CN107988205A - A kind of nucleic acid extraction and bisulfite conversion and the kit of nucleic acid purification - Google Patents
A kind of nucleic acid extraction and bisulfite conversion and the kit of nucleic acid purification Download PDFInfo
- Publication number
- CN107988205A CN107988205A CN201711240822.7A CN201711240822A CN107988205A CN 107988205 A CN107988205 A CN 107988205A CN 201711240822 A CN201711240822 A CN 201711240822A CN 107988205 A CN107988205 A CN 107988205A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- conversion
- dna
- edta
- tris
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 18
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 15
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 15
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 title claims abstract description 10
- 238000001821 nucleic acid purification Methods 0.000 title claims abstract description 5
- 238000000605 extraction Methods 0.000 title abstract description 10
- 230000007067 DNA methylation Effects 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 239000011324 bead Substances 0.000 claims abstract description 8
- 230000005291 magnetic effect Effects 0.000 claims abstract description 8
- 239000006166 lysate Substances 0.000 claims abstract description 6
- 230000011987 methylation Effects 0.000 claims description 11
- 238000007069 methylation reaction Methods 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 238000004140 cleaning Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000007983 Tris buffer Substances 0.000 claims description 7
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 7
- 238000005336 cracking Methods 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 2
- LSNNMFCWUKXFEE-UHFFFAOYSA-N Sulfurous acid Chemical class OS(O)=O LSNNMFCWUKXFEE-UHFFFAOYSA-N 0.000 claims description 2
- GLEVLJDDWXEYCO-UHFFFAOYSA-N Trolox Chemical class O1C(C)(C(O)=O)CCC2=C1C(C)=C(C)C(O)=C2C GLEVLJDDWXEYCO-UHFFFAOYSA-N 0.000 claims description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 2
- 239000004202 carbamide Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 238000004925 denaturation Methods 0.000 claims description 2
- 230000036425 denaturation Effects 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 239000002516 radical scavenger Substances 0.000 claims description 2
- 239000001509 sodium citrate Substances 0.000 claims description 2
- 235000011083 sodium citrates Nutrition 0.000 claims description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 2
- 229940123457 Free radical scavenger Drugs 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000010828 elution Methods 0.000 claims 1
- 230000005298 paramagnetic effect Effects 0.000 claims 1
- 239000002245 particle Substances 0.000 claims 1
- 238000002360 preparation method Methods 0.000 claims 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 abstract description 6
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 4
- 108091005804 Peptidases Proteins 0.000 abstract description 3
- 239000004365 Protease Substances 0.000 abstract description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract description 3
- 229940079827 sodium hydrogen sulfite Drugs 0.000 abstract description 3
- 229910052710 silicon Inorganic materials 0.000 abstract description 2
- 239000010703 silicon Substances 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 2
- 238000005406 washing Methods 0.000 abstract description 2
- FCSRWHZYWMEIMK-UHFFFAOYSA-N 2,5,7,8-tetramethyl-3,4-dihydro-2h-chromene Chemical compound C1=C(C)C(C)=C2OC(C)CCC2=C1C FCSRWHZYWMEIMK-UHFFFAOYSA-N 0.000 abstract 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 abstract 1
- 150000001735 carboxylic acids Chemical class 0.000 abstract 1
- 230000009089 cytolysis Effects 0.000 abstract 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract 1
- 238000005554 pickling Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 13
- 210000000349 chromosome Anatomy 0.000 description 10
- 210000002700 urine Anatomy 0.000 description 10
- 239000000523 sample Substances 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 8
- 230000003321 amplification Effects 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 239000003223 protective agent Substances 0.000 description 4
- 108091029523 CpG island Proteins 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000002779 inactivation Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 101000856723 Homo sapiens Cytochrome b-245 light chain Proteins 0.000 description 2
- 101001065272 Homo sapiens EGF-containing fibulin-like extracellular matrix protein 1 Proteins 0.000 description 2
- 101001088879 Homo sapiens Lysine-specific demethylase 5D Proteins 0.000 description 2
- 101000825071 Homo sapiens Sclerostin domain-containing protein 1 Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 210000002593 Y chromosome Anatomy 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- -1 6- hydroxyls -2 Chemical compound 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 102100025620 Cytochrome b-245 light chain Human genes 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical class OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 210000001766 X chromosome Anatomy 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000013010 irrigating solution Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000001035 methylating effect Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to Biotechniques Molecular diagnostic field, a kind of nucleic acid extraction is with as the bisulfite conversion reaction needed for DNA methylation research, and the nucleic acid purification after conversion, can fast and automatically change, high throughput.Methylated with human genome DNA based on research.Comprise the following steps：200 μ l liquid samples add 350 μ l lysates, 20 μ l protease, the sodium hydrogensulfite of 80 μ l 3M pH5.0,20 μ l, 6 hydroxyls 2,5,7,8 tetramethyl chroman, 2 carboxylic acid mixes, 90 DEG C of denatured lysis convert 10 minutes for 5 minutes, 60 DEG C, are repeated once, silicon substrate magnetic bead combination nucleic acid is added after converting completely, after washed 23 washings of liquid, magnetic bead coker pickling is taken off with alkaline eluant and is analyzed for DNA methylation.The kit significantly improves operation convenience compared with existing similar product, easy to be integrated with the self-reacting device of in the market, is diagnosed suitable for tumor cells.
Description
Technical field
The invention belongs to the research that methylates in biomedical sector, especially epigenetics, the promoter of specific gene
The methylation state on region CpG islands is related with diagnosis to tumor screening.
Background technology
The frequency that the CpG sequences of eukaryotic gene group especially vertebrate methylate is very high, but part
The promoter of gene (45% or so) nearby there are the CpG regions for being rich in nothing and methylating of one section of about 1000bp, is referred to as CpG
Island.CpG islands abnormal methylation is typically found in the normal dyeing of the promoter for the tissue-specific gene do not expressed, trace inactivation
On the X chromosome of promoter and the women inactivation of body allele in the promoter of each gene.This gene promoter methylation
Mode often make tumor suppressor gene transcriptional inactivation, the expression of a variety of cancer associated genes can be promoted, activate the propagation of tumour cell.
Due to DNA methylation and human developmental and the substantial connection of tumor disease, the apparent something lost using methylation analysis as representative in recent years
Pass and epigenomics is more active.
DNA methylation analysis usual way be：The good genomic DNA of extraction purification, through ultramicron uv-spectrophotometric
After meter is quantitative, DNA to be transformed is adjusted in suitable scope：1ng-2μg.Sodium hydrogensulfite and DNA are added under alkaline environment
Liquid is protected, is reacted 10-30 minutes by 95 DEG C of abundant denaturation, 60 DEG C, is repeated once to ensure that conversion is abundant.Product carries out again
Nucleic acid crosses column purification.Bisulf iotate-treated make it that unmethylated Cytosines are uracil, and 5 '-methylcystein is then
It is constant, it can also be identified by sequencing identification by methylation specific primer：Two pairs of primers are designed, it is sub- with weight respectively
Sequence complementary pairing after sulfuric acid salt treatment, i.e., a pair of methylate DNA chain combined after processing, another pair combines non-after handling
Methylate DNA chain.Analysing amplified product, if can amplify fragment with the primer for methylate DNA chain after processing, illustrates
The detected site, which exists, to methylate；Vice versa.Methylation sites PCR Taqman probes are directed in addition, can also use
High-resolution solubility curve (HRM) method based on method, PCR fluorescent dyes is identified.
There are step is more, process is cumbersome, DNA damages for the processing method of nucleic acid extraction and conversion in current methylation analysis
Consumption is big, time length when small (including the process such as sample extraction and measure, DNA conversions, purifying, at least 3), can not automate etc. and to lack
Point.
The content of the invention
For the deficiencies in the prior art, the present invention provides a kind of mankind or the genomic nucleic acids of higher mammal carry
Take, the bisulfite conversion reaction in methylation analysis, after conversion nucleic acid the rapid automated processes that are integrated of purifying, always
Time is within one hour.Its remarkable advantage is to automate, and is not susceptible to manual operation and repeatedly uncaps bring potential intersection
Pollution, than before traditional methylation analysis sample extraction and conversion need 6 it is small when more than shorten be the time more than 80%.In addition,
Conventional method is due to dividing three phases to complete, step is lost greatly more, and the present invention once completes, and DNA losses are few.Realize that the purpose is adopted
Technical key point is as follows：
1. Human Fluids' sample (serum, blood plasma, whole blood, urine, saliva, cerebrospinal fluid, irrigating solution, secretion etc.), oral cavity
Soak of swab or anal swab etc., (then reduces higher than this content and takes not higher than 10 μ g containing human genome DNA in theory
Sample amount, and certain volume is supplied with water), sample 200 μ l.
2. sequentially add 350 μ l lysates, 20 μ l liquid of protease, 80 μ l 3M sodium hydrogensulfites (pH5.0), 20 μ l DNA
Protective agent, after mixing, adds appropriate paraffin oil (Sigma) covering liquid level.
3.70-95℃2-10min、60℃10-30min,70-95℃2-10min、60℃10-30min。
4. adding the magnetic bead that 300 μ l are suspended in isopropanol, 5-10min is mixed.
5. being enriched with magnetic bead, it is transferred in 1ml cleaning solutions A and washes once, wash twice in 1ml cleaning solutions B, most washed after 0.1ml
In de- liquid, room temperature to 80 DEG C of 5-10min elutes.
Wherein lysate includes：5M GITC, 50mM sodium citrates, 5%Thesit, 5%Brij-58,1%DTT, 0.2M
Urea, 0.1%SDS, 10mM EDTA.Na2 (pH6.5)
Cleaning solution A is included：2M GITC, 0.1%SDS, 20mM Tris, 5mM EDTA.Na2,50% ethanol (pH7.5)
Cleaning solution B is included：2mM Tris, 2mM NaCL, 0.5mM EDTA.Na2,80% ethanol (pH7.5)
Eluent includes：50mM Tris
Protein enzyme solution is Proteinase K, is dissolved in 50% glycerine, 10mM Tris, 1mM EDTA.Na2,5mM CaCL2,5mM
Ca (OAc) 2,0.2%SDS, concentration are 20mg/ml (pH8.0)
3M sodium hydrogensulfites (pH5.0)：Contain 0.1%EDTA.Na2 as stabilizer
DNA protective agents：Comprising Trolox, i.e. 6- hydroxyls -2,5,7,8- tetramethyl chroman -2- carboxylic acids, are radicals scavenging
Agent
Magnetic bead：Silicon substrate magnetic bead is interpreted into 6.6mg/ml with isopropanol floride, can select Roche, Merck, Invitrogen, Kai Jie,
Hui Er, Jin Maige, profit difficult to understand, the product for Du Deng companies.
, can be with the EpiTect Bisulfite kit (article No.s of QIAGEN to understand whether conversion reaction is properly completed
59104) or EpiTect Fast Bisulfite kit (article No. 59824) compare same a sample.Can arbitrarily it choose several
A gene rich in CpG observes sequencing result after PCR amplification.
Compared with the prior art, the bisulfite conversion before human genome methylation analysis of the present invention and nucleic acid purification
Method has significant superiority：Fast and automatically change, be easy to standardization, the nucleic acid loss and friendship that effectively avoid multi-step from bringing
Fork pollution.
Brief description of the drawings：
Fig. 1 is that present invention collection elderly men urine is special to the EFEMP1 genes progress DNA methylation on No. 2 chromosomes
Fluorescent PCR amplification figure, abscissa represent amplification cycles number, and ordinate represents fluorescent value intensity.
Fig. 2 is that present invention collection elderly men urine carries out the SOSTDC1 genes on No. 7 chromosomes DNA methylation spy
Different fluorescent PCR amplification figure, abscissa represent amplification cycles number, and ordinate represents fluorescent value intensity.
Fig. 3 is that present invention collection elderly men urine carries out the RASSF1A genes on No. 3 chromosomes DNA methylation spy
Different fluorescent PCR amplification figure, abscissa represent amplification cycles number, and ordinate represents fluorescent value intensity.
Fig. 4 is that present invention collection elderly men urine is specifically glimmering to the GST1F genes progress DNA methylation of No. 11 chromosomes
Light PCR amplification result figure, abscissa represent amplification cycles number, and ordinate represents fluorescent value intensity.
Fig. 5 is that present invention collection elderly men urine is special to the CYBA genes progress DNA methylation on No. 16 chromosomes
Fluorescent PCR amplification figure, abscissa represent amplification cycles number, and ordinate represents fluorescent value intensity.
Fig. 6 is that present invention collection elderly men urine is specifically glimmering to the KDM5D genes progress DNA methylation on Y chromosome
Light PCR amplification result figure, abscissa represent amplification cycles number, and ordinate represents fluorescent value intensity.
Embodiment
The technical solution in the embodiment of the present invention will be clearly fully described by below, it is therefore apparent that described
Embodiment be only the present invention part of the embodiment instead of all the embodiments.Based on the embodiments of the present invention, this area
All other embodiment that those of ordinary skill is obtained without making creative work, belongs to protection of the present invention
Scope.
A kind of urine DNA methylation analysis method, comprises the following steps：
Urine specimen 1. (containing cast-off cells) 5-30ml, 3000rpm10min, supernatant discarding stay bottom liquid 1-5ml,
2. vibration takes 200 μ l after mixing, the cracking hole for the being filled with 350 μ l lysates in advance position in 96 hole depth orifice plates is added,
Suction makes a call to 1 time to mix, and adds the suction of 20 μ l liquid of protease and makes a call to 1 time, adds 80 μ l sodium hydrogensulfites, 20 μ l DNA protective agents, inhale
Beat and mix.
Note：The 1st row of 96 hole depth orifice plates of reagent are prefilled with added with 350 μ l lysates, the 2nd row added with 300 μ l magnetic beads, the
3 row show 100 μ l added with 950 μ l cleaning solutions B, the 5th row added with 950 μ l cleaning solutions A, the 4th row added with 950 μ l cleaning solutions B, the 6th
Eluent.The 1-6 row of deep-well plates, 7-12 row one extraction unit of each independent composition, each unit can do A-H totally 8 marks
This.
3. run according to following procedure, in day enterprising this cracking of rower of the automatic instrument for extracting nucleic acid of NP968 of grand biotechnology,
The conversion of DNA bisulfites, washing and purifying and etc., the purifying DNA after completing conversion can be obtained after end of run.
4. can also implement the comparative studies that is divided into two with portion sample, that is, carry out bisulfite conversion and do not convert
Compare.It is the nucleic acid extraction purifying that conversion is not carried out that sodium hydrogensulfite is not added with step 2 and is not added with DNA protective agents.
5. using multipair primer combination of probe, to the EFEMP1 genes on No. 2 chromosomes, (Illumina methylates core respectively
The site numbering cg05385513 of piece), the RASSF1A genes on No. 3 chromosomes, the SOSTDC1 genes on No. 7 chromosomes
CYBA on the GST1F genes of (cg06363129, cg11417025, cg07220448), No. 11 chromosome, No. 16 chromosomes
KDM5D genes (this research gathers elderly men urine) on gene (cg08843517), Y chromosome carry out DNA methyl
Change specific fluorescence PCR amplification.
The results show that EpiTect is first used again with the QIAamp DNA Blood Mini kit extractions of QIAGEN
Bisulfite kit are converted to be purified with DNA, to be sequenced after above-mentioned multipair primer amplification, is as a result consistent with the present invention 100%.
Claims (4)
1. the purification process of nucleic acid and examination after the sample preparation, bisulfite conversion, conversion before a kind of nucleic acid methylation analysis
Agent, it is characterised in that cracking, denaturation, conversion are in one automates step, plus nucleic acid combination magnetic bead and cleaning elution etc. certainly
Dynamicization nucleic acid purification so that sample process and DNA conversions before whole DNA methylation analysis are able to rapid automatized continuous complete
Into.
2. lysate described in is by 5M GITC, 50mM sodium citrates, 5%Thesit, 5%Brij-58,1%DTT, 0.2M
Urea, 0.1%SDS, 10mM EDTA.Na2 (pH6.5) etc. are formed, and the cleaning solution A is by 2M GITC, 0.1%SDS, 20mM
Tris, 5mM EDTA.Na2,50% ethanol (pH7.5) etc. form, the cleaning solution B by 2mM Tris, 2mM NaCL,
0.5mM EDTA.Na2,80% ethanol (pH7.5) etc. form, and the eluent includes 50mM Tris etc..
3. cracking according to the present invention and conversion reaction, it is characterized in that including using bisulfites as the conversion fluid of representative and
6- hydroxyl -2,5,7,8- tetramethyl chroman -2- carboxylic acids protect liquid for the free radical scavenger of representative.
4. method according to the present invention and reagent, it is characterized in that using paramagnetic particle method instrument for extracting nucleic acid, including the grand biological section in day
What the companies such as skill, Tiangeng are biochemical, Ai Kang is biological, large generation biology, Kai Jie, Roche, Hamilton, Supreme Being agree, Pa Jin-Ai Ermo produced
Self-reacting device, and not limited to this quasi-instrument.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711240822.7A CN107988205A (en) | 2017-12-01 | 2017-12-01 | A kind of nucleic acid extraction and bisulfite conversion and the kit of nucleic acid purification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711240822.7A CN107988205A (en) | 2017-12-01 | 2017-12-01 | A kind of nucleic acid extraction and bisulfite conversion and the kit of nucleic acid purification |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107988205A true CN107988205A (en) | 2018-05-04 |
Family
ID=62034746
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711240822.7A Pending CN107988205A (en) | 2017-12-01 | 2017-12-01 | A kind of nucleic acid extraction and bisulfite conversion and the kit of nucleic acid purification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107988205A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305935A (en) * | 2019-08-07 | 2019-10-08 | 湖南圣湘生物科技有限公司 | The full-automatic method for extracting nucleic acid of Septin9 gene methylation |
| CN111269963A (en) * | 2019-12-31 | 2020-06-12 | 广东凯普生物科技股份有限公司 | One-step nucleic acid extraction and transformation kit and use method thereof |
| CN111593092A (en) * | 2020-05-29 | 2020-08-28 | 武汉爱基百客生物科技有限公司 | Method for converting and purifying DNA bisulfite |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105506109A (en) * | 2015-12-31 | 2016-04-20 | 安诺优达基因科技(北京)有限公司 | Unicell simplified representative bisulfite sequencing method and kit |
| CN107043766A (en) * | 2017-02-08 | 2017-08-15 | 上海纽思格生物科技有限公司 | A kind of nucleic acid magnetic bead extracts reagent of instant |
-
2017
- 2017-12-01 CN CN201711240822.7A patent/CN107988205A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506109A (en) * | 2015-12-31 | 2016-04-20 | 安诺优达基因科技(北京)有限公司 | Unicell simplified representative bisulfite sequencing method and kit |
| 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 |
| CN107043766A (en) * | 2017-02-08 | 2017-08-15 | 上海纽思格生物科技有限公司 | A kind of nucleic acid magnetic bead extracts reagent of instant |
Non-Patent Citations (1)
| Title |
|---|
| GONZALGO等: "Methylation-sensitive single-nucleotide primer extension(Ms-SNuPE) for quantitative measurement of DNA methylation" * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305935A (en) * | 2019-08-07 | 2019-10-08 | 湖南圣湘生物科技有限公司 | The full-automatic method for extracting nucleic acid of Septin9 gene methylation |
| CN111269963A (en) * | 2019-12-31 | 2020-06-12 | 广东凯普生物科技股份有限公司 | One-step nucleic acid extraction and transformation kit and use method thereof |
| CN111269963B (en) * | 2019-12-31 | 2021-07-13 | 广东凯普生物科技股份有限公司 | One-step nucleic acid extraction and transformation kit and use method thereof |
| CN111593092A (en) * | 2020-05-29 | 2020-08-28 | 武汉爱基百客生物科技有限公司 | Method for converting and purifying DNA bisulfite |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7466580B2 (en) | Integrated purification and measurement of DNA methylation and simultaneous measurement of mutations and/or mRNA expression levels in an automated reaction cartridge | |
| CN103717750B (en) | The quantitation of minority nucleic acid substances | |
| JP7123050B2 (en) | Integrated Purification and Measurement of DNA Methylation and Simultaneous Measurement of Mutation and/or mRNA Expression Levels in Automated Reaction Cartridges | |
| US20120208711A1 (en) | Method for Analysis of DNA Methylation Profiles of Cell-Free Circulating DNA in Bodily Fluids | |
| CN106676169B (en) | Hybridization capture kit for detecting breast cancer susceptibility genes BRCA1 and BRCA2 mutation and method thereof | |
| CN107988205A (en) | A kind of nucleic acid extraction and bisulfite conversion and the kit of nucleic acid purification | |
| US10519484B2 (en) | Lytic composition and application thereof, kit, method for preparing nucleic acid by utilizing lytic composition, and nucleic acid analysis method | |
| CN103298955A (en) | Method for construction of plasma DNA sequencing library and kit thereof | |
| CN106929591A (en) | A kind of human HLA B*5801 genetic polymorphism detection kits | |
| CN106755496A (en) | Multiplex PCR specific primer, kit and method based on high throughput sequencing technologies detection hereditary hearing impairment gene | |
| WO2017143866A1 (en) | Kit and method for quantitative detection of dna methylation in rprm genes | |
| CA2905425A1 (en) | Methods of isolating nucleic acid using a lysis buffer containing ethanol | |
| CN107858409A (en) | A kind of micro degrading genes group DNA methylation builds storehouse sequence measurement and its kit | |
| CN113881739B (en) | Method for oxidizing nucleic acid molecules containing jagged ends, reduction method and library construction method | |
| EP3904516A1 (en) | Methylation modification-based tumor marker stamp-ep6 | |
| CN109837344B (en) | Methylated EphA7 nucleotide fragment, detection method and application thereof | |
| JP5258760B2 (en) | Method for amplifying methylated or unmethylated nucleic acid | |
| CN110452958B (en) | Joint, primer and kit for methylation detection of micro-fragmented nucleic acid and application of joint and primer and kit | |
| WO2020135347A1 (en) | Method for detecting dna methylation, test kit, device and application | |
| CN106609299B (en) | Kit for determining concentration of fetal free DNA in plasma of pregnant woman | |
| CN107227372A (en) | Primer, molecular beacon, kit and its detection method of ALDH2 gene pleiomorphism quick detections | |
| CN106868165A (en) | A kind of fast simple gene pleiomorphism detecting method and kit and application | |
| CN107090448A (en) | A kind of rapid extraction nuclei aoid methods detected for clinical sample PCR | |
| CN107988334B (en) | Method for SNP typing by direct PCR of oral swab | |
| CN114891886B (en) | Nucleic acid product, kit and application for diagnosing bladder cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180504 |