CN110079521A - The method and kit of DNA sulphite conversion processing - Google Patents

Abstract

Present disclose provides a kind of methods of DNA sulphite conversion processing, comprising: (a) prepares DNA solution to be processed；(b) DNA solution is added in transforming agent and protective agent; it is mixed to obtain DNA mixed solution; transforming agent be include the aqueous solution selected from least one of Sodium Metabisulfite, sodium hydrogensulfite, bisulfite amine, magnesium bisulfite, ammonium sulfite, protective agent is the aqueous solution for including at least one of quinhydrones, watermiscible vitamin E component and trehalose or agarose；And (c) DNA mixed solution is set and is converted under prescribed conditions, DNA is obtained and converts solution.According to the disclosure, be capable of providing it is a kind of can either efficiently, fast implement the sulphite conversion of DNA, and the method that the integrality of DNA can be effectively ensured.

Description

The method and kit of DNA sulphite conversion processing

Technical field

The present invention is more particularly directed to the methods and kit of a kind of DNA sulphite conversion processing.

Background technique

Methylation modification is a kind of mode that gene realizes epigenetic regulation, right in the case where not changing DNA sequence dna The expression model and Genome stability of gene play important regulating and controlling effect.DNA methylation occurs mainly in the site CpG, warp It is 5-methylcytosine that dnmt rna, which is catalyzed Cytosines, and CpG methylation is inversely proportional with transcriptional activity, biology Body influences cell and entire by changing the CpG methylation of gene promoter region, the expression of controlling gene The character of organism.Therefore, the methylation level of genomic DNA and the proliferation of cell, differentiation and canceration are closely related.At present The study found that the methylation level of several genes is abnormal related to the generation of cancer, such as the hair of SEPT9 methylation and colorectal cancer Raw, the generation of SHOX2 gene and RASSF1A gene methylation and lung cancer is closely related, by detecting these cancer markers Methylation level can realize the Non-invasive detection of cancer, improve the early detection rate of cancer.

There are many method for detecting DNA methylation level, and one of the most common method is based on DNA sulphite conversion skill Art, cytimidine unmethylated in DNA can be changed into uracil by sulphite processing, and the cytimidine to methylate is kept not Become, to distinguish methylate DNA and non-methylate DNA.Pyrosequencing, methylation-specific after the conversion of DNA sulphite The analysis means such as PCR, restriction endonuclease analysis and methylation sensitive high-resolution solubility curve analytic approach, are determining The conventional means of DNA methylation level and methylation sites.Therefore, transformation efficiency, the rate of recovery, the mould of sulphite processing DNA Plate quality etc. directly affects subsequent detection effect.

Traditional sulphite method for transformation is the integrality for guaranteeing DNA, and conversion condition is more mild, and required time is longer (such as need 20 hours or so).With the improvement of technology, the sulphite conversion reagent box being commercialized at present can realize DNA Rapid conversion, effectively shorten the time of gene DNA sulphite conversion, but significantly improve the fragmentation and drop of DNA Solution is horizontal, so that the DNA of long segment shortens, causes the verification and measurement ratio of the methylation sites of long segment to decline, limits to gene first The application of research and the detection of baseization level, thus there is an urgent need to can efficiently, fast implement DNA sulphite conversion and it is pure The method of change.

Summary of the invention

The disclosure in view of the above-mentioned prior art situation and complete, its purpose is to provide one kind can either efficiently, it is fast Speed realizes the sulphite conversion of DNA, and the DNA sulphite conversion processing of the integrality of the DNA of long segment can be effectively ensured Method.

For this purpose, disclosure first aspect provides a kind of method of DNA sulphite conversion processing, may include: (a) Prepare DNA solution to be processed；(b) DNA solution is added in transforming agent and protective agent, is mixed to obtain DNA mixing Solution, the transforming agent can be to include selected from Sodium Metabisulfite, sodium hydrogensulfite, bisulfite amine, magnesium bisulfite, Asia The aqueous solution of at least one of ammonium sulfate, the protective agent can be include at least one of quinhydrones, watermiscible vitamin E component With the aqueous solution of trehalose or agarose；And (c) the DNA mixed solution is set and is converted under prescribed conditions, is obtained DNA converts solution.

In the method for DNA sulphite conversion processing involved in the disclosure, DNA is deposited in protective agent with transforming agent jointly It is converted in case.In this case, transforming agent can prevent DNA in conversion process under the action of protective agent Middle generation fragmentation and degradation reduce the damage that DNA is subject in the conversion process, therefore it is sub- that DNA long fragment can be promoted to carry out Sulfate conversion, so in subsequent detection carry out long segment methylation sites detection.

In addition, optionally, in the transforming agent, solute contains in the method involved in the first aspect of the disclosure Amount is 2 to 5mol/L, and the pH value of the transforming agent is 5.0 to 5.5, and in step (c), the rated condition is at 50 to 70 DEG C Lower heating 30 to 60min.In this case, the conversion of DNA sulphite can be accelerated, be conducive to that sulfurous acid is efficiently rapidly completed Salt conversion.

In addition, before step (c), the DNA can be mixed in the method involved in the first aspect of the disclosure Solution is placed in heating 5 to 10min at 95 to 98 DEG C and carries out DNA thermal denaturation.In this case, DNA unwinding can be made to be formed as Single stranded DNA is conducive to the progress of DNA sulphite conversion.

In addition, in the method involved in the first aspect of the disclosure, optionally, before step (c), by the way that pH is added Regulator carries out DNA alkaline denaturation to the DNA mixed solution, and the pH adjusting agent is in sodium hydroxide, magnesium hydroxide, ammonium hydroxide At least one, the additive amount of the pH adjusting agent is 0.2 to 0.3mol/L.It in this case, can be by DNA mixed solution Become alkalinity, so as to make DNA unwinding be formed as single stranded DNA, is conducive to the progress of DNA sulphite conversion.

In addition, the protective agent can be quinhydrones and trehalose in the method involved in the first aspect of the disclosure Aqueous solution, wherein the content of the quinhydrones can be 0.03 to 0.3mol/L, the content of the trehalose can for 0.3 to 0.5mol/L.In this case, quinhydrones can stablize above-mentioned transforming agent, make the not oxidized cracking of DNA, while trehalose or fine jade Lipolysaccharide can protect DNA, and thereby, it is possible to reduce DNA fragmentation and degradation occur in the conversion process, protection DNA carries out sulfurous Hydrochlorate conversion, so as to the detection for long segment methylation sites.

In addition, can also include: that (d) converts solution to the DNA in the method involved in the first aspect of the disclosure It is purified, to obtain DNA long fragment solution.In this case, the sulphite conversion in DNA conversion solution can be recycled DNA long fragment.

In addition, the step (d) may include: that (d1) will be combined in the method involved in the first aspect of the disclosure Liquid and magnetic bead are added in the DNA conversion solution, are stood after mixing, remove supernatant to obtain the first DNA conjugate；(d2) First cleaning solution is added to the first DNA conjugate, is stood after mixing, supernatant is removed, sulfurous acid group is then added Liquid is removed, is stood after mixing, then remove supernatant to obtain the 2nd DNA conjugate；(d3) the second cleaning solution is added to described It in 2nd DNA conjugate, is stood after mixing, removes supernatant, addition eluent is stood after mixing, removal magnetic bead, described in acquisition The DNA solution of purifying.In this case, the DNA removal after sulfurous acid group removal liquid can help sulphite to convert is sub- Sulfate group can be realized quickly using liquid, sulfurous acid group removal liquid etc., and combination magnetic bead progress paramagnetic particle method purifying is combined Obtain high conversion, the DNA that the sulphite of high quality converts.

In addition, in the method involved in the first aspect of the disclosure, the combination liquid can be include guanidine thiocyanate, sugar Former, sodium iodide and ethyl alcohol aqueous solution, wherein the content of the guanidine thiocyanate can be 2 to 6mol/L, and the glycogen content can Think 0.05 to 0.1mol/L, the content of the sodium iodide can be 0.5 to 2mol/L, and the volume fraction of the ethyl alcohol can be 10% to 40%.In this case, guanidine thiocyanate can make with functional group (hydroxy or carboxy) collective effect of magnetic bead surfaces For the DNA of sulphite conversion in conjunction with magnetic bead, glycogen and ethyl alcohol can more retain the DNA that sulphite converts.

In addition, the partial size of the magnetic bead can be 100 to 500nm in the method involved in the first aspect of the disclosure, And the silica modified by hydroxy or carboxy can be coated on the surface of the magnetic bead.In this case, magnetic bead energy Enough DNA preferably screened and purify the sulphite conversion in DNA conversion solution.

The second aspect of the disclosure provides a kind of kit of DNA sulphite conversion, and the kit can wrap Include transforming agent used in above-mentioned method and protective agent.In this case, it can be realized and be quickly obtained high conversion, height The DNA of the sulphite conversion of quality.

According to the disclosure, be capable of providing it is a kind of can either efficiently, fast implement the sulphite conversion of DNA, and can be effective Guarantee the method and kit of the DNA sulphite conversion processing of the integrality of DNA.

Detailed description of the invention

Fig. 1 shows the flow diagram of the method for DNA sulphite conversion processing involved in the disclosure.

Fig. 2 shows the flow diagrams of the method for purifying DNA sulphite conversion solution involved in the disclosure.

Fig. 3 shows the result figure of the detection of DNA sulphite transformation efficiency involved in the disclosure.

Fig. 4 shows the result figure of the detection of methylation status of PTEN promoter involved in the disclosure.

Fig. 5 shows the result figure of the detection of methylation status of PTEN promoter involved in the disclosure.

Fig. 6 shows the amplified fragments size detection of the DNA of the conversion of DNA sulphite involved in the disclosure and purifying Result figure.

Specific embodiment

Hereinafter, explaining the preferred embodiment of the disclosure in detail with reference to attached drawing.In the following description, for identical Component assign identical symbol, the repetitive description thereof will be omitted.Scheme in addition, attached drawing is only schematical, the mutual ruler of component Very little shape of ratio or component etc. can be with actual difference.

In the disclosure, DNA long fragment generally refers to the DNA fragmentation that length is 100bp to 1000bp.Preferably, this DNA long fragment involved in open is 400bp to 750bp.In the disclosure, the DNA long fragment of 400bp to 750bp is more advantageous In retaining methylation sites, facilitate the detection to methylation sites.

Fig. 1 shows the flow diagram of the method for DNA sulphite conversion processing involved in the disclosure.Fig. 2 shows The flow diagram of the method for purifying DNA sulphite conversion solution involved in open.

A kind of method of DNA sulphite conversion processing involved by present embodiment may include: that preparation is to be processed DNA solution (step S10)；DNA solution is added in transforming agent and protective agent, is mixed to obtain DNA mixed solution (step S20)；And DNA mixed solution is set and is converted under prescribed conditions, DNA conversion solution (step S30) is obtained.

In the method for DNA sulphite conversion processing involved in present embodiment, DNA is common with transforming agent in protective agent In the presence of converted.In this case, transforming agent can prevent DNA transformed under the action of protective agent Fragmentation and degradation occur in journey, reduces the damage that DNA is subject in the conversion process, therefore DNA long fragment can be promoted to carry out Sulphite converts, and then the detection of long segment methylation sites is carried out in subsequent detection.

In addition, in some instances, in step slo, can by Chelex100 method, organic method, paramagnetic particle method, saltout The methods of method, NaOH method, kit extraction extract DNA, to obtain DNA solution to be processed.

In addition, in some instances, in step S20, transforming agent can be to include selected from Sodium Metabisulfite, sulfurous acid The aqueous solution of at least one of hydrogen sodium, bisulfite amine, magnesium bisulfite, ammonium sulfite.In this case, sulphite energy It is enough that sulphite conversion is carried out to DNA.

In addition, in some instances, in transforming agent, the content of solute can be 2 to 5mol/L, and the pH value of transforming agent can 5.0 to 5.5 are thought, it is specified that condition can be the heating 30 to 60min at 50 to 70 DEG C in step (c).In such case Under, the conversion of DNA sulphite can be accelerated, be conducive to that sulphite conversion is efficiently rapidly completed.In addition, the sulfurous acid of high concentration Salt can accelerate the conversion of DNA sulphite, be conducive to efficiently to be rapidly completed sulphite conversion, and transforming agent pH value be 5.0 to 5.5 can sulphite ingredient in stable conversion agent, so that sulphite is not allowed facile hydrolysis, and suitable conversion condition also has It is converted conducive to the sulphite of DNA.

For example, in one example, transforming agent can be Sodium Metabisulfite containing 5mol/L and 1mol/L sodium hydrogensulfite Aqueous solution, pH value 5.0.In another example, transforming agent can also be the aqueous solution of the sodium hydrogensulfite containing 6mol/L, Or the aqueous solution of the magnesium bisulfite containing 5mol/L, and the pH value of transforming agent can be 5.1,5.2,5.3,5.4,5.5.

In addition, in some instances, in step S20, protective agent can be include quinhydrones, in watermiscible vitamin E extremely A kind of few component and trehalose or agarose.In this case, quinhydrones can stable conversion agent, make the not oxidized cracking of DNA, Trehalose or agarose can protect DNA simultaneously, and thereby, it is possible to reduce DNA fragmentation and degradation, energy occur in the conversion process The methylation sites of the DNA long fragment in DNA solution are enough protected not to be destroyed, it is thus possible to carry out long segment methylation sites Detection.

In addition, in some instances, protective agent can be the aqueous solution of quinhydrones and trehalose.In addition, the content of quinhydrones can Think 0.03 to 0.3mol/L, the content of trehalose can be 0.3 to 0.5mol/L.For example, in one example, protective agent can Think the aqueous solution of quinhydrones and 0.5mol/L trehalose including 0.1mol/L.In another example, protective agent can be packet Include the quinhydrones of 0.3mol/L and the aqueous solution of 0.3mol/L trehalose.In addition, in yet another example, protective agent can be to include The quinhydrones of 0.03mol/L and the aqueous solution etc. of 0.5mol/L trehalose.

In some instances, protective agent can be the aqueous solution of quinhydrones and agarose.In addition, the content of quinhydrones can be 0.03 to 0.3mol/L, the content of agarose can be 0.5%-1.5%.For example, in one example, protective agent can be packet Include the aqueous solution of the quinhydrones of 0.03mol/L and 1.5% agarose.In another example, protective agent can be to include The quinhydrones of 0.3mol/L and 0.5% agarose aqueous solution.In addition, in yet another example, protective agent can be to include The quinhydrones of 0.2mol/L and 1% agarose aqueous solution etc..

In addition, DNA solution can be added in transforming agent and protective agent in step S20, mixed mixed to obtain DNA Solution is closed, also DNA solution can be added with protective agent in transforming agent and mix acquisition DNA sulphite conversion reaction system.Separately Outside, in some instances, the reaction volume of DNA sulphite conversion reaction system can be 50 μ L to 200 μ L, such as 50 μ L, 100 μ L, 150 μ L or 200 μ L.

In addition, in some instances, in the reaction volume of DNA sulphite transformation system, transforming agent and protectant Volume ratio can be 13:1 to 26:1, and the volume ratio of protective agent and DNA solution to be processed can be 1:3 to 3:1.For example, turning Agent and protectant volume ratio can for 13:1,14:1,15:1,16:1,17:1,18:1,19:1,20:1,21:1,22:1, The volume ratio of 22:3,28:3 or 26:1, protective agent and DNA solution to be processed can be 1:3,1:2,1:1,2:3,2:1,3:2 Or 3:1.

In addition, in some instances, the sum of transforming agent, protective agent and volume of DNA solution to be processed can be not enough to Then the reaction volume of DNA sulphite conversion reaction system can supply reaction volume with deionized water.In addition, some In example, the volume ratio of deionized water and DNA solution to be processed can be 1:10 to 10:1, for example, 1:9,1:7,1:5,1:3, 1:2,1:1,2:3,2:1,3:2,3:1,5:1,6:1 etc..

In addition, in some instances, before step S30, DNA mixed solution can be placed at 95 to 98 DEG C and heat 5 DNA thermal denaturation is carried out to 10min.In this case, DNA unwinding can be made to be formed as single stranded DNA, is conducive to DNA sulfurous acid The progress of salt conversion.

For example, in one example, DNA mixed solution can be placed at 95 DEG C and heat 10min.In another example In, DNA mixed solution can be placed at 96 DEG C and heat 8min.In yet another example, DNA mixed solution can be placed in 97 6min is heated at DEG C.

In addition, in other examples, before step S30, can by be added pH adjusting agent to DNA mixed solution into Row DNA alkaline denaturation, pH adjusting agent can be selected from least one of sodium hydroxide, magnesium hydroxide, ammonium hydroxide, the addition of pH adjusting agent Amount can be 0.2 to 0.3mol/L.In this case, DNA mixed solution can be become to alkalinity, so as to solve DNA Chain is formed as single stranded DNA, is conducive to the progress of DNA sulphite conversion.

In addition, in one example, pH adjusting agent can be sodium hydroxide.In another example, pH adjusting agent can be with For magnesium hydroxide.In addition, in yet another example, pH adjusting agent can be ammonium hydroxide.

In addition, in some instances, the content of pH adjusting agent can be 0.20 to 0.30mol/L.Preferably, pH adjusting agent Content can be 0.20mol/L.It is highly preferred that the content of pH adjusting agent can be 0.24mol/L.It is further preferred that pH The content of regulator can be 0.28mol/L.Most preferably, the content of pH adjusting agent can be 0.30mol/L.

In addition, in some instances, in step s 30, the rated condition of conversion can be can be at 50 to 70 DEG C Heating 30 to 60min.In this case, DNA can be made to carry out sulphite conversion well.In some instances, it converts Heating condition can be carried out in PCR instrument.In addition, the heating condition of conversion can also be in metal bath, water-bath or baking oven It carries out.

In addition, in one example, it is specified that condition can be to heat 50min at 50 DEG C.In another example, it is specified that Condition can be to heat 40min at 60 DEG C.In addition, in yet another example, it is specified that condition can be to heat at 70 DEG C 30min etc..

In addition, can also include: to be purified to DNA conversion solution, to obtain DNA solution (step in some instances S40).In this case, the DNA of the sulphite conversion in DNA conversion solution can be recycled.

In addition, in some instances, step S40 may include: that will be added in conjunction with liquid and magnetic bead in DNA conversion solution, It is stood after mixing, removes supernatant to obtain the first DNA conjugate (step S41).In step S41, can will in conjunction with liquid and Magnetic bead is added to DNA conversion solution, is then uniformly mixed for example, by modes such as whirlpool, concussion, hand bullet, reverse, pressure-vaccums, then 5 to 15min are stored at room temperature, then is placed in progress magnetic bead adsorption of DNA on magnetic frame, outwells supernatant, the first DNA is then obtained and combines Object.

In addition, in some instances, can be in conjunction with liquid includes glycogen, sodium iodide, guanidinium isothiocyanate, guanidine thiocyanate, salt The aqueous solution of at least one of sour guanidine and at least one of isopropanol or ethyl alcohol.In this case, sulphite can be made to turn The DNA of change reduces the loss of the DNA of sulphite conversion in conjunction with magnetic bead.

In addition, can be the aqueous solution for including guanidine thiocyanate, glycogen, sodium iodide and ethyl alcohol in conjunction with liquid in some instances. In this case, DNA and magnetic bead that guanidine thiocyanate can be such that sulphite converts with functional group's collective effect of magnetic bead surfaces In conjunction with the DNA precipitating that ethyl alcohol can be such that sulphite converts, glycogen can help to retain the DNA of more sulphite conversion. In addition, the content of guanidine thiocyanate can be 2 to 6mol/L, glycogen content can be 0.05 to 0.1mol/L, the content of sodium iodide It can be 0.5 to 2mol/L, the volume fraction of ethyl alcohol can be 10% to 40%.

For example, in one example, in conjunction with liquid can be the guanidine thiocyanate for including 3.5mol/L, the glycogen of 0.05mol/L, The aqueous solution for the ethyl alcohol that the sodium iodide and volume fraction of 2mol/L is 40%.It in another example, can be to include in conjunction with liquid The aqueous solution for the ethyl alcohol that the guanidine thiocyanate of 2mol/L, the glycogen of 0.1mol/L, the sodium iodide of 1mol/L and volume fraction are 10%. In addition, can be the guanidine thiocyanate for including 6mol/L, the glycogen of 0.08mol/L, 0.5mol/L in conjunction with liquid in yet another example Sodium iodide and volume fraction be 25% ethyl alcohol aqueous solution etc..

In addition, in some instances, the partial size of magnetic bead can be 100 to 500nm, and can coat on the surface of magnetic bead There is the silica modified by hydroxy or carboxy.In this case, magnetic bead can be screened preferably and purify DNA conversion solution In sulphite conversion DNA long fragment.In some instances, the partial size of magnetic bead can be 100 to 500nm, for example, magnetic bead Partial size can be 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm.

In addition, in some instances, the content of magnetic bead can be 25 to 50mg/ml.In this case, magnetic bead can be more The DNA of the sulphite conversion in DNA conversion solution is purified well.In some instances, the content of magnetic bead can be 30mg/ Ml, 35mg/ml, 40mg/ml, 45mg/ml etc..

In addition, in step s 40, may include that the first cleaning solution is added to the first DNA conjugate, stood after mixing, Supernatant is removed, sulfurous acid group is then added and removes liquid, is stood after mixing, then removes supernatant to obtain the 2nd DNA combination Object (step S42).In step S42, the first cleaning solution can be entered into the first DNA conjugate and be mixed, be subsequently placed in magnetic force Beads enrichment is carried out on frame, outwells supernatant, is added sulfurous acid group removal liquid, is then passed through whirlpool, concussion, hand bullet, top , the modes such as pressure-vaccum are uniformly mixed, and are stored at room temperature 15-20min, then be placed on magnetic frame and carry out adsorption of DNA, are outwelled supernatant, Then the 2nd DNA conjugate is obtained.

In addition, in some instances, the first cleaning solution can be include ethyl alcohol and guanidinium isothiocyanate, guanidine thiocyanate, hydrochloric acid The solution of at least one of guanidine component.In this case, the DNA long fragment of sulphite conversion can be further purified.

In addition, in some instances, the first cleaning solution can be the solution of ethyl alcohol and guanidine thiocyanate.In this case, The DNA that ethyl alcohol can precipitate sulphite conversion makes it desalt, and functional group's collective effect of guanidine thiocyanate and magnetic bead surfaces can More, the DNA long fragment for preferably combining sulphite conversion, thus, it is possible to the DNA of sulphite conversion is further purified.

In addition, in some instances, the volume fraction of ethyl alcohol can be 70% to 80%, and the content of guanidine thiocyanate can be 0.5 to 2.0mol/L.For example, in one example, the first cleaning solution can be the ethyl alcohol and 1mol/ for including volume fraction 70% The solution of the guanidine thiocyanate of L.In another example, the first cleaning solution can be include volume fraction 80% ethyl alcohol with The solution of the guanidine thiocyanate of 0.5mol/L.In addition, in yet another example, the first cleaning solution can be to contain volume fraction 75% Ethyl alcohol and 2.0mol/L guanidine thiocyanate solution etc..

In addition, in some instances, sulfurous acid group removal liquid can be include 0.05 to 0.5mol/L sodium hydroxide with The solution of 70% to 90% ethyl alcohol.In this case, it can help to remove the sulfurous acid on the DNA of the sulfurous acid conversion of purifying The DNA of sulphite conversion is further purified in group.

For example, in one example, it includes 0.5mol/L sodium hydroxide and 90% second that sulfurous acid group, which removes liquid can be, The solution of alcohol.In another example, it includes 0.05mol/L sodium hydroxide and 70% ethyl alcohol that sulfurous acid group, which removes liquid can be, Solution, in addition, in yet another example, sulfurous acid group removal liquid can also be include 0.2mol/L sodium hydroxide and 80% The solution etc. of ethyl alcohol.

In addition, in step s 40, the second cleaning solution can be added in the 2nd DNA conjugate and be mixed, to After Beads enrichment, supernatant is removed, eluent is added, is stood after mixing, after Beads enrichment, to obtain DNA solution (step S43).In step S43, the second cleaning solution can be added in the first DNA conjugate and be mixed, it is enterprising to be subsequently placed in magnetic frame Row Beads enrichment, outwells supernatant, adds eluent, is stored at room temperature 3-10min after mixing, is subsequently placed in 0.5- on magnetic frame Supernatant after magnetic bead adsorption of DNA, is transferred in new no DNase enzyme and RNase enzyme centrifuge tube that can be obtained DNA molten by 2min Liquid purified product, then can immediately using or be placed in -20 and saved with refrigerator.

In addition, in some instances, the second cleaning solution can be the ethyl alcohol of volume fraction 70% to 80%.In such case Under, the second cleaning solution can precipitate the DNA long fragment of sulphite conversion, and the DNA long fragment for enabling sulphite to convert is gone Except salt ion caused by step S43, thus, it is possible to the DNA long fragment of sulphite conversion is further purified.In some examples In, the second cleaning solution can be the ethyl alcohol of volume fraction 70%.In another example, the second cleaning solution can be volume fraction 80% ethyl alcohol.In addition, in yet another example, the second cleaning solution can be the ethyl alcohol etc. containing volume fraction 70%.

In addition, in some instances, eluent can be sterile water, TE buffer or the Tris of no DNase and RNase enzyme Buffer.In this case, the DNA of the sulphite conversion of purifying can be dissolved in eluent, and will not be damaged, is dirty Dye.For example, in one example, eluent can be TE buffer.In another example, eluent can be no DNase With the sterile water of RNase enzyme.In addition, in some instances, TE buffer can be include Tris-HCl buffer and EDTA Solution, pH value can be 7.5 to 9.0.

In addition, step S44 can be repeated once before step S45, in this case, sulfurous can be further purified The DNA of hydrochlorate conversion.

In step S41 into step S45, the DNA after sulfurous acid group removal liquid can help sulphite to convert is removed Sulfurous acid group can be realized using liquid, sulfurous acid group removal liquid etc., and combination paramagnetic particle method purifying is combined and be quickly obtained height Conversion ratio, high quality sulphite conversion DNA.

In addition, may include above-mentioned DNA sub- present embodiments provide for a kind of kit of DNA sulphite conversion Transforming agent and protective agent used in the method for sulfate conversion processing.In this case, it can be realized and be quickly obtained height Conversion ratio, high quality sulphite conversion DNA.

In addition, in some instances, which may include in the purification process of above-mentioned DNA sulphite conversion processing Used reagent.In this case, the DNA of purer sulphite conversion can be obtained.

In addition, in some instances, kit may include in conjunction at least one of liquid, sulfurous acid group removal liquid. In addition, in some instances, kit can also include the first cleaning solution, the second cleaning solution, eluent and magnetic bead.

In addition, in some instances, kit can using above-mentioned DNA sulphite conversion processing method to DNA into The conversion of row sulphite.In addition, handling the methylation sites that DNA obtained can be used to detect long segment by kit.

In addition, in some instances, can be used to detect by method or the kit DNA obtained of present embodiment The methylation sites of the long segment of about 100bp to 1000bp particularly can be used to detect the lengthy motion picture of 400bp to 1000bp The methylation sites of section, it is preferable that can be used to detect the methylation sites of the long segment of 400bp to 750bp.For example, can be with For detecting the long segment of 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 750bp, 900bp or 1000bp Methylation sites.In some instances, it can also be used to detect the length of 80bp, 1050bp, 1100bp, 1150bp or 1200bp The methylation sites of segment.

Hereinafter, in order to further illustrate the disclosure, in conjunction with the embodiments to DNA long fragment sulfurous acid involved by present embodiment The method and kit of salt conversion processing are described in detail, and the beneficial effect realized in conjunction with attached drawing and comparative example to the disclosure It is absolutely proved.Fig. 4 shows the result figure of the detection of methylation status of PTEN promoter involved in the disclosure.Fig. 5 shows this public affairs The result figure of the detection of methylation status of PTEN promoter involved in opening.Fig. 6 shows the conversion of DNA sulphite involved in the disclosure And the result figure of the amplified fragments size detection of the DNA of purifying.

[embodiment 1]

In the present embodiment, sulphite conversion and purifying are carried out to DNA to be processed, obtains DNA and converts solution, then, Transformation efficiency detection and methylation status of PTEN promoter detection are carried out to obtained DNA conversion solution.In the present embodiment, pass through quotient Use the DNA of kit extraction cell line HCT116 and Hela genome as DNA to be processed.

(conversion and purifying cells genomic DNA)

(1) cell line HCT116 genome is extracted using the genome DNA extraction kit of Quan Shi King Company commercialization DNA, concrete operations are carried out by shop instruction, and the DNA of extraction takes 5 μ L to wait for after UV spectrophotometer measuring nucleic acid concentration Handle the centrifuge tube that 200 μ L are added in DNA.

(2) 120 μ L Sodium Metabisulfites containing 2.5mol/L, 1mol/L sodium hydrogensulfite, mono- water sulfurous acid of 0.5mol/L are taken Step (1) is added in the protective agent of ammonium, the conversion fluid and 10 μ L that pH is 5.0 quinhydrones and 0.5mol/L trehalose containing 0.1mol/L Centrifuge tube in, then supply reaction volume to 150 μ L with deionized water, uniformly mix.

(3) reaction system in step (2) is placed in progress sulphite conversion processing, processing routine are as follows: 95 in PCR instrument DEG C, 10min, 50 DEG C, 40min, until restoring to room temperature.

(4) DNA after conversion is transferred in 1.5ml centrifuge tube, the guanidine thiocyanate containing 3.5mol/L of 400 μ L of addition, The partial size of the combination liquid for the ethyl alcohol that the glycogen of 0.05mol/L, the sodium iodide of 2mol/L and volume fraction are 40% and 15 μ L is 300nm, surface are by being modified with the silicon dioxide coated magnetic bead of hydroxyl, room temperature rotation mixing 5min.

(5) centrifuge tube is placed in 30s on magnetic frame, after Beads enrichment, abandons supernatant.

(6) sulphur for the ethyl alcohol and 1mol/L for being 70% containing volume fraction that 400 μ L are added into the centrifuge tube in step (5) First cleaning solution of cyanic acid guanidine, vortex mixed 20s are placed on magnetic frame, after Beads enrichment, abandon supernatant.

(7) 200 μ L sulfurous acid groups removal liquid is added into the centrifuge tube in step (6), stands 15min at room temperature, then Centrifuge tube is placed on magnetic frame, abandons supernatant after to be separated.

(8) be added into the centrifuge tube in step (7) 400 μ L volume fraction be 70% ethyl alcohol, vortex mixed 20s, It is placed on magnetic frame, after Beads enrichment, abandons supernatant.It is repeated once.

(9) 5min is stood on magnetic frame, it is dry.

(10) eluent for the TE buffer that the pH that 30 μ L are added is 8.5, after mixing well, is stored at room temperature 5min.

(11) centrifuge tube is placed in magnetic frame 30s, after to be separated, supernatant be transferred in new centrifuge tube, converted And the DNA solution of purifying.

(transformation efficiency detection)

(a) prepare blank control group

In order to obtain conversion and purifying DNA solution DNA transformation efficiency, using unconverted DNA solution as blank pair Its sulphite transformation efficiency is calculated according to group.Specifically, in above-mentioned conversion and purifying cells genomic DNA, only spend from Sub- water supplies the step alternative steps (2) of reaction volume to 150 μ L, other steps and above-mentioned conversion and purifying cells genome DNA is identical, obtains unconverted DNA solution.

(b) DNA solution and unconverted DNA solution each 1 μ L of above-mentioned conversion and purifying are taken, it is molten according to unconverted DNA The sequence information of DNA in liquid, the design site reference gene ACTB 1 (site sequence is SEQ ID NO:1) specific primer Forward primer sequence (AC-F) and reverse primer sequences (AC-R), and the DNA (referred to as " conversion of the DNA solution to conversion and purifying DNA ") and the DNA (referred to as " unconverted DNA ") of unconverted DNA solution carry out fluorescent PCR quantitative analysis, specific primer sequence It is shown in Table 1.

1 primer sequence of table

(c) reaction system then is prepared to be expanded and be detected according to table 2, amplification condition is the first stage: 95 DEG C, 10min, 1 circulation；Second stage: 95 DEG C, 10s, 60 DEG C, 35s, 40 circulations, and fluorescence is collected since second stage Signal.

2 amplification reaction system of table

According to the variation judgement conversion DNA's of the ct value (the amplification cycles number passed through when reaching fluorescence threshold) of the two Sulphite transformation efficiency, testing result are shown in Fig. 3.Choosing fluorescence threshold is 10000, and the ct value for obtaining conversion DNA is 15, is not turned The ct value for changing DNA is 24, so Δ ct is 9, thus judges that conversion ratio is greater than 99%.

(methylation status of PTEN promoter detection)

[can A. preliminary experiment carry out methylation status of PTEN promoter detection for judging]

It is not methylated with the site Septin-9 of the DNA of Hela genome and detects methylation status as a control group.Specifically For, in above-mentioned conversion and purifying, the DNA of the extraction cell line HCT116 genome in step (1) is by extraction cell line The DNA of Hela genome is replaced, other steps are identical as above-mentioned conversion and purifying cells genomic DNA, are converted and purified Cell line Hela genome DNA solution.

The DNA of the DNA solution of the conversion and purifying that take 1 μ L above-mentioned respectively and the Cell line Hela genome of conversion and purifying Solution carries out real-time fluorescence PCR detection gene Septin-9 methylation sites and house-keeping gene ACTB.Design Septin-9 methyl Change the forward primer sequence (Septin-9-F) and reverse primer of site 1 (site sequence is SEQ ID NO:4) specific primer Sequence (Septin-9-R), probe sequence (Septin-9-probe), and design 2 (site sequence of the site house-keeping gene ACTB For the forward primer sequence (ACTB-F1) and reverse primer sequences (ACTB-R1), probe sequence of SEQ ID NO:5) specific primer It arranges (ACTB-probe), the particular sequence of primer and probe is shown in Table 3.

3 primer and probe sequence of table

Then, reaction system is prepared according to table 4 to carry out real-time fluorescent PCR amplification and detection, amplification condition are as follows: first Stage: 95 DEG C, 10min, 1 circulations；Second stage: 95 DEG C, 10s, 60 DEG C, 35s, 40 circulations, and opened from second stage Begin to collect fluorescence signal.

4 reaction system of table (20 μ L)

As a result as shown in Figure 4 and Figure 5, go out from the experimental results, the site the Septin-9 methyl of the DNA of HCT116 genome There are signals to belong to methylation state in real-time fluorescent PCR amplification and detection process for change, the DNA's of Hela genome The site Septin-9, which does not methylate, there is not signal in real-time fluorescent PCR amplification and detection process.In addition, HCT116 genome The house-keeping gene ACTB of DNA of DNA and Hela genome there is signal in real-time fluorescent PCR amplification and detection process. Therefore, using the sulphite conversion of the present embodiment and purification process processing conversion and the DNA of the DNA solution purified, first can be used Base specificity real-time fluorescence PCR detection goes out the methylation status of Septin-9 and house-keeping gene ACTB.

[detection of B. methylation status of PTEN promoter]

The DNA solution of the conversion and purifying that take 10 μ L above-mentioned, then carries out methylation-specific in the same manner described above PCR detection, testing result is as shown in table 10,.

(the amplified fragments size detection of the DNA of conversion and purifying)

It takes the DNA solution of the above-mentioned conversion of 1 μ L and purifying to carry out real-time fluorescence PCR, detects the amplification water of different length segment It is flat.Design the forward primer sequence of Septin-9 methylation sites 2 (site sequence is SEQ ID NO:12) specific primer (Septin-9-F1) and reverse primer sequences (Septin-9-R1), (site sequence is SEQ ID in the site house-keeping gene ACTB 3 NO:13) the forward primer sequence (ACTB-F2) and reverse primer sequences (ACTB-R2) of specific primer, MLH1 gene loci 1 The forward primer sequence (MLH1-F2) and reverse primer sequences (MLH1- of (site sequence is SEQ ID NO:14) specific primer ) and the forward primer sequence (MLH1- of MLH1 gene loci 2 (site sequence be SEQ ID NO:15) specific primer R2 F3) and reverse primer sequences (MLH1-R3), the particular sequence of primer are shown in Table 5.

5 primer sequence of table

Then, reaction system is prepared to carry out real-time fluorescent PCR amplification respectively according to table 6,7,8 and 9 respectively, expand item Part are as follows: first stage: 95 DEG C, 10min, 1 circulations；Second stage: 95 DEG C, 10s, 60 DEG C, 35s, 40 circulations, and from the Two-stage starts to collect fluorescence signal.

6 reaction system of table (20 μ L)

7 reaction system of table (20 μ L)

8 reaction system of table (20 μ L)

9 reaction system of table (20 μ L)

As a result as shown in fig. 6, wherein DNA Marker is the 1kb DNA loading of Tiangeng.From the point of view of testing result, this implementation The fragmentation degree of the DNA for the sulphite conversion that the conversion of example and purification process obtain is lower, and amplifiable 100bp out is extremely The nucleic acid fragment of 1000bp.In addition, as can be seen from Fig. 4, the effect of the nucleic acid fragment amplification of 400bp to 750bp is more obvious.By This is as it can be seen that illustrate the method DNA obtained of DNA sulphite conversion processing involved by the disclosure suitable for Long fragment gene Methylation level detection.

[embodiment 2]

In the present embodiment, compared with Example 1, it has used comprising 2.5mol/L Sodium Metabisulfite, 1mol/L sulfurous The conversion fluid of sour hydrogen sodium, mono- water ammonium sulfite of 0.5mol/L and 0.3mol/L sodium hydroxide (pH adjusting agent), in PCR instrument into The program of row sulphite conversion processing are as follows: 60 DEG C, 40min, then handled, turned in such a way that embodiment 1 is identical The DNA solution changed and purified.Then 10 μ L are taken, methylation status of PTEN promoter detection is carried out in such a way that embodiment 1 is identical, are detected It the results are shown in Table 10.

[comparative example 1]

In this comparative example, using the kit of scientific and technological (Beijing) Co., Ltd of prune carry out the conversion of DNA sulphite and Purifying then, carries out methylation status of PTEN promoter detection to obtained DNA conversion solution to obtain DNA conversion solution.

(a1) DNA to be processed of the Conversion Reagent and 20 μ L of 130 μ L are added in the PCR pipe of 200 μ L, mixes It is even.

(b1) sulphite processing, processing routine are carried out in PCR instrument are as follows: 98 DEG C, 8min；54 DEG C, 60min.

(c1) 600 μ L Binding Buffer are added into sample, after mixing in adsorption column, 12000rpm is centrifuged 30s, Discard efflux.

(d1) 100 μ L Wash Buffer, 12000rpm centrifugation 30s are added, discard efflux.

(e1) 200 μ L Desulphonation Buffer are added, are placed at room temperature for 10min, 12000rpm is centrifuged 30s, abandons Remove efflux.

(f1) 200 μ L Wash Buffer, 12000rpm centrifugation 30s are added, discard efflux.It is repeated once.

(g1) adsorption column is placed in the 1.5ml centrifuge tube of no DNase and RNase enzyme, adds 10 μ L Elution Buffer, 2000rpm are centrifuged 30s, the DNA solution for being converted and being purified.

The DNA solution for taking 10 μ L to convert and purify, carries out carry out methylation status of PTEN promoter in such a way that embodiment 1 is identical Detection, testing result are compared with the result of embodiment 1, and comparison result is shown in Table 10.

[comparative example 2]

In this comparative example, DNA sulphite is carried out using the kit of TIANGEN Biotech (Beijing) Co., Ltd. and is turned Change and purifying then, converts solution to obtained DNA and carry out methylation status of PTEN promoter detection to obtain DNA conversion solution.

(a2) 90 μ L sulfite solutions are added in 200 μ L centrifuge tubes, 10 μ L buffer DP and 20 μ L's is to be processed DNA is mixed.

(b2) sulphite conversion, specific procedure are as follows: 95 are carried out on the instrument of the settable temperature change program such as PCR instrument DEG C, 10min, 64 DEG C, 60min.

(c2) reaction system in pipe is transferred in clean 1.5ml centrifuge tube, the combination liquid PB of 600 μ L is added, fills Divide and mixes.

(d2) acquired solution is added in adsorption column, is stored at room temperature 2min, 12000rpm is centrifuged 30~60s, abandons collecting pipe In waste liquid.

(e2) 600 μ L rinsing liquid PW, 12000rpm are added into adsorption column and are centrifuged 30~60s, abandon the waste liquid in collecting pipe.

(f2) 600 μ L solution D B are added into adsorption column, are placed at room temperature for 15min, 12000rpm is centrifuged 30~60s, abandons and receives Waste liquid in collector.

(g2) 600 μ L rinsing liquid PW, 12000rpm are added into adsorption column and are centrifuged 30~60s, abandon the waste liquid in collecting pipe, Thoroughly dry.

(h2) adsorption column is put into clean centrifuge tube, 30 μ L elution buffers is vacantly added dropwise in adsorbed film middle position EB, is placed at room temperature for 2min, and 12000rpm is centrifuged 2min, the DNA solution for being converted and being purified.

The DNA solution for taking 10 μ L to convert and purify, carries out carry out methylation status of PTEN promoter in such a way that embodiment 1 is identical Detection, testing result are compared with the result of embodiment 1, and comparison result is shown in Table 10.

10 testing result of table

As can be seen from Table 10, embodiment 1 and embodiment 2 are compared with comparative example 1 and comparative example 2, and Ct value is small, transformation efficiency Height, and embodiment 1 and embodiment 2 are all made of paramagnetic particle method purifying, therefore DNA mass obtained is high, facilitate automation behaviour Make.

Although above combine drawings and embodiments the disclosure is illustrated, it will be appreciated that on state It is bright not limit the disclosure in any form.Those skilled in the art are without departing from the connotation and range of the disclosure It can according to need and the disclosure is deformed and is changed, these deformations and variation are each fallen in the scope of the present disclosure.

Sequence table

<110>Shanghai Ai Wen Biotechnology Co., Ltd

<120>method and kit of DNA sulphite conversion processing

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 98

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

agacagtgtt gtgggtgtag gtactaacac tggctcgtgt gacaaggcca tgaggctggt 60

gtaaagcggc cttggagtgt gtattaagta ggtgcaca 98

<210> 2

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

agacagtgtt gtgggtgtag gt 22

<210> 3

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

tgtgcaccta cttaatacac actccaagg 29

<210> 4

<211> 89

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gatagtgttg tgggtgtagg tattaatatt ggtttgtgtg ataaggttat gaggttggtg 60

taaagtggtt ttggagtgtg tattaagta 89

<210> 5

<211> 85

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

tcgattttcg gttgtgttcg gcgtcgtcgc ggtgttcggc gtcgtcgttt cgttcggcgg 60

ggtcgttcgg agcgttcgta ttttc 85

<210> 6

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gatagtgttg tgggtgtagg 20

<210> 7

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

tacttaatac acactccaaa acc 23

<210> 8

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

ggttcgtgtg ataaggttat gagg 24

<210> 9

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

tcgattttcg gttgtgttcg gc 22

<210> 10

<211> 24

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

gaaaatacga acgctccgaa cgac 24

<210> 11

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

tcgtcgcggt gttcggcgtc gtcgtttc 28

<210> 12

<211> 102

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

tcgattttcg gttgtgttcg gcgtcgtcgc ggtgttcggc gtcgtcgttt cgttcggcgg 60

ggtcgttcgg agcgttcgta ttttcgttcg tttttatttg gt 102

<210> 13

<211> 405

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

ttgggtgtta attgtgtgtg tgttgggaat tggtgttaat tgtgtgtgtg tgttgggatt 60

taaggtgtta attgtgtgtg tgttttgggg tttggggtgt tgtggtttgg gttggggtga 120

aggtgggttt ggttggaagg ggtggggttg ttgtggtttt tgggtgtttg tgtgtatttt 180

ttgtttgagt tgttggttgt ttgagggtgt ggttgttgtg tgtgtgtgtg ttgatttggt 240

gttgtttgaa ttgggtggag gtggggttgg tgtttggttg ggagggggtt ggggtttggt 300

tttttgttgt gtgttgtggg gatgtttttg attagtgttt gttttttatg gtaataatgt 360

ggttggtttg gttttttttg tttttaattt gggtgtgtgt tggtg 405

<210> 14

<211> 750

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

gtatgggtcg tggttagttt aattattttt tgtaagttaa gttttgttgt ttgtagggat 60

tttaggattg tcgatatgag cgtattaata ttgaaatgat gagttaggtt gattatggtt 120

agaagatttt tttgtatttt taatttaggg tttatatcgc ggataaagat taggaggtag 180

tttttatagg ttataaaagt ttggtcgttt aaggtaagag aataggtttt aaagtttttg 240

gttcggttaa aaagttggtt gcgtagattt ttgttaatgc tcaggatttt ttgttttgtg 300

atatttggag ataagttaac gttttgtagg acgtttatat gttcgggtag tatttttttt 360

agtaatattt ttatgtattg gtatataaag ttttttttat tttagtcgcg attttttaag 420

gttaagaggc ggtagagttt gaggtttgta cgagtagttt tttttttagg agtgaaggag 480

gccacgggta agtcgttttg acgtagacgt tttattaggg tcgcgcgttc gtcgttcgtt 540

atatatcgtt cgtagtattc gtgtttagtt tcgtagtggc gtttgacgtc gcgttcgcgg 600

gtagttacga tgaggcggcg atagattagg tatagggttt tatcgttttt cggaggcttt 660

attattaaat aacgttgggt ttattcgggt cggaaaatta gagtttcgtc gatttttatt 720

ttgttttttt tgggcgttat ttatattttg 750

<210> 15

<211> 1007

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

tttaggattg tcgatatgag cgtattaata ttgaaatgat gagttaggtt gattatggtt 60

agaagatttt tttgtatttt taatttaggg tttatatcgc ggataaagat taggaggtag 120

tttttatagg ttataaaagt ttggtcgttt aaggtaagag aataggtttt aaagtttttg 180

gttcggttaa aaagttggtt gcgtagattt ttgttaatgc tcaggatttt ttgttttgtg 240

atatttggag ataagttaac gttttgtagg acgtttatat gttcgggtag tatttttttt 300

agtaatattt ttatgtattg gtatataaag ttttttttat tttagtcgcg attttttaag 360

gttaagaggc ggtagagttt gaggtttgta cgagtagttt tttttttagg agtgaaggag 420

gccacgggta agtcgttttg acgtagacgt tttattaggg tcgcgcgttc gtcgttcgtt 480

atatatcgtt cgtagtattc gtgtttagtt tcgtagtggc gtttgacgtc gcgttcgcgg 540

gtagttacga tgaggcggcg atagattagg tatagggttt tatcgttttt cggaggcttt 600

attattaaat aacgttgggt ttattcgggt cggaaaatta gagtttcgtc gatttttatt 660

ttgttttttt tgggcgttat ttatattttg cgggaggtta taagagtagg gttaacgtta 720

gaaaggtcgt aaggggagag gaggagtttg agaagcgtta agtatttttt tcgttttgcg 780

ttagattatt ttagtagagg tatataagtt cggtttcggt atttttgttt ttattggttg 840

gatatttcgt atttttcgag tttttaaaaa cgaattaata ggaagagcgg atagcgattt 900

ttaacgcgta agcgtatatt tttttaggta gcgggtagta gtcgttttag ggagggacga 960

agagatttag taatttatag agttgagaaa tttgattggt atttaag 1007

<210> 16

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

accaaataaa aacgaacgaa aatacg 26

<210> 17

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

ttgggtgtta attgtgtgtg tgttggg 27

<210> 18

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

aacactatct taaacaccta atc 23

<210> 19

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

gtatgggtcg tggttagttt aattattt 28

<210> 20

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

ccgcaaaata taaataacgc ca 22

<210> 21

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

tttaggattg tcgatatgag cgtatt 26

<210> 22

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

cttaaatacc aatcaaattt ctcaactc 28

Claims (10)

1. a kind of method of DNA sulphite conversion processing, it is characterised in that:

Include:

(a) prepare DNA solution to be processed；

(b) DNA solution is added in transforming agent and protective agent, is mixed to obtain DNA mixed solution, the transforming agent is Water including being selected from least one of Sodium Metabisulfite, sodium hydrogensulfite, bisulfite amine, magnesium bisulfite, ammonium sulfite Solution, the protective agent are that include at least one of quinhydrones, watermiscible vitamin E component water-soluble with trehalose or agarose Liquid；And

(c) the DNA mixed solution is set and is converted under prescribed conditions, obtained DNA and convert solution.

2. the method as described in claim 1, it is characterised in that:

In the transforming agent, the content of solute is 2 to 5mol/L, and the pH value of the transforming agent is 5.0 to 5.5,

In step (c), the rated condition is that 30 to 60min are heated at 50 to 70 DEG C.

3. method according to claim 1 or 2, it is characterised in that:

Before step (c), the DNA mixed solution is placed in heating 5 to 10min at 95 to 98 DEG C and carries out DNA thermal denaturation.

4. method according to claim 2, it is characterised in that:

Before step (c), DNA alkaline denaturation, the pH adjusting agent are carried out to the DNA mixed solution by the way that pH adjusting agent is added Selected from least one of sodium hydroxide, magnesium hydroxide, ammonium hydroxide, the additive amount of the pH adjusting agent is 0.2 to 0.3mol/L.

5. the method as described in claim 1, it is characterised in that:

The protective agent is the aqueous solution of quinhydrones and trehalose, wherein the content of the quinhydrones is 0.03 to 0.3mol/L, described The content of trehalose is 0.3 to 0.5mol/L.

6. method according to claim 1 or 2, it is characterised in that:

Further include: (d) purifies DNA conversion solution, to obtain the DNA solution of purifying.

7. method as claimed in claim 6, it is characterised in that:

The step (d) includes:

(d1) it will be added in conjunction with liquid and magnetic bead in the DNA conversion solution, be stood after mixing, and remove supernatant to obtain first DNA conjugate；

(d2) the first cleaning solution is added to the first DNA conjugate, is stood after mixing, remove supernatant, be then added sub- Sulfate group removes liquid, stands after mixing, then remove supernatant to obtain the 2nd DNA conjugate；

(d3) the second cleaning solution is added in the 2nd DNA conjugate, is stood after mixing, remove supernatant, elution is added Liquid is stood after mixing, is removed magnetic bead, is obtained the DNA solution of the purifying.

8. the method for claim 7, it is characterised in that:

The combination liquid is the aqueous solution for including guanidine thiocyanate, glycogen, sodium iodide and ethyl alcohol, wherein the guanidine thiocyanate contains Amount is 2 to 6mol/L, and the glycogen content is 0.05 to 0.1mol/L, and the content of the sodium iodide is 0.5 to 2mol/L, described The volume fraction of ethyl alcohol is 10% to 40%.

9. the method for claim 7, it is characterised in that:

The partial size of the magnetic bead is 100 to 500nm, and is coated with two modified by hydroxy or carboxy on the surface of the magnetic bead Silica.

10. a kind of kit of DNA sulphite conversion, it is characterised in that:

The kit includes transforming agent used in method described in any one of claim 1 to 9 and protective agent.