CN102807975A - Method for rapidly extracting nucleic acid from biological sample - Google Patents
Method for rapidly extracting nucleic acid from biological sample Download PDFInfo
- Publication number
- CN102807975A CN102807975A CN2011101432418A CN201110143241A CN102807975A CN 102807975 A CN102807975 A CN 102807975A CN 2011101432418 A CN2011101432418 A CN 2011101432418A CN 201110143241 A CN201110143241 A CN 201110143241A CN 102807975 A CN102807975 A CN 102807975A
- Authority
- CN
- China
- Prior art keywords
- sample
- chelex
- pcr reaction
- extracting
- aqueous solution
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 61
- 108020004707 nucleic acids Proteins 0.000 title abstract description 7
- 102000039446 nucleic acids Human genes 0.000 title abstract description 7
- 150000007523 nucleic acids Chemical class 0.000 title abstract description 7
- 239000012472 biological sample Substances 0.000 title abstract 3
- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 37
- 239000007864 aqueous solution Substances 0.000 claims abstract description 30
- 239000012188 paraffin wax Substances 0.000 claims abstract description 21
- 238000003757 reverse transcription PCR Methods 0.000 claims abstract description 18
- 238000000605 extraction Methods 0.000 claims abstract description 13
- 238000010438 heat treatment Methods 0.000 claims abstract description 12
- 239000008280 blood Substances 0.000 claims description 14
- 210000004369 blood Anatomy 0.000 claims description 14
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 7
- 210000000582 semen Anatomy 0.000 claims description 6
- 241000521257 Hydrops Species 0.000 claims description 5
- 206010030113 Oedema Diseases 0.000 claims description 5
- 230000001575 pathological effect Effects 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- 210000002700 urine Anatomy 0.000 claims description 5
- 230000004888 barrier function Effects 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 55
- 239000003960 organic solvent Substances 0.000 abstract description 5
- 229940057995 liquid paraffin Drugs 0.000 abstract description 4
- 230000007062 hydrolysis Effects 0.000 abstract description 2
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 abstract 1
- 108091005804 Peptidases Proteins 0.000 abstract 1
- 239000004365 Protease Substances 0.000 abstract 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 230000002411 adverse Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 abstract 1
- 230000014759 maintenance of location Effects 0.000 abstract 1
- 239000012528 membrane Substances 0.000 abstract 1
- 239000008096 xylene Substances 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 15
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 12
- 230000008569 process Effects 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 239000000284 extract Substances 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 7
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 7
- 238000000246 agarose gel electrophoresis Methods 0.000 description 7
- 235000019441 ethanol Nutrition 0.000 description 6
- 230000008520 organization Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 108010067770 Endopeptidase K Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000009514 concussion Effects 0.000 description 4
- 239000003599 detergent Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000000227 grinding Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 239000001993 wax Substances 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- RIZUCYSQUWMQLX-UHFFFAOYSA-N 2,3-dimethylbenzoic acid Chemical compound CC1=CC=CC(C(O)=O)=C1C RIZUCYSQUWMQLX-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229910000906 Bronze Inorganic materials 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000010974 bronze Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- KUNSUQLRTQLHQQ-UHFFFAOYSA-N copper tin Chemical compound [Cu].[Sn] KUNSUQLRTQLHQQ-UHFFFAOYSA-N 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000007479 molecular analysis Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- KAKZBPTYRLMSJV-UHFFFAOYSA-N vinyl-ethylene Natural products C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a method for rapidly extracting nucleic acid from a biological sample. The method comprises: taking a biological sample, placing the sample in an aqueous solution of chelex-100 or a DEPC aqueous solution of chelex-100, and heating for 1-50 minutes at a temperature of 80-100 DEG C; placing an extraction column into a centrifuge tube, adding the resulting liquid to the extraction column, and centrifugating for 2-120 seconds at a rotation speed of 1000-20000 rotation/min; and taking the centrifugated liquid to carry out a PCR reaction or a RT-PCR reaction. According to the present invention, chelex-100 and a high temperature heating method are adopted to extract so as to eliminate a protease hydrolysis step, eliminate a paraffin section dewaxing step, remove an organic solvent xylene, and substantially shorten a time; and a retention membrane is placed in the extraction column so as to remove adverse effects on the PCR reaction or the RT-PCR reaction by the liquid paraffin and the chelex-100, and improve sensitivity of the PCR reaction or the RT-PCR reaction.
Description
Technical field
The present invention relates to a kind of from biological specimen the method for rapid extraction nucleic acid.
Background technology
Dna molecular is the basic material of molecular biology research, can take different method for extracting to obtain the DNA that quality and quantity does not wait according to different experiment purposes.The method of extracting DNA generally has following several kinds at present:
(1) anionic detergent method: make protein denaturation with stain removers such as SDS or xylic acid sodium, can directly from biomaterial, extract DNA.In the cell between DNA and the protein normal electrostatic attraction or the co-ordination bond of borrowing combines because anionic detergent can destroy this valence link,, react but anionic detergent usually influences subsequent P CR so anionic detergent commonly used extracts DNA.
(2) water extraction process: utilize nucleic acid to be dissolved in the character of water, after the histocyte fragmentation, remove RNA with low salts solution, will precipitate soluble in waterly then, DNA fully is dissolved in the water, supernatant is collected in centrifugal back.In supernatant, add solid sodium chloride and be adjusted to 2.6M.Add 2 times of volume 95% ethanol, stir out with paddling process immediately, use the washing of 66%, 80% and 95% ethanol and third bronze medal then respectively, at last at air drying, both got the DNA sample, protein contnt is higher among the DNA that this method is extracted, so generally need not.
(3) phenol extraction process: phenol has suppressed the Degradation of DNase simultaneously as protein denaturant.When handling homogenate with phenol, because albumen and DNA link button are disconnected, the protein molecular surface contains again that a lot of polar groups are similar with phenol to mix.Protein molecular is dissolved in the phenol phase, and DNA is soluble in the aqueous phase.Take out water layer after the centrifugal layering, repeatedly repetitive operation remerges the water that contains DNA, utilizes nucleic acid to be insoluble to the character of alcohol, uses ethanol sedimentation DNA.This moment, DNA was a ten minutes heavy-gravity material, and one of the very long coiled of useable glass takes out.The characteristics of this method are to make the DNA of extraction keep native state.
The phenol extraction process is the most conventional method of extracting DNA, but because will use phenol, harmful to the experimenter, therefore the commercial kit of utilizing the method for adsorbing post to extract DNA is also arranged at present.Because need the Proteinase K treating processes, generally need extractive process through 3-4 hour, could get into subsequent P CR reaction.
There is a large amount of various diseases formaldehyde fixed paraffin-embedded tissues (formalin fixed and paraffin embedded tissues in hospital pathology department; FFPET); For providing great deal of information source, targeted therapy in recent years, molecular pathology research make from paraffin-embedded tissue extracting DNA become the focus of research.Yet the degraded of the wax embedding tissue DNA after the formalin fixed is serious; The file FFPET tissue slice amount of getting is limited; Tradition phenol chloroform extraction DNA step is numerous and diverse; The serious grade of leaching process loss all restricted the Application Research of paraffin organization in molecular pathology, and how efficient, high quality extracts that DNA carries out molecular studies and diagnoses most important utilizing paraffin organization in the micro-paraffin organization.
Tradition extracting DNA in the paraffin organization needs earlier to remove YLENE with absolute ethyl alcohol then with the YLENE dewaxing, and dewaxing process is loaded down with trivial details, needs 3～4 hours at least, and YLENE is organic solvent, and is harmful to experimental implementation person's health.After having taken off wax, sample also needs could get into subsequent P CR reaction again through 3～4 hours extracting.
Chelex-100 is a kind of chelate resin of being made up of vinylbenzene and benzene divinyl interpolymer; Its suspension is under alkaline environment (pH10～11) and 100 ℃ of conditions; Can cause membranolysis and the DNA sex change is discharged; But and the combination polyvalent cation of Chelex-100 highly selective, remove the polyvalent metal ion in sample and the damping fluid, avoid the degraded of template DNA in the boiling part.Chelex-100 extracts DNA and has economy, advantage such as easy, efficient, has been used at present extract DNA from whole blood or blood, seminal fluid or seminal stain, mixed stain, hair, culturing cell etc.But generally also need could get into subsequent P CR reaction through 2～3 hours extractive process.
The report that adopts Chelex-100 to extract the genetic material in the paraffin section has been arranged at present; In article " comparisons of four a kinds of micro-paraffin organization DNA extraction methods " literary composition of delivering on Hainan medical science institute journal in 2010, just adopted Chelex-100 to extract the DNA in the paraffin section, but in the document; Before adopting Chelex-100 to extract; Still adopt the step of YLENE dewaxing, washing with alcohol, thereby made complex steps, operated not enough simple and fast.For this reason, the contriver has proposed a kind of simple and fast, the respond well method of from biological specimen, extracting DNA.
But still do not adopt Chelex-100 to extract the report of RNA at present, for this reason, the contriver has proposed simply quick, the respond well method of from biological specimen, extracting RNA of a kind of Chelex-100 of employing.
Summary of the invention
Goal of the invention of the present invention is to propose a kind of rapid extracting method of nucleic acid.
In order to realize goal of the invention of the present invention, the technical scheme that is adopted is:
The present invention relates to a kind of from biological specimen the method for rapid extraction DNA, described method may further comprise the steps:
1. the biological specimen that takes a morsel, said biological specimen is selected from pathological tissue paraffin section sample, blood cake seminal stain or semen sample, tissue juice sample, CSF sample, blood sample, serum sample, plasma sample, urine specimen, hydrops sample, histocyte sample;
2. biological specimen is placed the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 1～80g/100ml;
3.80 heated 1～50 minute under～100 ℃ of conditions;
4. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 1000～20000 rev/mins, centrifugal 2～120 seconds;
5. collect the liquid under leaving, be used for the PCR reaction.
The invention still further relates to a kind of from biological specimen the method for rapid extraction RNA, it is characterized in that described method may further comprise the steps:
1. the biological specimen that takes a morsel, said biological specimen is selected from pathological tissue paraffin section sample, blood cake seminal stain or semen sample, tissue juice sample, CSF sample, blood sample, serum sample, plasma sample, urine specimen, hydrops sample, sample of hair, histocyte sample;
2. place the DEPC aqueous solution of 1～80%chelex-100;
3.80 heated 1～50 minute under～100 ℃ of conditions;
4. the extracting column jecket is put into centrifuge tube, the liquid that obtains in the step (3) is joined in the extracting column jecket, 1000～20000 rev/mins, centrifugal 2～120 seconds;
5. collect the liquid under leaving, be used for the RT-PCR reaction.
Wherein, first optimal technical scheme of the present invention is that described method extracting post is to have one deck to play the film of physical barriers in the extracting column bottom.
Second optimal technical scheme of the present invention is that the concentration of the aqueous solution of described chelex-100 is 2～50g/100ml, more preferably 3～10g/100ml.
The 3rd optimal technical scheme of the present invention is that the concentration of the EDPC aqueous solution of described chelex-100 is 2～50g/100ml, preferred 3～10g/100ml.
The 4th optimal technical scheme of the present invention is that described Heating temperature is 90～100 ℃, preferred 90～95 ℃.
The 5th optimal technical scheme of the present invention is that be 5～30 minutes described heat-up time, preferred 8～10 minutes.
The 6th optimal technical scheme of the present invention is that the described centrifugal time is 2～120 seconds, preferred 10～30 seconds.
The 7th optimal technical scheme of the present invention does, described centrifugal rotation speed is 5000～20000 rev/mins, preferred 10000～20000 rev/mins, and more preferably 15000～20000 rev/mins.
Wherein, the biological specimen among the present invention is selected from pathological tissue paraffin section sample, blood cake seminal stain or semen sample, tissue juice sample, CSF sample, blood sample, serum sample, plasma sample, urine specimen, hydrops sample, sample of hair, histocyte sample; When biological specimen is histocyte sample or paraffin section, in step 1, adopt grinding rod that biological tissue is ground, when extracting RNA; Then need grind on ice; To reduce the RNA degraded, the release that improves RNA in the biological tissue improves checkability.
Technical superiority of the present invention is:
1. the inventive method is simple, and the time is short, and does not use harmful organic solvent.Generally extracting DNA needs are first from paraffin-embedded tissue in the traditional method remove YLENE with absolute ethyl alcohol then with the YLENE dewaxing, and the used time is long; Process is loaded down with trivial details; At least need 3～4 hours ability to accomplish, and YLENE is organic solvent, harmful.After having taken off wax, sample need pass through the processing of Proteinase K again, and to remove protein, this process also needs 3～4 hours, so DNA in the usual method extracting paraffin-embedded tissue gets into follow-up PCR reaction, needs more than 6～8 hours.Of the present invention method is simple, do not use volatility harmful organic solvent YLENE, can not damage the experimenter, and required time shortens dramatically, and minute can accomplish surplus only needing 10;
2. the present invention adopts heat to extract, and not only makes the paraffin liquefy, and makes the protein denaturation in the tissue, and DNA or RNA are released out, and having saved needs 3～4 hours process of Proteinase K hydrolysis needs;
3. liquid paraffin and chelex-100 can cause detrimentally affect to PCR or RT-PCR reaction usually; Through the present invention; Can liquid paraffin and chelex-100 be trapped on the film of extracting post; Thereby eliminated liquid paraffin and chelex-100 detrimentally affect, improved the sensitivity of PCR or RT-PCR reaction PCR or RT-PCR reaction;
4. still do not adopt at present Chelex-100 to extract the report of RNA, the contriver has proposed simply quick, the respond well method of from biological specimen, extracting RNA of a kind of Chelex-100 of employing in the present invention.
Description of drawings
Fig. 1 adopts the two kinds of extractive genomic dna agarose gel electrophoresis of method figure among the embodiment 1; Wherein, M represents molecular weight marker; 1 representative be with the extractive genomic dna agarose gel electrophoresis of test kit figure, 2 what represent is with the extractive genomic dna agarose gel electrophoresis of method of the present invention figure.
Following specific embodiments of the invention is done further explanation and explanation, but content of the present invention is not constituted restriction.The used reagent of the present invention is the commercial reagent, and used laboratory apparatus is the laboratory common instrument.
Embodiment
Embodiment 1
1. extract the DNA in the paraffin-embedded tissue section, 3 samples of every kind of method extracting, parallel comparison with two kinds of methods.
1) DNA during the paraffin organization section DNA extraction test kit extracting paraffin-embedded tissue of producing with Beijing hundred Tyke Bioisystech Co., Ltd is cut into slices
A. get medical paraffin (model: model: BX12M311398, the long-range Science and Technology Ltd. in Chinese and Western, Beijing produces) investing tissue's section of 2 5um and put into the centrifuge tube of 1.5mL.
B. add 1ml YLENE, concussion mixing, 55 ℃ of water-baths 10 minutes.13000 changeed 5 minutes, inhaled and abandoned supernatant.
C. repeating the 2nd goes on foot one to three time.
D. add the 1ml absolute ethyl alcohol, the concussion mixing.13000 changeed 5 minutes, inhaled and abandoned supernatant.
E. repeated for the 4th step once.
F. with the sample oven dry that settles down.
G. add 200 μ l Buffer 1, mixing.
H. add 25 μ l Proteinase K, the concussion mixing.55 ℃ of water-bath digestion are spent the night.
I.13000 left the heart 5 minutes, supernatant is transferred in the clean 1.5mL centrifuge tube.
J. add 220 μ l Buffer 2, the concussion mixing.70 ℃ of water-baths 10 minutes
K. add 220 μ l absolute ethyl alcohols, mixing.
L. the extracting post is put into collection tube, move into the mixing liquid in the 5th step, room temperature left standstill 3 minutes.
M.13000 leave the heart 2 minutes, and changed liquid over to pillar again, cross post again.
N.13000 leave the heart 2 minutes, and abandoned liquid.
O. move into pillar in the new collection tube, add 500 μ l Buffer 3.
P. move into pillar in the new collection tube, 13000 left the heart 1 minute, abandoned liquid.Add 600 μ l Wash Buffer.(Wash Buffer adds an amount of absolute ethyl alcohol in advance by packing)
Q.13000 leave the heart 1 minute, and abandoned liquid.Add 600 μ l DNA Wash Buffer.
R.13000 leave the heart 1 minute, and abandoned liquid.13000 left the heart 2 minutes, volatilization 4-5 minute of uncapping.
S., move into pillar in the centrifuge tube with cover of 1.5mL, in the adding 40 μ l Elution Buffer of film central authorities (70 ℃ of preheatings of palpus).Room temperature left standstill 3 minutes.
T.13000 left the heart 1 minute, and collected liquid, get 5ul and be used for the PCR reaction.
2) DNA in the method extracting paraffin-embedded tissue section of the present invention
A. get medical paraffin (model: model: BX12M311398, the long-range Science and Technology Ltd. in Chinese and Western, Beijing produces) investing tissue's section of 2 5um;
B. place the solution 1 of 200ul, described solution is the aqueous solution of 10%chelex-100 (sigma company);
C.90 heated 20 minutes under ℃ condition;
D. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 12000 rev/mins centrifugal 30 seconds; Described extracting post is:
E. collect the liquid under leaving, get 5ul and be used for the PCR reaction.
2.DN A authentication method:
2.1 the agarose gel electrophoresis of DNA quality is identified:
Because the dna content in the paraffin section is lower; Can not detect the amount of DNA with plain agar sugar gel electrophoresis method, so will use every kind of extractive three sample genomic dnas of method, respectively get 20 μ L and mix; After vacuum is taken out part moisture; Add appearance on the DNA loading buffer mixing of 5 μ L, 80V electrophoresis 30min in 2% sepharose, observation post extracts genomic quality of DNA and mug under ultraviolet gel imaging appearance.Fig. 1 is the two kinds of extractive genomic dna agarose gel electrophoresis of method figure; Wherein, M represents molecular weight marker, 1 representative be with the extractive genomic dna agarose gel electrophoresis of test kit figure, 2 what represent is with the extractive genomic dna agarose gel electrophoresis of method of the present invention figure.
2.2 the genomic concentration of DNA that fluorescence quantitative PCR detection is extracted
Select primer and TaqMan fluorescent probe, adopt Primer Express software to design voluntarily to people β-2actin genomic dna, synthetic by Shanghai bio-engineering corporation (Sangong).Primer sequence is seen table 1.
Table 1: the sequence table of primer and probe
| Upstream primer | 5′-GGACCTGACTGACTACCTCATGAA-3′ |
| Downstream primer | 5′-CTTAATGTCACGCACGATTTCC-3′ |
| Probe | 5′Fam-CACCGAGCGCGGCTACAGCTTC-TAMARA?3′ |
PCR reaction system: template DNA 2 μ l, 10 * buffer (no Mg2+) 2.5ul, 2.5mmoL/L dNTP 2ul; 25mmoL/L MgCl2 1.5ul, primer 10mmol/L 0.5ul, 10mmol/L probe 0.4ul; 5U/ul Taq polysaccharase 0.5 μ l, remainder is supplied by the sterilization deionized water
The pcr amplification condition: 94 ℃ of 3min, 94 ℃ of 15s, 60 ℃ of 60s, 40 circulations, 60 ℃ are detected fluorescence, and the positive, negative control are all established in each PCR reaction.
Selected three samples altogether, each sample repeats 5 times, uses the amount of β in each sample of fluorescence quantitative PCR detection people-2actin genomic dna respectively, uses the Ct value representation, compares the quality and quantity of two kinds of DNA that method for extracting obtained, and experimental result is as shown in table 2.
Visible from table 2, the extractive Ct value of Ct value comparison reagent kit of the β-2actin that genomic dna increased that obtains with the inventive method extracting is little, and all comparison reagent kit is extractive good to point out the genomic dna content that obtains with the inventive method extracting and quality.
Table 2: adopt two kinds of methods to extract the Ct value of DNA
Embodiment 2
1. the seminal stain sample is placed the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 20g/100ml;
2.95 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 20000 rev/mins, centrifugal 20 seconds;
4. collect the liquid under leaving, be used for the PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 1 adopted in PCR reaction, and obtained similar experimental result.
Embodiment 3
1. the tissue juice sample is placed the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 10g/100ml;
2.100 heating is 50 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 15000 rev/mins, centrifugal 30 seconds;
4. collect the liquid under leaving, be used for the PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 1 adopted in PCR reaction, and obtained similar experimental result.
Embodiment 4
1. after the histocyte sample being ground with grinding rod, place the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 15g/100ml;
2.80 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 15000 rev/mins, centrifugal 30 seconds;
4. collect the liquid under leaving, be used for the PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 1 adopted in PCR reaction, and obtained similar experimental result.
Embodiment 5
1. the blood cake sample is placed the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 20g/100ml;
2.95 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 20000 rev/mins, centrifugal 20 seconds;
4. collect the liquid under leaving, be used for the PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 1 adopted in PCR reaction, and obtained similar experimental result.
Embodiment 6
1. the RNA during the rapid extraction paraffin-embedded tissue is cut into slices:
Medical paraffin (model: BX12M311398, the long-range Science and Technology Ltd. in Chinese and Western, Beijing produces) investing tissue's section of a) getting 3 5um is put into the centrifuge tube of 1.5mL on ice after grinding;
B) place the solution 1 of 200ul, described solution is the DEPC aqueous solution of 10%chelex-100;
C) heated 20 minutes under 90 ℃ of conditions;
D) the extracting column jecket is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting column jecket, 12000 rev/mins centrifugal 30 seconds;
E) liquid under collection is left is got 5ul and is used for the RT-PCR reaction.
2. fluorescence quantitative RT-RCR detects the concentration of the RNA that extracts
Select to people GAPDH primer and TaqMan fluorescent probe, adopt Primer Express software to design voluntarily, synthetic by Shanghai bio-engineering corporation (Sangong).Primer sequence is seen table 1.
Table 1 is the nucleotide sequence of primer, probe:
| Upstream primer | 5′-CATCTTCCAGGAGCGAGA-3′ |
| Downstream primer | 5′-TGTTGTCATACTTCTCAT-3′ |
| Probe | 5′Fam-CCTCACCACCATGGAGAAGGCT-TAMARA?3′ |
Contain in the 40 μ L reaction systems: 2.5 μ L, 10 * PCR reaction buffer (Mg2+free), 3mmol/L Mg2+, 0.125mmol/L dNTP; 0.25 μ mol/L primer, 0.156 μ mol/L probe, polysaccharase 2U; UNG enzyme 0.2U; RT enzyme 100u, the template amount is 3 μ L, make up water to 40 μ L.
Pcr amplification condition: 42 ℃ of 30min, 94 ℃, 5min; 94 ℃ of 15s, 60 ℃ of 60s, 40 circulations, 60 ℃ are detected fluorescence, and the positive, negative control are all established in each PCR reaction.
The mRNA amplification of RT-PCR to GAPDH carried out in 8 paraffin-embedded tissue sections of picked at random, and each sample can both amplify GAPDH, and it is feasible fast pointing out this method, sees table 2.
Table 2 is the Ct value of sample of the present invention:
| Mark this shop | The Ct value |
| 1 | 32.1382 |
| 2 | 32.694 |
| 3 | 29.1025 |
| 4 | 28.9604 |
| 5 | 24.662 |
| 6 | 24.5847 |
| 7 | 21.0877 |
| 8 | 21.1414 |
Embodiment 7
1. the seminal stain sample is placed the DEPC aqueous solution of 100 μ l chelex-100, the concentration of the DEPC aqueous solution of described chelex-100 is 20g/100ml;
2.95 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 20000 rev/mins, centrifugal 20 seconds;
4. collect the liquid under leaving, be used for the RT-PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 6 adopted in RT-PCR reaction, and obtained similar experimental result.
Embodiment 8
1. the tissue juice sample is placed the DEPC aqueous solution of 200 μ l chelex-100, the concentration of the DEPC aqueous solution of described chelex-100 is 10g/100ml;
2.100 heating is 50 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 15000 rev/mins, centrifugal 30 seconds;
4. collect the liquid under leaving, be used for the RT-PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 6 adopted in RT-PCR reaction, and obtained similar experimental result.
Embodiment 9
1. after the histocyte sample being ground with grinding rod on ice, place the DEPC aqueous solution of 400 μ l chelex-100, the concentration of the DEPC aqueous solution of described chelex-100 is 15g/100ml;
2.80 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 15000 rev/mins, centrifugal 30 seconds;
4. collect the liquid under leaving, be used for the RT-PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 6 adopted in RT-PCR reaction, and obtained similar experimental result.
Embodiment 10
1. the blood cake sample is placed the DEPC aqueous solution of 800 μ l chelex-100, the concentration of the DEPC aqueous solution of described chelex-100 is 20g/100ml;
2.95 heating is 10 minutes under ℃ condition;
3. the extracting post is put into centrifuge tube, the liquid that obtains in the step 3 is joined in the extracting post, 20000 rev/mins, centrifugal 20 seconds;
4. collect the liquid under leaving, be used for the RT-PCR reaction.
Wherein, the extracting post is commercially available; The condition identical with embodiment 6 adopted in RT-PCR reaction, and obtained similar experimental result.
Claims (4)
1. the method for a rapid extraction DNA from biological specimen is characterized in that, described method may further comprise the steps:
(1) biological specimen that takes a morsel, said biological specimen is selected from pathological tissue paraffin section sample, blood cake seminal stain or semen sample, tissue juice sample, CSF sample, blood sample, serum sample, plasma sample, urine specimen, hydrops sample, sample of hair, histocyte sample;
(2) biological specimen is placed the aqueous solution of chelex-100, the concentration of the aqueous solution of described chelex-100 is 1～80g/100ml;
Heated 1～50 minute under (3) 80～100 ℃ of conditions;
(4) the extracting post is put into centrifuge tube, the liquid that obtains in the step (3) is joined in the extracting post, 1000～20000 rev/mins, centrifugal 2～120 seconds;
(5) liquid under collection is left is used for the PCR reaction.
2. the method for a rapid extraction RNA from biological specimen is characterized in that, described method may further comprise the steps:
(1) biological specimen that takes a morsel, said biological specimen is selected from pathological tissue paraffin section sample, blood cake seminal stain or semen sample, tissue juice sample, CSF sample, blood sample, serum sample, plasma sample, urine specimen, hydrops sample, sample of hair, histocyte sample;
(2) place the DEPC aqueous solution of 1～80%chelex-100;
Heated 1～50 minute under (3) 80～100 ℃ of conditions;
(4) the extracting column jecket is put into centrifuge tube, the liquid that obtains in the step (3) is joined in the extracting column jecket, 1000～20000 rev/mins, centrifugal 2～120 seconds;
(5) liquid under collection is left is used for the RT-PCR reaction.
3. method according to claim 1 and 2 is characterized in that, described extracting post is to have one deck to play the film of physical barriers in the extracting column bottom.
4. method according to claim 1 and 2 is characterized in that, the concentration of the aqueous solution of described chelex-100 is 2～50g/100ml, preferred 3～10g/100ml; The concentration of the DEPC aqueous solution of described chelex-100 is 2～50g/100ml, preferred 3～10g/100ml.
A) method according to claim 1 and 2 is characterized in that, described Heating temperature is 90～100 ℃, preferred 90～95 ℃.
B) method according to claim 1 and 2 is characterized in that, be 5～30 minutes described heat-up time, preferred 8～10 minutes.
C) method according to claim 1 and 2 is characterized in that, the described centrifugal time is 2～120 seconds, preferred 10～30 seconds.
D) method according to claim 1 and 2 is characterized in that, described centrifugal rotation speed is 5000～20000 rev/mins, preferred 10000～20000 rev/mins, and more preferably 15000～20000 rev/mins.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110143241.8A CN102807975B (en) | 2011-05-30 | 2011-05-30 | Method for rapidly extracting nucleic acid from biological sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110143241.8A CN102807975B (en) | 2011-05-30 | 2011-05-30 | Method for rapidly extracting nucleic acid from biological sample |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102807975A true CN102807975A (en) | 2012-12-05 |
| CN102807975B CN102807975B (en) | 2014-10-29 |
Family
ID=47231846
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110143241.8A Active CN102807975B (en) | 2011-05-30 | 2011-05-30 | Method for rapidly extracting nucleic acid from biological sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102807975B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757095A (en) * | 2013-10-19 | 2014-04-30 | 中国医科大学 | Method for distinguishing individual in mixed seminal stain by single sperm capture and mitochondrial DNA typing |
| CN103882012A (en) * | 2014-04-21 | 2014-06-25 | 山东省农业科学院生物技术研究中心 | Method for rapidly and efficiently extracting DNA (Desoxvribose Nucleic Acid) from milk products such as liquid milk and milk powder |
| CN104232616A (en) * | 2013-06-06 | 2014-12-24 | 芮宝生医股份有限公司 | Method for extracting nucleic acid from formalin-fixed paraffin-embedded sample |
| CN104531682A (en) * | 2015-01-23 | 2015-04-22 | 四川农业大学 | Kit for extracting hair genome DNA (deoxyribonucleic acid) through PCR (polymerase chain reaction) instrument method and application thereof |
| CN105779438A (en) * | 2016-03-22 | 2016-07-20 | 北京交通大学 | Method for quickly separating and detecting crosslinking DNA in paraffin-embedded tissue sample |
| CN106086166A (en) * | 2016-06-07 | 2016-11-09 | 苏州贝斯派生物科技有限公司 | Reagent of detection paraffin section DNA mass and preparation method and application |
| CN106350512A (en) * | 2016-11-24 | 2017-01-25 | 深圳华大基因股份有限公司 | Eukaryotic cell nucleic acid extraction method |
| CN106755467A (en) * | 2017-01-12 | 2017-05-31 | 天津诺禾医学检验所有限公司 | A kind of method of detection FFPE sample DNAs content and integrality |
| CN106987587A (en) * | 2017-04-11 | 2017-07-28 | 江苏睿玻生物科技有限公司 | A kind of rapid extraction nuclei aoid methods for fluorescence quantitative PCR detection |
| CN107271239A (en) * | 2017-07-06 | 2017-10-20 | 浙江大学 | A kind of method for dissolving paraffin tissue sections and application |
| CN110819622A (en) * | 2019-10-09 | 2020-02-21 | 广州达正生物科技有限公司 | Method for rapidly extracting RNA (ribonucleic acid) of paraffin tissue |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2595816C1 (en) * | 2015-11-09 | 2016-08-27 | государственное бюджетное образовательное учреждение высшего профессионального образования "Кемеровская государственная медицинская академия" Министерства здравоохранения Российской Федерации | Method of preparing suspension of human lymphocytes in ethanol-acetic retainer for dna extraction |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101586162A (en) * | 2009-06-10 | 2009-11-25 | 戴立忠 | Method of extracting target nucleic acid and performing PCR augmentation |
-
2011
- 2011-05-30 CN CN201110143241.8A patent/CN102807975B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101586162A (en) * | 2009-06-10 | 2009-11-25 | 戴立忠 | Method of extracting target nucleic acid and performing PCR augmentation |
Non-Patent Citations (4)
| Title |
|---|
| C S EDWIN SHIAW ET AL.: "Evaluation of DNA and RNA Extraction Methods", 《MED J MALAYSIA》, vol. 65, no. 2, 30 June 2010 (2010-06-30), pages 134 - 4 * |
| C. ARNAL ET AL.: "Comparison of seven RNA extraction methods on stool and shellfish samples prior to hepatitis A virus amplification", 《JOURNAL OF VIROLOGICAL METHODS》, vol. 77, 31 December 1999 (1999-12-31), pages 20 - 6 * |
| 周月琴等: "用chelex-100快速提取微量血痕中的DNA", 《复旦学报（医学版）》, vol. 30, no. 4, 31 July 2003 (2003-07-31), pages 379 - 100 * |
| 张幼芳等: "微量体表脱落上皮细胞的DNA检验", 《法律与医学杂志》, vol. 10, no. 2, 31 December 2003 (2003-12-31) * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232616A (en) * | 2013-06-06 | 2014-12-24 | 芮宝生医股份有限公司 | Method for extracting nucleic acid from formalin-fixed paraffin-embedded sample |
| CN103757095B (en) * | 2013-10-19 | 2016-08-17 | 中国医科大学 | Method individual in monosperm captured line mitochondrial DNA typing difference mixing seminal stain |
| CN103757095A (en) * | 2013-10-19 | 2014-04-30 | 中国医科大学 | Method for distinguishing individual in mixed seminal stain by single sperm capture and mitochondrial DNA typing |
| CN103882012A (en) * | 2014-04-21 | 2014-06-25 | 山东省农业科学院生物技术研究中心 | Method for rapidly and efficiently extracting DNA (Desoxvribose Nucleic Acid) from milk products such as liquid milk and milk powder |
| CN103882012B (en) * | 2014-04-21 | 2016-10-05 | 山东省农业科学院生物技术研究中心 | A kind of quickly method of high efficiency extraction DNA from the milk product such as cattle and sheep liquid milk and milk powder |
| CN104531682A (en) * | 2015-01-23 | 2015-04-22 | 四川农业大学 | Kit for extracting hair genome DNA (deoxyribonucleic acid) through PCR (polymerase chain reaction) instrument method and application thereof |
| CN105779438A (en) * | 2016-03-22 | 2016-07-20 | 北京交通大学 | Method for quickly separating and detecting crosslinking DNA in paraffin-embedded tissue sample |
| CN106086166A (en) * | 2016-06-07 | 2016-11-09 | 苏州贝斯派生物科技有限公司 | Reagent of detection paraffin section DNA mass and preparation method and application |
| CN106350512A (en) * | 2016-11-24 | 2017-01-25 | 深圳华大基因股份有限公司 | Eukaryotic cell nucleic acid extraction method |
| CN106755467A (en) * | 2017-01-12 | 2017-05-31 | 天津诺禾医学检验所有限公司 | A kind of method of detection FFPE sample DNAs content and integrality |
| CN106987587A (en) * | 2017-04-11 | 2017-07-28 | 江苏睿玻生物科技有限公司 | A kind of rapid extraction nuclei aoid methods for fluorescence quantitative PCR detection |
| CN107271239A (en) * | 2017-07-06 | 2017-10-20 | 浙江大学 | A kind of method for dissolving paraffin tissue sections and application |
| CN110819622A (en) * | 2019-10-09 | 2020-02-21 | 广州达正生物科技有限公司 | Method for rapidly extracting RNA (ribonucleic acid) of paraffin tissue |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102807975B (en) | 2014-10-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102807975B (en) | Method for rapidly extracting nucleic acid from biological sample | |
| CN102807976B (en) | Rapid nucleic acid extraction kit and applications thereof | |
| KR20100001826A (en) | Nucleic acid extraction apparatus | |
| US20160237496A1 (en) | Methods for assessing status of post-transplant liver and determining and administering specific treatment regimens | |
| KR20100001827A (en) | Nucleic acid extraction method | |
| TWI462930B (en) | A method for isolating nucleic acids from formalin-fixed paraffin embedded tissue samples | |
| CN107201361A (en) | A kind of extracting method of excretion body total serum IgE | |
| CN104561360A (en) | TOP2A gene abnormality detection probe, kit and method | |
| CN103820320A (en) | Non-freezing type RNA (Ribonucleic Acid) protection fluid | |
| CN111487404A (en) | Kit for extracting DNA of body fluid tumor cells | |
| CN105950740B (en) | LAMP kit for early diagnosis of candida krusei and special primer thereof | |
| Liu et al. | The use of combined PCR, fluorescence in situ hybridisation and immunohistochemical staining to diagnose mucormycosis from formalin‐fixed paraffin‐embedded tissues | |
| CN107345246A (en) | A kind of diatom rbcL genetic analysis method and its application in legal medical expert detects | |
| CN109913556A (en) | A kind of primer, kit and its method for Rapid identification dove gender | |
| CN101875980B (en) | Kit and method for detecting Macrobrachium rosenbergii Nodavirus | |
| CN107475441A (en) | It is a kind of to predict reactive biomarker of the patient with breast cancer to AT scheme new adjuvant chemotherapies | |
| CN110864939A (en) | Protein extraction method and application | |
| CN105385699A (en) | cytB specific sequences of four rats in river ports of three gorges area and molecular identification method | |
| CN112195239B (en) | Esophageal squamous carcinoma metastatic tissue and serum exosome marker and application thereof | |
| CN106591426A (en) | COI gene standard complete sequence and molecular identification method of hoolock leuconedys | |
| CN103952399A (en) | Method of extracting ribonucleic acid by using phenol guanidine salt lysate containing indicator | |
| CN110257477A (en) | Nucleic acid liquid phase is extracted and detection method | |
| CN110387370A (en) | It is a kind of to extract micro adder gene and its method for detecting specificity | |
| CN103173524A (en) | Application of KIF20A gene | |
| CN114622010B (en) | Marker for identifying lethal hypothermia based on brown fat and forensic application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20121205 Assignee: SUZHOU YUNTAI BIOLOGICAL PHARMACEUTICAL CO., LTD. Assignor: Xiong Hui Contract record no.: 2014320010178 Denomination of invention: Method for rapidly extracting nucleic acid from biological sample Granted publication date: 20141029 License type: Exclusive License Record date: 20141226 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160120 Address after: 201403 Building 5, building 2066, 3 hope road, Shanghai, Fengxian District Patentee after: Shanghai Rui Rui Biological Technology Co., Ltd. Address before: 201101, room 99, No. 88, Lane 1102, Bao Ming Road, Shanghai, Minhang District Patentee before: Xiong Hui |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20160621 Address after: 201403 Building 5, building 2066, 3 hope road, Shanghai, Fengxian District Patentee after: Shanghai Rui Rui Biological Technology Co., Ltd. Address before: 201101, room 99, No. 88, Lane 1102, Bao Ming Road, Shanghai, Minhang District Patentee before: Xiong Hui |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20180326 Address after: Xiangcheng District Economic Development Zone, Suzhou City, Jiangsu province 215131 Huayuan Road No. 9 Building No. 6 Patentee after: SUZHOU YUNTAI BIOLOGICAL PHARMACEUTICAL CO., LTD. Address before: 201403 Building 5, building 2066, 3 hope road, Shanghai, Fengxian District Patentee before: Shanghai Rui Rui Biological Technology Co., Ltd. |
|
| TR01 | Transfer of patent right |