims description 7
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims description 4
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- 230000008859 change Effects 0.000 claims description 4
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000012154 double-distilled water Substances 0.000 claims description 2
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- 230000011987 methylation Effects 0.000 abstract description 12
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- 238000011529 RT qPCR Methods 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 abstract 1
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- 238000007710 freezing Methods 0.000 abstract 1
- 238000004153 renaturation Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 56
- 238000001514 detection method Methods 0.000 description 15
- 230000007067 DNA methylation Effects 0.000 description 7
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- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 4
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- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
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- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
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- 238000003753 real-time PCR Methods 0.000 description 1
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- 229940079827 sodium hydrogen sulfite Drugs 0.000 description 1
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- 229940035893 uracil Drugs 0.000 description 1
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Abstract
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.
Description
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.
Example 1 bisulfite conversion and purification of DNA Using different conversion solutions
1. Preparation of bisulfite conversion solution
(1) Preparation of mother solution of each reagent
3M sodium hydroxide solution: 3g of sodium hydroxide granules, adding ddH2And O is metered to 25 mL.
320mM hydroquinone solution: 0.88g of hydroquinone, plus ddH2And O is metered to 25 mL.
100mM tetraethylammonium chloride solution: 0.42g of tetraethylammonium chloride, ddH2And O is metered to 25 mL.
5M ammonium acetate solution: 19.25g ammonium acetate, plus ddH2And O is metered to 50 mL.
(2) Preparation of transformation solution (System 10mL)
Transformation liquid A: 4.8g of sodium bisulfite, 800. mu.L of 3M sodium hydroxide solution, 200. mu.L of 320mM hydroquinone solution, 0.148g of water-soluble vitamin C, 100. mu.L of 100mM tetraethylammonium chloride solution and 0.288g of guanidine hydrochloride are mixed, stirred until completely dissolved, and water is added to make up to 10 mL.
And (3) conversion solution B: mixing 4.5g of sodium bisulfite, 600. mu.L of 3M sodium hydroxide solution, 300. mu.L of 320mM hydroquinone solution, 0.1g of water-soluble vitamin C, 150. mu.L of 100mM tetraethylammonium chloride solution and 0.2g of guanidine hydrochloride, stirring until completely dissolved, and adding water to make up to 10 mL.
And (3) conversion solution C: 4.6g of sodium bisulfite, 900. mu.L of 3M sodium hydroxide solution, 250. mu.L of 320mM hydroquinone solution, 0.3g of water-soluble vitamin C, 200. mu.L of 100mM tetraethylammonium chloride solution and 0.4g of guanidine hydrochloride are mixed, stirred until completely dissolved, and water is added to make up to 10 mL.
2. DNA bisulfite conversion and purification
The DNA sample used in this example was human genomic DNA extracted by a commercial human genome extraction kit.
The method for converting and purifying the bisulfite comprises the following specific implementation steps:
(1) taking 3 centrifuge tubes of 1.5mL, and respectively adding 25 mu L of DNA to be processed;
(2) incubating at 95 ℃ for 5min, immediately taking out each centrifuge tube, placing on ice, respectively adding 150 mu L of prepared transformation liquid A, B, C, inverting or shaking for uniform mixing, and centrifuging for a short time to collect reaction liquid to the bottom of the tube;
(3) placing each centrifuge tube in a PCR instrument, and carrying out thermal cycling reaction according to the conditions set in the following table:
(4) to each reaction product, 350. mu.L (2X) of a magnetic bead solution was added, and the mixture was incubated at room temperature for 5min to bind DNA to the magnetic beads;
(5) keeping each centrifuge tube on a magnetic frame, and carefully removing supernatant after the solution in the centrifuge tube is clarified (about 5 min);
(6) keeping each centrifuge tube on a magnetic frame, adding 800 μ L of freshly prepared 70% ethanol solution to rinse the magnetic beads, incubating at room temperature for 60s, and carefully removing the supernatant;
(7) directly adding 60 mu L of TE buffer solution into each rinsed centrifugal tube, gently blowing and uniformly mixing by using a pipette gun, then adding 6 mu L of 3M sodium hydroxide solution, reversing or uniformly mixing by oscillation, and incubating for 15min at 42 ℃;
(8) respectively adding 16.5 μ L of 5M ammonium acetate solution (the amount of the added 5M ammonium acetate solution is 0.25 times of the total solution volume), inverting or shaking for mixing, correspondingly adding 165 μ L of magnetic bead solution into the obtained solution, gently blowing and mixing by using a pipette, and incubating at room temperature for 10min to bind the DNA to the magnetic beads;
(9) keeping each centrifuge tube on a magnetic frame, and carefully removing supernatant after the solution in the centrifuge tube is clarified (about 5 min);
(10) keeping each centrifuge tube on a magnetic frame, adding 800 μ L of freshly prepared 70% ethanol solution to rinse the magnetic beads, incubating at room temperature for 60s, and carefully removing the supernatant;
(11) repeating the step (10) once;
(12) keeping each centrifugal tube on a magnetic frame, and opening the cover to dry for 10min at room temperature;
(13) the tubes were removed from the magnetic stand and 62.5. mu.L of ddH was added to each tube2And O, lightly blowing and uniformly mixing by using a pipette gun, keeping each centrifuge tube on a magnetic frame, and carefully sucking 60 mu L of supernatant into a new centrifuge tube after the solution in the centrifuge tube is clarified (about 5 min).
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.
The TE buffer contained 10mM Tri-HCl and 1mM EDTA, and had a pH of 7.5 to 8.5.
Methylation detection was performed by real-time fluorescence PCR on 1. mu.L of DNA transformed with the three different transformation solutions (A, B, C).
And (3) detection results: the conversion solution A, B, C is used for carrying out bisulfite conversion of human genome DNA, and MSP primers are used for carrying out real-time fluorescence PCR detection on methylation conditions, and the results show that Ct values of all groups are basically equivalent. The method for converting and purifying the DNA bisulfite has high conversion efficiency and recovery rate, and is simple and easy to operate.
Example 2 comparison of the method of the invention with the ZYMO RESEARCH commercial kit method
The DNA sample used in this example was human genomic DNA extracted by a commercial human genome extraction kit.
1. The method for converting and purifying the bisulfite comprises the following specific implementation steps:
(1) adding 20 mu L of sample to be processed into a 1.5mL centrifuge tube;
(2) incubating at 95 ℃ for 3min, immediately taking out the centrifuge tube, placing on ice, adding 100 mu L of the prepared transformation liquid A (same as in example 1), reversing or shaking for uniform mixing, and centrifuging for a short time to collect the reaction liquid to the bottom of the tube;
(3) the centrifuge tube was placed in a PCR instrument and the thermocycling reaction was performed according to the conditions set forth in the following table:
(4) adding 240. mu.L (2X) of a magnetic bead solution to the reaction product, and incubating at room temperature for 10min to bind the DNA to the magnetic beads;
(5) the centrifuge tube was held on a magnetic rack and after the solution in the centrifuge tube cleared (about 5min), the supernatant was carefully removed;
(6) the tube was held in a magnetic rack, 500 μ L of freshly prepared 80% ethanol solution was added to rinse the beads, incubated at room temperature for 30s, and the supernatant carefully removed;
(7) directly adding 50 μ L of TE buffer solution into the rinsed centrifugal tube, gently blowing and uniformly mixing by using a pipette gun, adding 5 μ L of 3M sodium hydroxide solution, reversing or shaking for uniformly mixing, and incubating for 20min at 42 ℃;
(8) adding 13.75 μ L of 5M ammonium acetate solution (the amount of the added 5M ammonium acetate solution is 0.25 times of the total solution volume), reversing or shaking for mixing, adding 137.5 μ L of magnetic bead solution into the obtained solution, gently blowing and mixing by using a pipette, and incubating at room temperature for 10min to bind the DNA to the magnetic beads;
(9) the centrifuge tube was held on a magnetic rack and after the solution in the centrifuge tube cleared (about 5min), the supernatant was carefully removed;
(10) the tube was held in a magnetic rack, 500 μ L of freshly prepared 80% ethanol solution was added to rinse the beads, incubated at room temperature for 30s, and the supernatant carefully removed;
(11) repeating the step (10) once;
(12) keeping the centrifugal tube on a magnetic frame, and uncovering and drying for 5min at room temperature;
(13) will be centrifugedThe tube was removed from the magnetic stand and 52.5. mu.L of ddH was added2And O, lightly blowing and uniformly mixing by using a pipette gun, keeping the centrifugal tube on a magnetic frame, and carefully sucking 50 mu L of supernatant into a new centrifugal tube after the solution in the centrifugal tube is clarified (about 5 min).
The conversion solution A contained 4.61M sodium hydrogen sulfite, 0.24M sodium hydroxide, 6.4mM hydroquinone, 84.0mM water-soluble vitamin C, 1.0mM tetraethylammonium chloride and 0.30M guanidine hydrochloride.
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.
The TE buffer contained 10mM Tri-HCl and 1mM EDTA, and had a pH of 7.5 to 8.5.
2 sets of parallel experiments were performed according to the above experimental procedure, and two sets of DNA obtained after transformation were named BS-1 and BS-2, respectively, and 1. mu.L of each DNA was subjected to methylation real-time fluorescence PCR detection (note: the detection index T16953 is linked to the base T after transformation, and the detection index C7302 is linked to the base C not transformed).
2. ZYMO RESEARCH commercialized kit method
Bisulfite conversion and purification of human genomic DNA were performed according to the protocol of ZYMO RESEARCH commercial kit instructions. 1 μ L of the transformed DNA was subjected to methylation real-time fluorescence PCR detection, and 2 sets of replicates were performed.
The results are shown in the following table:
in the above table, the methylation detection index and the unmethylated detection index are the detection of methylation of different treated DNA templates using methylation specific primers and unmethylated specific primers, respectively, wherein the negative control template is water and the positive control template is human genomic DNA that has not been transformed. The combination of FIG. 1 and FIG. 2 shows that the conversion and purification method of the invention has the same detection result as ZYMO RESEARCH kit, but the method of the invention has the advantages of simpler operation, short time consumption, low cost of used reagents, high DNA conversion efficiency and purification rate after treatment, high detection sensitivity, and contribution to full-automatic and standardized operation of methylation RESEARCH.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (7)
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.
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