CN105907854A - Method for methylation and sulfurization transformative modification of genes and kit using method - Google Patents
Method for methylation and sulfurization transformative modification of genes and kit using method Download PDFInfo
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- CN105907854A CN105907854A CN201610272926.5A CN201610272926A CN105907854A CN 105907854 A CN105907854 A CN 105907854A CN 201610272926 A CN201610272926 A CN 201610272926A CN 105907854 A CN105907854 A CN 105907854A
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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a method for methylation and sulfurization transformative modification of genes and a kit using the method. The method includes: preparing a sulfurization transformative reaction system of 100 Mul of a DNA sample, 190 Mul of a sulfurization reagent and 30 Mul of a DNA protectant, reacting at 80 DEG C for 1 h, and storing at 20 DEG C for 20 hours; the sulfurization reagent is a mixed solution of ammonium bisulfite and anhydrous sodium sulfite, and the DNA protectant is a mixed solution of tetrahydrofuran and VE. The kit includes a sulfurization reagent and a DNA protectant, the sulfurization reagent is mixed solution of ammonium bisulfite and anhydrous sodium sulfite, and the DNA protectant is a mixed solution of tetrahydrofuran and VE. The mixed solution of ammonium bisulfite and anhydrous sodium sulfite is first used as DNA methylation and sulfurization reagent. The method and kit have the advantages such as sulfurization completeness, low pollution, operational simplicity and time efficiency, and safety.
Description
Technical field
The present invention relates to gene sulfuration conversion field, particularly relate to a kind of method and test kit thereof sulfuration in methylation analysis being converted and modifying.
Background technology
DNA methylation refer to organism under the catalysis of dnmt rna (DNMT), S-adenosylmethionine is methyl donor, and methyl is transferred to the process in specific base.In mammal, DNA methylation occurs mainly on 5 '-CpG-3 ', wherein, the CpG dinucleotide of 80% is modified by methyl, remaining 20% non-methylated CpG dinucleotide high aggregation, is distributed widely in the First Exon district of promoter region and encoding gene, named CpG island.CpG island abnormal methylation is a key factor of tumor development, due to the local height on CpG island methylates will be early than malignant hypertrophy, therefore its methylated detection can be used for the prediction of tumor.Methylating can be as the biomarker of the early diagnosiss such as tumor and prognosis evaluation index, and examination and risk assessment, early diagnosis, by stages typing, Index for diagnosis and Treatment monitoring to tumor all have great importance.
DNA methylation analysis typically requires and DNA carries out sulfuration conversion modification.The dissociative DNA extracted is through sulfiding reagent effect, the upper unmethylated cytosine deamination of DNA is transformed into uracil, and methylated cytosine keeps constant, then carry out PCR by specific primer and probe and i.e. can get methylation status and the level of genes of interest.
The most common vulcanization process major part that methylates is all to use sodium sulfite as sulfiding reagent.Sodium sulfite extremely indissoluble, preparation is inconvenient and loses time, and its sulfurating strength is more weak, and sulfuration required time is longer, it is generally required to 2-3 hour the most overnight.
Summary of the invention
In order to solve the deficiencies in the prior art, it is an object of the invention to provide a kind of method and test kit thereof converting gene methylation sulfuration and modifying, curing efficiency is high, vulcanize complete, simple to operate saving time.
A kind of method that gene methylation sulfuration is converted modification, prepares sulfuration conversion reaction system: 100ul DNA sample+190ul sulfiding reagent+30ul DNA protects liquid, 80 ° of C to react 1 hour, and 20 ° of C preserved to 20 hours afterwards；Described sulfiding reagent is the mixed solution of ammonium bisulfite and anhydrous sodium sulfite, and described DNA protection liquid is the mixed liquor of oxolane and VE.
Described DNA sample is human genome DNA.
Described DNA sample is mankind's normal gene group DNA or pathological changes genomic DNA；Derive from histiocyte DNA, peripheral blood cells DNA, peripheral blood serum or plasma dna, body fluid DNA or Excreta cell DNA.
A kind of employing converts, according to the vulcanizing gene methylation of described method, the test kit modified; liquid is protected including sulfiding reagent and DNA; described sulfiding reagent is the mixed solution of ammonium bisulfite and anhydrous sodium sulfite, and described DNA protection liquid is the mixed liquor of oxolane and VE.
Described test kit, farther include negative quality-control product, positive quality control product and no template control, described positive quality control product is bisulphite modified permethylated human genome DNA, and described negative quality-control product is the bisulphite modified non-human genome DNA that methylates.
The invention has the beneficial effects as follows: the present invention converts, to sulfuration in gene methylation detection, the method modified, have that curing efficiency is high, sulfuration completely, preparation of reagents save time cheap, simple to operate quickly, the advantage such as safety, testing result has preferable accuracy and repeatability, gene methylation sulfuration is converted modification and provides a kind of better method.
Accompanying drawing explanation
Fig. 1 be in the present invention gene methylation is detected sulfuration convert modify after in methylate template and the schematic diagram of the non-template that methylates；
Fig. 2 is to utilize the present invention's gene methylation sulfuration is converted septin9 DNA methylation assay amplification curve diagram in method of modifying one preferred embodiment.
Fig. 3 is to utilize the present invention's gene methylation sulfuration converts septin9 DNA methylation assay amplification curve comparison diagram in sulfuration conversion method of modifying one preferred embodiment conventional on method of modifying and market.
Detailed description of the invention
Technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, all other embodiments that those of ordinary skill in the art are obtained under not making creative work premise, broadly fall into the scope of protection of the invention.
As it is shown in figure 1, after over cure converts and modifies, the upper unmethylated cytosine deamination of DNA is transformed into uracil, and the holding of methylated cytosine is constant, then carries out PCR by specific primer and probe and i.e. can get methylation status and the level of genes of interest.
Embodiment
Material: plasma sample to be checked, positive quality control product, negative quality-control product.
Instrument: Lightcycler 480, nanodrop 1000, high speed centrifuge, water-bath, whirlpool concussion instrument, autoclave.
Reagent: ammonium bisulfite (WAKO company of Japan), anhydrous sodium sulfite (Aladdin), oxolane (Aladdin), VE(Aldrich)；
The system of vulcanization reaction and reaction condition: consisting of of described vulcanization reaction system: 100ul DNA+190ul sulfiding reagent+30ul DNA protects liquid.Described DNA is generally 0.5ng ~ 2ng.Sulfiding reagent is 90%(v/v) ammonium bisulfite and the mixed liquor of anhydrous sodium sulfite of 0.1g/mL.Described DNA protection liquid is the mixed liquor of oxolane and VE.
The actual conditions of described sulfuration conversion reaction is: 80 ° of C react 1 hours, 20 ° of C and preserve to 20 hours.
After carrying out above-mentioned sulfuration conversion reaction, DNA after modifying being purified recovery, finally carries out PCR detection, septin9 methylated amplification curve is as shown in Figure 2.
The vulcanization process that zymo vulcanization process conventional on this technological invention people market and the present invention provide is contrast experiment, and the DNA methylation sulfuration that demonstrating the present invention provides converts modification reaction and will not have a negative impact subsequent reactions, as shown in Figure 3.
The advantage that sulfuration in gene methylation detection method converts method of modifying in the present invention has: (1) anhydrous sodium sulfite easily dissolves in ammonium bisulfite, and sulfiding reagent preparation simply saves time.(2) the method overall process only needs 1 hour, and need not 98 DEG C of high temperature and unwind, and experimental implementation is simple and efficient.(3) sulfuration is completely, remains to methylating of genes of interest be detected under the background that (90%) non-methylate DNA exists；(4) curing efficiency is high, and 0.1ng positive DNA converts method of modifying through the sulfuration that the present invention provides and remains to all detect.(5) safety, whole system does not comprise poisonous and harmful substance, to testing crew and environment all non-hazardous.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every equivalent structure utilizing description of the invention content to be made or equivalence flow process conversion; or directly or indirectly it is used in other relevant technical field, the most in like manner it is included in the scope of patent protection of the present invention.
Claims (5)
1. one kind converts, to gene methylation sulfuration, the method modified, it is characterised in that prepare sulfuration conversion reaction system: 100ul
DNA sample+190ul sulfiding reagent+30ul DNA protects liquid, 80 ° of C to react 1 hour, and 20 ° of C preserved to 20 hours afterwards；Described sulfiding reagent is the mixed solution of ammonium bisulfite and anhydrous sodium sulfite, and described DNA protection liquid is the mixed liquor of oxolane and VE.
Method the most according to claim 1, it is characterised in that described DNA sample is human genome DNA.
Method the most according to claim 2, it is characterised in that described DNA sample is mankind's normal gene group DNA or pathological changes genomic DNA；Derive from histiocyte DNA, peripheral blood cells DNA, peripheral blood serum or plasma dna, body fluid DNA or Excreta cell DNA.
4. the test kit that gene methylation sulfuration is converted modification using method according to claim 1; it is characterized in that; liquid is protected including sulfiding reagent and DNA; described sulfiding reagent is the mixed solution of ammonium bisulfite and anhydrous sodium sulfite, and described DNA protection liquid is the mixed liquor of oxolane and VE.
Test kit the most according to claim 4, it is characterized in that, farther include negative quality-control product, positive quality control product and no template control, described positive quality control product is bisulphite modified permethylated human genome DNA, and described negative quality-control product is the bisulphite modified non-human genome DNA that methylates.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755391A (en) * | 2016-12-16 | 2017-05-31 | 江苏为真生物医药技术股份有限公司 | A kind of gene methylation detects vulcanizing agent and its application |
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| CN102399862A (en) * | 2010-09-16 | 2012-04-04 | 上海迦美生物科技有限公司 | Methylated DNA detection method based on melting curve |
| CN103667432A (en) * | 2012-09-20 | 2014-03-26 | 中国科学院沈阳应用生态研究所 | Improved sulfite sequencing method |
| CN104131070A (en) * | 2014-05-22 | 2014-11-05 | 苏州工业园区为真生物医药科技有限公司 | Gene methylation detection method |
| CN105462960A (en) * | 2016-01-08 | 2016-04-06 | 杭州千基生物科技有限公司 | DNA sulfite conversion and purification method |
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2016
- 2016-04-28 CN CN201610272926.5A patent/CN105907854A/en active Pending
Patent Citations (5)
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| CN102272331A (en) * | 2009-01-05 | 2011-12-07 | 生物梅里埃公司 | Method for amplifying and/or detecting nucleic acids, kits and uses of said method |
| CN102399862A (en) * | 2010-09-16 | 2012-04-04 | 上海迦美生物科技有限公司 | Methylated DNA detection method based on melting curve |
| CN103667432A (en) * | 2012-09-20 | 2014-03-26 | 中国科学院沈阳应用生态研究所 | Improved sulfite sequencing method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755391A (en) * | 2016-12-16 | 2017-05-31 | 江苏为真生物医药技术股份有限公司 | A kind of gene methylation detects vulcanizing agent and its application |
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Application publication date: 20160831 |