CN107058293A - A kind of method that 96 orifice plate magnetic bead extracts buccal swab DNA - Google Patents
A kind of method that 96 orifice plate magnetic bead extracts buccal swab DNA Download PDFInfo
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- CN107058293A CN107058293A CN201710353091.0A CN201710353091A CN107058293A CN 107058293 A CN107058293 A CN 107058293A CN 201710353091 A CN201710353091 A CN 201710353091A CN 107058293 A CN107058293 A CN 107058293A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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Abstract
The present invention relates to biological technical field, especially, it is related to a kind of method that 96 orifice plate magnetic bead extracts buccal swab DNA.The method that 96 orifice plate magnetic bead of the present invention extracts buccal swab DNA, when handling swab samples, directly using 96 deep-well plates as carrier, and handle acquisition DNA enzymatic solution liquid, directly the DNA enzymatic solution liquid can be placed on new Tissue Culture Plate and carry out conventional subsequent treatment, the risk of sample mixing when being shifted from centrifuge tube is preferably minimized by whole process, while reducing operating procedure, saves the operating time.
Description
Technical field
The present invention relates to biological technical field, especially, it is related to the side that a kind of 96 orifice plate magnetic bead extracts buccal swab DNA
Method.
Background technology
DNA extract be molecular biology basic experiment, it is most basic side in the genechip detection continued to develop
Method, is also the premise that genetic chip is prepared, analyzes and diagnosed, and therefore, it is one of most basic technology of biology that DNA, which is extracted,.
Successful nucleic acid isolates and purifies four important steps of needs：The broken and cracking of tissue or cell, nucleoprotein are multiple
Fit denaturation, the inactivation of nuclease and the removal of pollutant, high-quality target can be obtained by preferable method for extracting
Nucleic acid, while without albumen, sugar, fat and other pollution of nucleic acid etc..
At present, conventional DNA extraction method mainly has the physics sides such as glass bead method, supercritical ultrasonics technology, polishing, freeze-thaw method
The biological extracting mode such as method, the chemical mode such as CTAB methods, guanidine isothiocyanate method, alkaline lysis, and enzyme extraction method；According to nucleic acid
Isolating and purifying the difference of mode has siliceous material, anion exchange resin extraction etc..Wherein, the DNA extraction method of plant origin
Mainly there is cetyl triethyl group Australia by (CTAB) method, SDS methods and Low pH extraction with high salts method etc.；Bacterium, the DNA of originated from fungus are carried
It is alkaline lysis, boiling method, SDS cracking process and Triton- lysozyme lysis methods etc. to take method；The DNA of animal origin is the most
Classical extracting method mainly has phenol extraction method, isopropanol precipitating method, first phthalein amine cracking process, and existing many methods are all herein
On the basis of improve and get.
Mucous membrane of mouth is stratified squamous epithelium, and with vigorous, updating decision, caducous feature is metabolized, it is by multi-layer cellular
Composition, basalis has vigorous splitting ability, although its epithelium cast-off cells karyopyknosis, as other histocytes
With complete genomic DNA, so that the biological assays material as medical jurisprudence and science of heredity DNA polymorphic detection partings.
It is previously many using methods such as phenol chloroform method or Chelex-100, there is phenol, contamination with chloroform or yield rate are low, purity is not high lacks
Point.
Using buccal swab method (mouth desquamated cells) extract mouth epithelial cells be a kind of non-intervention, no pain, it is noninvasive
Wound, simple DNA methods of sample collection, this method are applied to gather the DNA sample of any age bracket crowd, while can also answer
It is used for the inconvenient blood sampling crowd such as neonate, infant or the old,weak,sick and disabled person.
At present, extracted more than buccal swab DNA extraction using 96 orifice plate magnetic beads, it is necessary to elder generation during processing buccal swab sample
Swab head is cut and is put into 1.5ml or so centrifuge tubes, digestive juice and Proteinase K is added, cracking and the enzymolysis for carrying out cell are anti-
Ying Hou, then it is transferred to the follow-up experiment of 96 deep-well plates progress.On the one hand, the mode of separate operations adds operating procedure, made twice
Wasted into the time, while during sample is shifted, also significantly increasing the risk of sample mixing.
The content of the invention
The invention provides a kind of method that 96 orifice plate magnetic bead extracts buccal swab DNA, oral cavity in the prior art is solved
The problem of swab DNA extracts sample easy sample mixing.
To achieve the above object, the present invention proposes a kind of method for cracking and digesting buccal swab DNA, including following step
Suddenly：
(1) Tissue Culture Plate is taken, room is corresponded in sequence, buccal swab head is placed in deep-well plates；
(2) digestive juice and Proteinase K are added into the Tissue Culture Plate；
(3) Tissue Culture Plate is sealed；
(4) Tissue Culture Plate is obtained into DNA enzymatic solution liquid in fully mixing simultaneously water bath processing on oscillator.
The Tissue Culture Plate is 96 orifice plates.
The Tissue Culture Plate is 96 deep-well plates.
In the step (3), sealed using the rubber pad special of the Tissue Culture Plate.
The temperature of the water bath processing step is 56 DEG C.
The digestive juice is BL Buffer.
The addition of the digestive juice is 500ul.
The addition of the Proteinase K is 20ul.
The invention also discloses a kind of method that 96 orifice plate magnetic bead extracts buccal swab DNA, comprise the following steps：
(A) according to the cracking and enzymolysis buccal swab DNA method, the DNA enzymatic solution liquid is obtained；
(B) the DNA enzymatic solution liquid is transferred on new Tissue Culture Plate, carried out according to prior art conventional method follow-up
DNA extract.
In the step (B), in addition to add the step of magnetic bead is extracted.
The method that 96 orifice plate magnetic bead of the present invention extracts buccal swab DNA, it is directly deep with 96 when handling swab samples
Orifice plate is carrier, and handles acquisition DNA enzymatic solution liquid, the DNA enzymatic solution liquid directly can be placed in into new Tissue Culture Plate enterprising
The risk of sample mixing when being shifted from centrifuge tube is preferably minimized by row routine subsequent treatment, whole process, while operating procedure is reduced, section
The operating time is saved.
Brief description of the drawings
Fig. 1 is the operation chart of 96 orifice plates extraction buccal swab DNA in the embodiment of the present invention 1；
Fig. 2 is another operating angle schematic diagram of 96 orifice plates extraction buccal swab DNA in the embodiment of the present invention 1；
Fig. 3 is the operation chart of single tube centrifuge tube extraction buccal swab DNA in comparative example of the present invention.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, it is below in conjunction with the accompanying drawings and specific real
Applying mode, the present invention is further detailed explanation.
Embodiment 1
As shown in Figure 1, 2, the method that 96 orifice plate magnetic beads described in the present embodiment extract buccal swab DNA is entered with 96 orifice plates
OK, comprise the following steps：
(A) buccal swab DNA cracking and enzymolysis, are specifically included：
(1) it is Tissue Culture Plate to take 96 deep-well plates, according to《Sample extraction table》Order, correspond room, use hand
Art, which is cut, cuts buccal swab head, is placed in 96 deep-well plates；
(2) 500ul digestive juice BL Buffer and 20ul Proteinase K are added into the Tissue Culture Plate；
(3) 96 deep-well plates are sealed with the 96 deep-well plates rubber pad special；
(4) 96 deep-well plates are obtained into DNA enzymolysis liquids in fully being mixed on oscillator and controlling 56 DEG C of water bath processings；
(B) the DNA enzymatic solution liquid is transferred on new Tissue Culture Plate, carried out according to prior art conventional method follow-up
DNA extract, specifically include：
(1) 350ul absolute ethyl alcohols are added, 30ul magnetic beads are added, 1200rpm on blending instrument is placed in, mixed 1 minute；Stand
Absorption 10 minutes, and mixed 1 time respectively standing the 3rd minute, the 6th minute；
(2) after absorption terminates, magnetic bead is mixed, deep-well plates are placed on magnetic frame, to solution clarification, torn rubber cushion off, abandon
Supernatant in deep-well plates is removed, is careful not to touch magnetic bead；
(3) the cleaning solution BW1 that 600ul has added ethanol is added, covers rubber cushion, be placed in 1350rpm on blending instrument, mix 1 point
Clock；And deep-well plates are placed on magnetic frame, to solution clarification, tear rubber cushion off, discard supernatant in deep-well plates, be careful not to touch
Encounter magnetic bead；
(4) the cleaning solution BW1 that 600ul has added ethanol is added, covers rubber cushion, washed once again according to the method described above；
(5) ethanol solutions of 500ul 80% are subsequently added, rubber cushion is covered, washed once according to the method described above；
(6) 96 deep-well plates are placed in block heater, 40 DEG C dry 5 minutes；
(7) 50ul eluent CE are added, 1600rpm on blending instrument is placed in, mixed 1 minute, 10 points are incubated in 56 DEG C of water-baths
Clock carries out DNA elutions, and is mixed 1 time at the 5th minute；
(8) it is incubated after terminating and mixes magnetic bead, is placed in magnetic frame up to solution and clarifies, draw supernatant；Surveyed using Nanodrop
Determine concentration, concentration is filled up to《Concentration of specimens table》In, it is stand-by in -20 DEG C of preservations.
Comparative example
The method that buccal swab DNA is extracted in the comparative example is same as Example 1, and it is differed only in, such as Fig. 3 institutes
Show, in the step (A), it is described cracking and enzymolysis DNA the step of in, handle buccal swab sample when, first by buccal swab
Head, which is cut, to be put into 1.5ml or so centrifuge tubes, adds identical digestive juice and Proteinase K, under the same conditions, carries out cell
Cracking and enzyme digestion reaction, obtain the DNA enzymatic solution liquid；After digestion is finished, then it is transferred in 96 deep-well plates, it is normal according to prior art
Rule method carries out follow-up DNA and extracted, the DNA extraction steps be the same as Example 1.
Experimental example
To in above-described embodiment 1 with 96 deep-well plates operated and comparative example in existing single tube extract buccal swab side
Obtained DNA sample concentration is extracted in method and purity is detected and contrasted, record data such as table 1 below.
The two methods DNA sample concentration of table 1 and comparison or purity
The concentration that sample obtains DNA sample is incubated using 96 deep-well plates and single tube as seen from the above table, its purity is approximate, does not have
There is significant difference, but the method for the invention carries out DNA using 96 deep-well plates and extracts operation compared to single tube extraction operation energy
The risk of sample mixing when greatly reducing the time of operation and effectively reducing transfer.
The embodiment of the present invention is described in detail above, specific case used herein to the principle of the present invention and
Embodiment is set forth, and the explanation of above example is only intended to the method and its core concept for helping to understand the present invention；
Simultaneously for those of ordinary skill in the art, according to the thought of the present invention, can in specific embodiments and applications
There is change part, in summary, this specification content should not be construed as limiting the invention.
Claims (10)
1. a kind of method for cracking and digesting buccal swab DNA, it is characterised in that comprise the following steps：
(1) Tissue Culture Plate is taken, room is corresponded in sequence, buccal swab head is placed in deep-well plates；
(2) digestive juice and Proteinase K are added into the Tissue Culture Plate；
(3) Tissue Culture Plate is sealed；
(4) Tissue Culture Plate is obtained into DNA enzymatic solution liquid in fully mixing simultaneously water bath processing on oscillator.
2. cracking according to claim 1 and enzymolysis buccal swab DNA method, it is characterised in that the cell culture
Plate is 96 orifice plates.
3. cracking according to claim 2 and enzymolysis buccal swab DNA method, it is characterised in that the cell culture
Plate is 96 deep-well plates.
4. the method for the cracking and enzymolysis buccal swab DNA according to claim any one of 1-3, it is characterised in that described
In step (3), sealed using the rubber pad special of the Tissue Culture Plate.
5. the method for the cracking and enzymolysis buccal swab DNA according to claim any one of 1-4, it is characterised in that described
The temperature of water bath processing step is 56 DEG C.
6. the method for the cracking and enzymolysis buccal swab DNA according to claim any one of 1-5, it is characterised in that described
Digestive juice is BL Buffer.
7. cracking according to claim 6 and enzymolysis buccal swab DNA method, it is characterised in that the digestive juice
Addition is 500ul.
8. the method for the cracking and enzymolysis buccal swab DNA according to claim any one of 1-7, it is characterised in that described
The addition of Proteinase K is 20ul.
9. a kind of method that 96 orifice plate magnetic bead extracts buccal swab DNA, it is characterised in that comprise the following steps：
(A) according to any one of claim 1-8 methods described, the DNA enzymatic solution liquid is obtained；
(B) the DNA enzymatic solution liquid is transferred on new Tissue Culture Plate, carries out follow-up according to prior art conventional method
DNA is extracted.
10. the method that 96 orifice plate magnetic bead according to claim 9 extracts buccal swab DNA, it is characterised in that the step
(B) in, in addition to the step of magnetic bead is extracted is added.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710353091.0A CN107058293A (en) | 2017-05-18 | 2017-05-18 | A kind of method that 96 orifice plate magnetic bead extracts buccal swab DNA |
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| CN201710353091.0A CN107058293A (en) | 2017-05-18 | 2017-05-18 | A kind of method that 96 orifice plate magnetic bead extracts buccal swab DNA |
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| CN201710353091.0A Pending CN107058293A (en) | 2017-05-18 | 2017-05-18 | A kind of method that 96 orifice plate magnetic bead extracts buccal swab DNA |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385682A (en) * | 2015-12-29 | 2016-03-09 | 杭州谷坤生物技术有限公司 | Simple method for fast extracting human fecal bacterium DNA |
-
2017
- 2017-05-18 CN CN201710353091.0A patent/CN107058293A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385682A (en) * | 2015-12-29 | 2016-03-09 | 杭州谷坤生物技术有限公司 | Simple method for fast extracting human fecal bacterium DNA |
Non-Patent Citations (3)
| Title |
|---|
| 刘超: "DNA据库建设中批量样本直接扩增检验法的应用", 《中国法医学杂志》 * |
| 杨建强等: "一种从口腔拭子中提取DNA的简易方法", 《第三军医大学学报》 * |
| 童大跃等: "《新编法医物证检验技术》", 30 November 2013, 中国医药科技出版社 * |
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Application publication date: 20170818 |
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