CN1634965A - Process for extracting DNA from biological samples - Google Patents
Process for extracting DNA from biological samples Download PDFInfo
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- CN1634965A CN1634965A CN 200410084445 CN200410084445A CN1634965A CN 1634965 A CN1634965 A CN 1634965A CN 200410084445 CN200410084445 CN 200410084445 CN 200410084445 A CN200410084445 A CN 200410084445A CN 1634965 A CN1634965 A CN 1634965A
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- dna
- magnetic bead
- polydimethylsiloxane
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Abstract
This invention relates to a biological sample DNA extraction method, which comprises the following steps: a, leading the micro magnetic leads with carboxyl mark into the micro channel of the chip and absorbing and fixing it inside the channel to form the micro flow chip of the sample; b, mixing the sample and DNA absorbing liquid and then leading into the chip and mixing it with the micro magnetic leads, wherein the DNA in sample is absorbed on the leads with buffer liquid flowing in for washing and then collecting the washing liquid to get the high purified DNA.
Description
Technical field
The present invention relates to the method for DNA extraction in a kind of biological sample, the method for using little magnetic bead that DNA in the biological sample is extracted in particularly a kind of micro-fluid chip.
Background technology
When the DNA in the biological sample is analyzed, often need carry out DNA extraction, DNA cloning (pcr amplification) and augmentation detection steps such as (electrophoresis detection or hybridization detect).The conventional DNA extraction method in laboratory is mainly still taked alkaline lysis or phenol chloroform method at present, be liquid phase extraction method (Liquid-liquid Methods), need equipment such as whizzer, and operation steps is more time-consuming, loaded down with trivial details, phenol, chloroform are also toxic to human body.
It is solid-phase extraction method that newer method is to use SPE (solid phase extraction) method.Compare advantage such as it has with low cost, easy to use, and contaminative is little with traditional liquid phase extraction method (Liquid-liquid Methods).In the DNA extraction experiment, the SPE method mainly is to utilize the surface of some solid matters, as silicon face, glass, ion exchange resin or the special adsorption of DNA such as magnetic bead through modifying.The benefit of this method is that the degraded of DNA is less, is used in combination protein denaturant and RNA degraded reagent, and the purity of DNA and output are improved, and SPE method operation steps is less, can adapt to different samples, as document [1] Marko, M.A., Chipperfield, R., and Birnboim, H.C.Anal.Biochem, 1982,121:382～387., [2] Matitashvili, E., and Zavizion, B.Anal.Biochem., 1997,246:260～262., [3] Walsh, P.S., Metzger, D.A., and Higuchi, R.BioTechniques, 1991, reported method such as 10:506～513..
The principle of using little magnetic bead to carry out DNA extraction in the biological sample is: little magnetic bead surfaces can be modified various chemical groups, utilizes DNA can carry out reversible bonded principle with the magnetic bead of carboxyl mark, and biomolecules in the sample is selected and separated.Because it has superparamagnetism, can handle simultaneously by externally-applied magnetic field.Therefore more convenient operation, this technology be also referred to as the reversible fixation method of solid phase (solid-phase reversible immobilization, SPRI).With method such as original phenol chloroform relatively, reagent is safer, and is easy and simple to handle, need not equipment such as whizzer.In some biology laboratories, being substituted original separating and purifying method by the test kit of some companies preparation becomes breadboard routine techniques, as: the content of http://www.bangslabs.com/ and [5] http://www.abgene.com/ report.
But at present report to magnetic bead method of operating still be unrealized miniaturization and portability, need bigger equipment, reagent consumption is also more.
As can be seen, traditional method reagent consumes more, and equipment is relied on height, and it is also higher to detect cost; Because the existence of some dangerous malignant bacterias and virus makes disposable detection necessitate, and conventional method cost is all higher, and equipment can't disposablely use, and makes the chance of polluting and infecting increase.
Development along with micro mechanical technology, make the microminiaturization and the automatization of Biomedical Instruments become possibility, the chip that making can be extracted DNA in the biological sample easily is automated and portability owing to can reduce reagent consumption, has therefore caused people's attention.As Christel et al. (Christel, L.A., Petersen, K., Mcwilliam, W., Northrup, M.A., Transact.ASME 1999,121:272～279.) utilize deep etching technology on silicon chip, to carve many pillars, utilize this structures capture DNA.But the chip of present report mainly is based on traditional bulk silicon process and is made, and the chip cost of silicon processing is still higher, and the complete processing complexity is unfavorable for detecting the reduction of cost.
The microchip made of plastic material is because its attention that causes people with low cost, easy to process in recent years, but plastic material can't be finished extraction to DNA as solid support because the restriction of itself material behavior.Therefore use plastic material manufactured samples DNA extraction chip not appear in the newspapers.
Summary of the invention
The technical issues that need to address of the present invention be to disclose a kind of novel in the micro-fluid chip that polydimethylsiloxane is made, use little magnetic bead to biological sample in the method extracted of DNA, to overcome the above-mentioned defective that prior art exists.
Method of the present invention comprises the steps:
(1) the little magnetic bead with the carboxyl mark imports in the fluid channel of chip, and the permanent magnet of placing by the chip below is absorbed and fixed in the fluid channel of chip, forms the micro-fluid chip that can be used for the biological sample DNA extraction;
Little magnetic bead of said carboxyl mark refers to little magnetic bead surfaces sign carboxyl, can adopt the commercially available prod; Product Dynabeads MyOneTM Carboxylic Acid as Dynabeads company, the Magnetic XM200 MicroSphere of ABgene company, or use (Biotechnol Bioeng 60:419-424,1998) open introduction method such as Hwee Peng Khng to make.
Said chip comprises chip body and is arranged on main intravital fluid channel with import and outlet, the material selection polydimethylsiloxane of said chip body;
(2) will import in the chip after biological sample and the mixing of DNA adsorption liquid, mix with the little magnetic bead that is marked with carboxyl that is fixed in the chip, because the existence of polyoxyethylene glycol, DNA in the sample is adsorbed on little magnetic bead, and adsorption time is 15～25 minutes, and Xi Fu impurity such as protein etc. then do not flow out chip, feed damping fluid again, DNA is eluted from magnetic bead, collect wash-out, obtain the DNA of separation and purification;
Said biological sample comprises intestinal bacteria nutrient solution, PCR product etc.;
Said DNA adsorption liquid is that the weight concentration that contains 0.5M NaCl is the aqueous solution of 20% polyoxyethylene glycol, and the molecular weight of polyoxyethylene glycol is 8000 polyoxyethylene glycol.
Said damping fluid is 10mM Tris-Acetate, pH7.8.
According to the present invention, said chip production method comprises the steps:
The precursor of polydimethylsiloxane and the mixture of solidifying agent are cast on the mould, hot setting, the demoulding, be formed with the polydimethylsiloxane thin slice of fluid channel, by carrying out bonding encapsulation with another sheet polydimethylsiloxane thin slice or sheet glass, forming at last and adopting polydimethylsiloxane is the chip of material preparation.
The precursor of polydimethylsiloxane and solidifying agent are available from dow corning company;
The precursor of polydimethylsiloxane and the ratio of solidifying agent are 10: 1;
Solidification value is 65 ℃, and be 1.5 hours set time;
Advantage of the present invention and effect are as follows: use the plastic material polydimethylsiloxane to make and be used for the chip that sample DNA extracts, adopt speed forming method on the technology, make the with low cost of chip manufacturing, technology is simple.The little processing chip cost of traditional silicon is about 50-100 yuan, and the chip cost that uses PDMS to make is lower than 1 yuan, the extraction of DNA need not to rely on the extraction that the chip manufacturing material carries out DNA during the little magnetic bead of use was finished biological sample as solid phase carrier in the PDMS chip; Little magnetic bead in the chip can be controlled by externally-applied magnetic field easily, and the handiness of chip design is improved and methods such as traditional phenol/chloroform, alkaline lysis are compared, and it is little to use the solid-phase extraction method of little magnetic bead to have a reagent toxicity, and security is than advantages such as height; The PDMS material also has good biocompatibility, therefore the security of experiment is strengthened, and little magnetic bead solid-phase extraction method of in chip, implementing, except characteristics such as reagent consumption are few, also because bigger specific surface area is arranged, promoted speed of response, the time of finishing DNA extraction in the biological sample is in 25 minutes.And methods such as traditional phenol/chloroform, alkaline lysis need 1.5 hours, and little magnetic bead solid phase extractions rule of not using chip was finished and once tested required cost reduction in 30-40 minute.The little magnetic bead solid-phase extraction method cost that does not use chip is about 10 yuan, and traditional phenol/chloroform, alkaline lysis cost are about 15 yuan, and method therefor cost of the present invention is about 3 yuan.
In a word, method of the present invention combines the advantage of plastic material chip and little magnetic bead solid-phase extraction method, chip manufacturing is simple, with low cost, experiment reagent consumption is few, need not complex instrument equipment, operational safety, help disposable use, can reduce the DNA extraction cost effectively, shorten the operating time, step simplifies the operation, be low cost, fast, the disease detection of safety lays the foundation, and helps micro-total analysis system (micro total analytical system, realization u-TAS) simultaneously, plastic material polydimethylsiloxane (Polydimethylsiloxane is used in the making of micro-fluid chip, PDMS), its advantage is low price, can be mass-produced; Preparation is easy, weak point consuming time; Encapsulation easily, wearing quality is good, and is reusable; Have good biological fitness and gas permeability; Good insulation performance, calorifics stability and optical characteristics.Use rapid shaping complete processing (Rapid Prototyping) on technology is made, so chip manufacturing is with low cost, technology is simple; Little magnetic bead of chip internal fixing carboxyl mark, use the reversible fixation method of solid phase that the DNA in the biological sample is extracted, utilize the paramagnetism of magnetic bead to control magnetic bead in the chip easily by externally-applied magnetic field, use reagent safety, easy and simple to handle, reagent consumption is few, easily is automated and portability.
Description of drawings
Fig. 1 is the technical process of the polydimethylsiloxanechip chip of speed forming method making.
Fig. 2 is the PDMS chip structure synoptic diagram of producing.
Fig. 3 is that magnetic bead imports CCD photo in the chip runner.
Fig. 4 is for using the sample of collecting behind 70% alcohol flushing.
Fig. 5 carries out the sample collected behind the wash-out for the TE damping fluid that uses pH8.0.
Embodiment
Referring to Fig. 1, the preparation method of polydimethylsiloxanechip chip comprises the steps:
(1) preparation of mould: the chip structure that designs transferred to adopt silicon chip or sheet glass be base material and be coated with the covering on the plate 1 of photoresist material, photoetching 4 then, adopt conventional method to develop 5, obtain to be provided with the mould of projection runner 2;
Said photoresist material is a kind of material that is used for photoetching of routine, as the SU8 negative photoresist; (SU-8,2025, Microchem).
(2) preparation of chip: the top that little glass column 3 is placed on the projection runner 2 of mould, then the mixture of polydimethylsiloxane and solidifying agent is cast on the mould 6, solidify, the demoulding 7, promptly obtain to have the polydimethylsiloxane thin slice of fluid channel, by carrying out bonding encapsulation with another sheet polydimethylsiloxane thin slice or sheet glass, forming at last and adopting polydimethylsiloxane is the chip of material preparation, as Fig. 2, the duct that little glass column 3 forms is import and outlet.
Embodiment 1
DNA in the intestinal bacteria nutrient solution is extracted, and step is as follows:
1) the little magnetic bead diluent of 5ul imports in the chip, and magnet absorption magnetic bead is placed in the raceway groove below, adsorb that pressurization makes liquid discharge chip after three minutes, and the absorption by magnet is fixed in the chip magnetic bead, as Fig. 3;
2) intestinal bacteria Ecoli DH5 α inoculation, the shaking table overnight incubation.
3) take magnet away, bacterium liquid is got 20 μ L and is joined (4%Triton X-100 in the 50 μ L adsorption-buffering liquid, 0.5M NaCl, 20%PEG 8000), slowly import in the chip, mixing the back imports in the chip, placed 15 minutes for 37 ℃, place magnet absorption magnetic bead then below raceway groove, pressurization simultaneously makes the liquid discharge make flow velocity at 2 μ L/min, collect and reclaim liquid, be sample 1
4) still below chip, place magnet, import the mobile cleaning of 20ul70% ethanol in the raceway groove.
5) chip was placed on 37 ℃ of thermostat containers interior 2 minutes, made the magnetic bead drying.
6) take magnet away, 5 μ l DNA lysates import in the chip, mix 10 minutes, are placing magnet absorption magnetic bead below the raceway groove after three minutes then, and pressurization is flowed out liquid, collects the DNA that this liquid is purifying.
Experimental result is measured:
1. purity testing: utilize spectrophotometer to carry out A260/280 to the purify DNA of collecting and measure, value is 1.878, illustrate that the purity of purification is higher;
2. adopting fluorescence dye when control experiment detects is SYBR-green-I, it be in dna double chain dyestuff and the sample double-stranded DNA in conjunction with after, its fluorescence intensity is directly proportional with the quantity of DNA.Specimen inspection system is: the reflective fluorescent microscope of OLYMPUS BX-51; Fluorescent signal is gathered by cooled CCD (Cooled Charge-coupled Devices, Cooled CCD) OLYMPUSDP50.The results are shown in Figure 4 and Fig. 5.Fig. 4 is for using the sample of collecting behind 70% alcohol flushing, and Fig. 5 carries out the sample collected behind the wash-out for the TE damping fluid that uses pH8.0, and as can be seen, sample 1 does not have fluorescent signal, and sample 2 then has very strong fluorescence; Because the content of fluorescent signal and DNA is relevant, DNA under the wash-out then is described when 70% alcohol flushing not, use 10mMTris-Acetate, pH7.8 then is adsorbed on the DNA on the magnetic bead smoothly under the wash-out.Contain DNA in the interpret sample 2; This explanation can effectively be carried out DNA extraction to magnetic bead employing this method of carboxyl mark.
Embodiment 2
DNA in the PCR product is extracted, and step is as follows:
1) the little magnetic bead diluent of 5ul imports in the chip, and magnet absorption magnetic bead is placed in the raceway groove below, adsorb that pressurization makes liquid discharge chip after three minutes, and the absorption by magnet is fixed in the chip magnetic bead, as Fig. 3;
2) take magnet away, the PCR product is got 10 μ L and is joined in the 20 μ L adsorption-buffering liquid and slowly import in the fluid channel of chip, mixes, and places 10 minutes for 37 ℃, place magnet absorption magnetic bead then below fluid channel, pressurization simultaneously makes the liquid discharge make flow velocity at 2 μ L/min;
3) still below chip, place magnet, import the mobile cleaning of 20ul70% ethanol in the fluid channel;
4) chip was placed on 37 ℃ of thermostat containers interior 2 minutes, made the magnetic bead drying;
5) take magnet away, 5 μ l DNA lysates import in the chip, mix 5 minutes, are placing magnet absorption magnetic bead below the raceway groove after three minutes then, and pressurization is flowed out liquid, collects the DNA that this liquid is purifying.
Purity testing: utilize spectrophotometer to carry out A260/280 to the purify DNA of collecting and measure, value is 1.924, illustrates that the purity of purifying is higher.
Claims (9)
1. the method for DNA extraction in the biological sample is characterized in that, comprises the steps:
(1) the little magnetic bead with the carboxyl mark imports in the fluid channel of chip, and is absorbed and fixed in the fluid channel of chip, forms the micro-fluid chip that can be used for the biological sample DNA extraction;
Said chip comprises chip body and is arranged on main intravital fluid channel with import and outlet, the material selection polydimethylsiloxane of said chip body;
(2) will import in the chip after biological sample and the mixing of DNA adsorption liquid, mix with the little magnetic bead that is marked with carboxyl that is fixed in the chip, DNA in the sample is adsorbed on little magnetic bead, Xi Fu impurity such as protein etc. then do not flow out chip, feed damping fluid again, DNA is eluted from magnetic bead, collect wash-out, obtain the DNA of separation and purification.
2. method according to claim 1 is characterized in that, in the fluid channel of permanent magnet with little magnetic bead importing chip of carboxyl mark by the placement of chip below.
3. method according to claim 1 is characterized in that, said biological sample comprises inoculum or PCR product etc.
4. method according to claim 1 is characterized in that, said DNA adsorption liquid is the aqueous solution that contains the polyoxyethylene glycol of 0.5M NaCl, and molecular weight polyethylene glycol is 8000.
5. method according to claim 4 is characterized in that, the weight concentration of the polyoxyethylene glycol aqueous solution is 20%.
6. method according to claim 1 is characterized in that, said damping fluid is 10mMTris-Acetate, pH7.8.
7. method according to claim 1 is characterized in that, adsorption time is 15～25 minutes.
8. method according to claim 1, it is characterized in that, said chip is preparation like this: the mixture of polydimethylsiloxane and solidifying agent is cast on the mould, hot setting, the demoulding, be formed with the polydimethylsiloxane thin slice of fluid channel, by carrying out bonding encapsulation with another sheet polydimethylsiloxane thin slice or sheet glass, forming at last and adopting polydimethylsiloxane is the chip of material preparation.
9. a micro-fluid chip that is used for the biological sample DNA extraction comprises chip body and is arranged on main intravital fluid channel with import and outlet, it is characterized in that the material selection polydimethylsiloxane of said chip body.
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| CNB2004100844459A CN1314700C (en) | 2004-11-22 | 2004-11-22 | Process for extracting DNA from biological samples |
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| CNB2004100844459A CN1314700C (en) | 2004-11-22 | 2004-11-22 | Process for extracting DNA from biological samples |
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| CN1314700C CN1314700C (en) | 2007-05-09 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153281B (en) * | 2006-09-29 | 2011-01-19 | 中国科学院大连化学物理研究所 | DNA on-line separating microcurrent control chip and analytical method thereof |
| CN102174383A (en) * | 2011-02-15 | 2011-09-07 | 福州大学 | DNA (deoxyribonucleic acid) biosensor chip based on nanometer magnetic bead technique and experimental method thereof |
| CN102250878A (en) * | 2011-06-03 | 2011-11-23 | 吉林大学 | Reverse DNA extraction method based on solid phase medium surface charges |
| CN105372415A (en) * | 2015-11-17 | 2016-03-02 | 武汉金开瑞生物工程有限公司 | Method for labeling antibody with DNA |
| CN106805954A (en) * | 2017-02-28 | 2017-06-09 | 华中科技大学 | A kind of Wearable pliable pressure sensor and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5523231A (en) * | 1990-02-13 | 1996-06-04 | Amersham International Plc | Method to isolate macromolecules using magnetically attractable beads which do not specifically bind the macromolecules |
| JPH08154678A (en) * | 1994-12-08 | 1996-06-18 | Hitachi Electron Eng Co Ltd | Purification of pcr amplified product |
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2004
- 2004-11-22 CN CNB2004100844459A patent/CN1314700C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153281B (en) * | 2006-09-29 | 2011-01-19 | 中国科学院大连化学物理研究所 | DNA on-line separating microcurrent control chip and analytical method thereof |
| CN102174383A (en) * | 2011-02-15 | 2011-09-07 | 福州大学 | DNA (deoxyribonucleic acid) biosensor chip based on nanometer magnetic bead technique and experimental method thereof |
| CN102250878A (en) * | 2011-06-03 | 2011-11-23 | 吉林大学 | Reverse DNA extraction method based on solid phase medium surface charges |
| CN105372415A (en) * | 2015-11-17 | 2016-03-02 | 武汉金开瑞生物工程有限公司 | Method for labeling antibody with DNA |
| CN105372415B (en) * | 2015-11-17 | 2017-05-10 | 武汉金开瑞生物工程有限公司 | Method for labeling antibody with DNA |
| CN106805954A (en) * | 2017-02-28 | 2017-06-09 | 华中科技大学 | A kind of Wearable pliable pressure sensor and preparation method thereof |
| CN106805954B (en) * | 2017-02-28 | 2020-02-14 | 华中科技大学 | Wearable flexible pressure sensor and preparation method thereof |
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| CN1314700C (en) | 2007-05-09 |
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