CN101705225A - High-efficiency separation method for lower molecular multiplex PCR-amplified fragments - Google Patents
High-efficiency separation method for lower molecular multiplex PCR-amplified fragments Download PDFInfo
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- CN101705225A CN101705225A CN200910236730A CN200910236730A CN101705225A CN 101705225 A CN101705225 A CN 101705225A CN 200910236730 A CN200910236730 A CN 200910236730A CN 200910236730 A CN200910236730 A CN 200910236730A CN 101705225 A CN101705225 A CN 101705225A
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Abstract
The invention provides a high-efficiency separation method for lower molecular multiplex PCR-amplified fragments, which comprises: firstly, purifying lower molecular multiplex PCR-amplified fragments by using a magnetic bead purification method; and secondly, separating the lower molecular multiplex PCR-amplified fragments by using a capillary electrophoresis method. The method combining a multiplex PCR method and capillary electrophoresis can quickly screen transgenic plants. The multiplex PCR has high template-saving, reagent-saving and time-saving performance; when used to process the products of the multiplex PCR, the magnetic bead method can effectively remove the impurities from a PCR reaction system to make the PCR reaction system more suitable for performing capillary electrophoresis; and the capillary electrophoresis has the characteristics of low detection limit, high sensitivity and short detection time. The method can be safely used, successfully separate and identify the lower molecular multiplex PCR-amplified fragments, keep the detection time within 10.6 minutes and be widely used in fields such as food sanitation quarantine and clinical examination.
Description
Technical field
The present invention relates to a kind of method, specifically, relate to a kind of method.The present invention can success lower molecular multiplex PCR-amplified fragments is carried out high efficiency separation, multiple PCR products can better separate it with capillary electrophoresis through behind the magnetic beads for purifying.
Background technology
The security of genetically modified food has become one of hot issue of paying close attention in the whole world, in order to identify genetically modified crops, usually not only to screen and detect CaMV35S promotor and the NOS terminator of genetically modified crops, also will identify the strain and the quantitative transgene component of transgenic crop.
The research of deep processed product PCR molecular detection technology is the difficult point and the focus of detection technique research always; because its genomic DNA fragment is because the destruction of course of processing mesohigh high temperature and mechanical force; be degraded into very small segment (＜100bp); and content is very small; therefore traditional PCR-agarose gel electrophoresis can't be realized detecting effectively to these little purpose fragments, false-negative result occurs through regular meeting.
At present, the existing multi-PRC reaction technology that adopts increases to these small segment genes, to improve detection specificity.But because the multi-PRC reaction system is comparatively complicated, follow-up separation method is required strictness more, traditional agarose gel electrophoresis carries out compartment analysis, and its resolving power is limited, can't reach the requirement that lower molecular multiplex PCR-amplified fragments is detected.
Therefore, need a kind of energy of research quickness and high efficiency ground carry out isolating method to lower molecular multiplex PCR-amplified fragments.
Summary of the invention
The objective of the invention is to overcome the defective of the method for existing detection PCR small molecules amplified fragments, a kind of method of easy and simple to handle, separation lower molecular multiplex PCR-amplified fragments rapidly and efficiently is provided.
The efficient separation method that is used for lower molecular multiplex PCR-amplified fragments provided by the invention, it carries out purifying with the magnetic beads for purifying method to lower molecular multiplex PCR-amplified fragments earlier, and then adopts capillary electrophoresis to separate.
Sample of the present invention is any raw produce and deep processed product that contains the corn composition in the field of food.
Because the multi-PRC reaction system is comparatively complicated, has the impurity that some may interfere with follow-up capillary electrophoresis, therefore need carry out purification process to it.The present invention adopts the magnetic beads for purifying method.Magnetic bead is a kind of functional supports that is coated with bio-active group, can be scattered in to form the magnetic liquid material in the base fluid, and it has the flowability of liquid and the dual characteristics of solid magnetic particulate material concurrently, thereby makes the separation of solid-liquid phase become very convenient quick.The magnetic beads for purifying method is organically integrated DNA isolation technique and beneficiation technologies specifically, promptly can obtain the very high target matter DNA of purity by simple wash-out.
Capillary electrophoresis (Capillary Electrophoresis, CE) also be HPCE (HPCE), capillary electrophoresis is development in recent years one of separate analytical technique rapidly, it is lower that capillary electrophoresis has overcome traditional agarose gel electrophoresis resolving power, the process complexity, the sample requirement is big, poor reproducibility, its topmost characteristics are efficient, quick and micro-. that the present invention adopts is non gel sieving capillary gel electrophoresis (NGE), its adopts is can form gel-free but non gel sieving medium that sieving action is arranged, it can avoid the formation of cavity, preparation than gel column is simple, convenient. as far back as nineteen ninety, existing polyacrylamide (LPA) but as the report of DNA separating medium. its viscosity is big, operate more loaded down with trivial details again, and not very stable. so at several years thereafter, PEO, PDMA, PVP is in the news successively at the linear polymeric material that the fused quartz capillary wall has dynamic coating effect separately. and through experimental study, it is Natvosol (HEC) that the present invention selects separating medium.
The magnetic bead that described magnetic beads for purifying method adopts BIO-V company to buy carries out purifying.Adopt magnetic bead absorption, washing and elution step successively.
The step of described magnetic beads for purifying method is:
1) get multiple PCR products add magnetic bead in conjunction with liquid (to 100mmol/L Tris-Cl, add 2.0mol/L NaCl, 1.0mol/L MgCl2,15% PEG 6000 and 15% PEG 8000 in the 10mmol/L EDTA solution, the pH value is 6.5) and the DTT solution (dithiothreitol (DTT) solution) of 30uL 1mol/L;
2) to wherein adding the magnetic bead that keeps even suspended state, make magnetic bead keep suspended state 5min;
3) centrifuge tube is placed carry out magnetic resolution on the magnet stand, inhale and remove liquid;
4) add 70% ethanol then and wash mixing; Carry out magnetic resolution, inhale and remove liquid;
5) repeating step 4) once;
6) under the magnetic condition, room temperature is dried, and inhales and remove liquid;
7) add TE (pH 8.0 for 100mmol/L Tris-Cl, 10mmol/L EDTA) solution, mixing;
8) magnetic resolution, imbitition, gained liquid are the PCR product of purifying.
The concrete operations step is as follows:
1) gets multiple PCR products in 1.5ml Eppendorf centrifuge tube.Add isopyknic magnetic bead and (, add 2.0mol/L NaCl, 1.0mol/L MgCl in the 10mmol/L EDTA solution to 100mmol/L Tris-Cl in conjunction with liquid
2, 15% PEG 6000 and 15% PEG 8000, the pH value is 6.5) and the DTT solution of 30uL 1mol/L.
2) add the 200uL magnetic bead.Before drawing magnetic bead, the reagent bottle 30s of necessary first vortex vibration dress magnetic bead, even to guarantee the magnetic bead suspended state.Blow and beat repeatedly or, make magnetic bead remain suspended state 5min with liquid getting device with the abundant mixing of whirlpool mixed instrument.
3) centrifuge tube is positioned over leaves standstill 30s on the magnet stand, treat can inhale liquid when magnetic bead is adsorbed in centrifuge tube fully by magnet stand one side, liquid feed is drawn clean.
4) take off centrifuge tube from magnet stand, the ethanol that adds 1mL 70% in the centrifuge tube washs, concussion mixing 1min.Centrifuge tube is positioned on the magnet stand, and magnetic resolution 30s inhales as far as possible and removes liquid.
5) repeating step 4) once.
6) centrifuge tube is placed on the magnet stand all the time, and room temperature was dried 10～15 minutes, every 5min, may be from magnetic bead liquid body exudate, accumulate in the centrifuge tube bottom, with liquid getting device these liquid are in time taken out.
7) (pH8.0) solution shakes mixing 5～10min, makes magnetic bead remain suspended state for 100mmol/L Tris-Cl, 10mmol/L EDTA to add TE to centrifuge tube.
8) magnetic separates, and centrifuge tube is positioned over leaves standstill about 30s on the magnet stand, and careful imbitition is put into new centrifuge tube when making magnetic bead be adsorbed in centrifuge tube by magnet stand one side fully, and gained liquid is the PCR product of purifying.
Natvosol (HEC) is a main research object of the present invention.The present invention uses the linear polymeric solution of HEC as the filled media in the kapillary.Utilization kapillary non gel sieving and capillary dynamic are coated with stain zone electrophoresis pattern and set up the scheme that multiple PCR products is sieved.
The used screening solution system of capillary electrophoresis separation method of the present invention is made up of sieving medium and matrix damping fluid.Sieving medium is Natvosol (HEC), and the matrix damping fluid is made up of 89mol/L Tris base (Tutofusin tris), 332mol/L boric acid and 2mol/L EDTA (ethylenediamine tetraacetic acid (EDTA)), pH between 7.2～7.5, more preferably 7.4.Wherein, Tris, boric acid and EDTA are the analytical pure rank at least.
Described sieving medium HEC is a water-soluble high-molecular substance, and preferred mass concentration scope is 1.0～1.5%, more preferably 1.2%.Capillary inner diameter is 75 μ m.Determine that through optimizing the back use length to be 48.5cm, detection window to the distance of electrode end is 8.5cm, so the kapillary useful length is 40cm at every turn.Running voltage is a negative pressure, and size further is optimized for-16.5kv between 15kv～20kv.
Specifically, Capillary Electrophoresis Separation Process of the present invention is:
1) preparation electrophoretic buffer: with 30mL solution is example, takes by weighing the Tris of 323.4mg, the boric acid of 615.9mg, and the EDTA of 22.5mg is settled to 30mL with deionized water, regulates pH value to 7.2～7.5 with the boric acid of 1.0mol/L.
2) with 1) in the electrophoretic buffer branch take on 15mL and prepare dissociating buffer.To wherein adding mass concentration is 1.0～1.5% HEC because the viscosity of HEC is bigger, so be difficult for dissolving, can adopt the concussion of whirlpool oscillator after, put into water-bath temperature again and bathe and make it to be dissolved in fully electrophoretic buffer, be cooled to 4 ℃ and seal preservation.
3) with 1) and 2) solution cross 45 μ m filter membranes, 4 ℃ of preservations.
4) internal diameter capillaceous is 75 μ m, and at 8.2～8.5cm place calcination kapillary, as detection window, capillary pipe length is 48.5cm, and useful length is 40cm.
5) calcination head and tail capillaceous place makes it pass electrode and immerses in the electrophoretic buffer.
6) condition of new pre-treatment capillaceous is as follows: the sodium hydroxide operation 5min of 0.10mol/L; Deionized water operation 10min; Dissociating buffer operation 10min.
7) parameter of capillary electrophoresis apparatus operation is as follows: capillary temperature is 20 ℃, sample introduction 20s under the-5kv, and the operation down of the voltage of-16.5kv detects under Sig.260/8nm and Ref.350/80nm condition.
The efficient separation method that is used for lower molecular multiplex PCR-amplified fragments of the present invention can extract Plant Genome earlier from sample; Carry out the multiplex PCR amplification then.
The present invention extracts plant genome DNA and adopts CTAB (bromohexadecane base Trimethylamine 99) method from sample.Concrete steps are as follows:
1) the crispy rice sample that will contain the corn composition is ground into powder in liquid nitrogen.
2) taking by weighing 200mg joins in the 1.5mL Eppendof centrifuge tube (safe Eppendorf tube).
3) add 1000 μ L CTAB solution, vibration evenly, 65 ℃ of incubation 40min, mixing 5 times at least therebetween was centrifugal 10 minutes of 4 ℃ of following 12000rpm.
4) carefully draw supernatant in another new pipe, add and the saturated phenol of the isopyknic Tris of supernatant: trichloromethane: primary isoamyl alcohol (25: 24: 1), fully vibration is even, 4 ℃ of centrifugal 15min of following 12000rpm.
5) repeating step 4) once.
6) carefully draw supernatant in another new pipe, add and the isopyknic trichloromethane of supernatant: primary isoamyl alcohol (24: 1), vibration is even, at 4 ℃ of centrifugal 15min of following 12000rpm.
7) carefully draw supernatant in another new pipe, add the Virahol of 2/3 volume, the sodium acetate of 1/10 volume.Leave standstill 2h under-20 ℃ of conditions.At 4 ℃ of centrifugal 15min of following 12000rpm.
8) abandoning supernatant adds 70% an amount of ethanolic soln washing precipitation.
9) abandoning supernatant dries up precipitation.With the aseptic ultrapure water dissolution precipitation of 100uL.
10) add 3 μ l RNA enzyme solution, digest 30min in 37 ℃ of water-bath environment.
Sample of the present invention can be crispy rice, also can be any deep processed product and deep processing transgenic product that contains the corn composition in the field of food.
The segmental amplification procedure of lower molecular multiplex PCR of the present invention is:
PCR reaction system following (30 μ L): 10 * PCR damping fluid: 3 μ L (10 * PCR damping fluid: 100mmol/L Tris-HCl, 500mmol/L KCl, 15mmol/L MgCl
2); 2.5mmol/L dNTP:2.4 μ L; Primer concentration is 10 μ M, totally three pairs of primers, and the upstream and downstream primer of every pair of primer adds 1 μ L respectively; The Taq archaeal dna polymerase: 0.4 μ L, the DNA of extraction add 1 μ l as template, use ddH
2O water complements to 30 μ l.
The amplification condition of PCR is as follows: pre-sex change: 94 ℃ of 5min; Sex change: 94 ℃ of 30s; Annealing: 60 ℃ of 30s; Extend: 72 ℃ of 30s; Extend at last: 72 ℃ of 7min; Establish 35 circulations altogether.
The present invention adopts the paramagnetic particle method purified pcr product, efficiently removed the impurity in the reaction system fast, make it be suitable for carrying out capillary electrophoresis, to go up sample through the dilution of the PCR product behind purifying back, sharp peak has appearred, as seen magnetic beads for purifying helps the operation of capillary electrophoresis, has improved the purity of sample greatly.
The magnetic beads for purifying method that adopts among the present invention is different from common DNA test kit method of purification, the principle that common method of purification utilizes the silicon class matrix under the high salt concentration that the DNA uniqueness is adsorbed, remove gel and other impurity, lower to the rate of recovery less than the dna fragmentation of 100bp.And the magnetic beads for purifying method of using among the present invention is foundation with the centrifugation of magnetic bead uniqueness, provides an extremely easy schedule of operation: magnetic bead absorption, washing and wash-out.Under the suitableeest reagent and operational condition, can obtain high-quality DNA.The present invention adopts the magnetic beads for purifying method to have easy, quick and characteristics of high efficiency, need not use organic solvents such as phenol, chloroform in the whole extraction purge process.
The invention provides the capillary electrophoresis that is used to separate small segment PCR product, this method can be in 10.6min under the ultraviolet detection condition success isolate the lower molecular multiplex PCR-amplified fragments that size is respectively 51bp, 74bp and 88bp.
The implementation result of table 1 the present invention and traditional agarose gel electrophoresis relatively
The present invention has remedied the deficiency that prior art detects lower molecular multiplex PCR-amplified fragments, successful foundation a kind of quick screening method that is used for deep processed product PCR Molecular Detection.Multiple PCR technique has the template of saving, saves reagent and the high efficiency of time; Paramagnetic particle method is handled the impurity that can effectively remove in the PCR reaction system to multiple PCR products, and it is more suitable in carrying out capillary electrophoresis; Capillary electrophoresis has the advantages that detectability is low, highly sensitive and detection time is short.Application safety of the present invention, can success lower molecular multiplex PCR-amplified fragments is carried out isolation identification, be controlled at 10.6min detection time, can be widely used in the fields such as food sanitation quarantine and Clinical Laboratory.
Description of drawings
Fig. 1 is agarose gel electrophoresis checking extracting genome DNA figure of the present invention; M: λ DNA/EcoR I+Hind III Marker, 1,2 are the crispy rice food DNA electrophoretogram that the CTAB method is extracted
Fig. 2 detects multiple PCR products for agarose gel electrophoresis of the present invention; M:DL 2000DNA Marker, 1,2 are multiple PCR products
Fig. 3 is a capillary electrophoresis separation multiple PCR products of the present invention;
Fig. 4 goes out the enlarged view of peak part for Fig. 3.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
1) the crispy rice sample that will contain the corn composition is ground into powder in liquid nitrogen.
2) take by weighing 200mg and join in the 1.5mL Eppendof centrifuge tube, need set up negative control simultaneously.
3) add 1000 μ L CTAB solution, vibration evenly, 65 ℃ of incubation 40min, mixing 5 times at least therebetween was centrifugal 10 minutes of 4 ℃ of following 12000rpm.
4) carefully draw supernatant in another new pipe, add and the saturated phenol of the isopyknic Tris of supernatant: trichloromethane: primary isoamyl alcohol (25: 24: 1), fully vibration is even, 4 ℃ of centrifugal 15min of following 12000rpm.
5) repeating step (4) once.
6) carefully draw supernatant in another new pipe, add and the isopyknic trichloromethane of supernatant: primary isoamyl alcohol (24: 1), vibration is even, at 4 ℃ of centrifugal 15min of following 12000rpm.
7) carefully draw supernatant in another new pipe, add the Virahol of 2/3 volume, the sodium acetate of 1/10 volume.Leave standstill 2h under-20 ℃ of conditions.At 4 ℃ of centrifugal 15min of following 12000rpm.
8) abandoning supernatant adds 70% an amount of ethanolic soln washing precipitation.
9) abandoning supernatant dries up precipitation.With the aseptic ultrapure water dissolution precipitation of 100uL.
10) add 3 μ l RNA enzyme solution, digest 30min in 37 ℃ of water-bath environment.
11) after the digestion, run the agarose gel electrophoresis of 1% concentration the genome that is extracted is verified.
As shown in Figure 1, the result shows, finds after during through agarose gel electrophoresis, adopts the CTAB method can extract genomic dna in the crispy rice, and applied sample amount is 5 μ L.The position of wherein genomic size about the 21kb of λ DNA/EcoR I+Hind III Marker.Genomic band disperse, this is the result who adopts after deep process technology is handled sample, does not have albumen residual substantially in the sample point sample hole, extracts the genome that obtains with method of the present invention and all can be used for pcr amplification.
The DNA that 2 couples of embodiment of embodiment 1 extract is PCR and detects
The DNA that extracts with embodiment 1 is a template, with the genome of primer (as table 2) the amplification crispy rice of design.PCR reaction system following (30 μ L): 10 * PCR damping fluid: 3 μ L (10 * PCR damping fluid: 100mmol/L Tris-HCl, 500mmol/L KCl, 15mmol/L MgCl
2); 2.5mmol/L dNTP:2.4 μ L; Primer concentration is 10 μ M, totally three pairs of primers, and the upstream and downstream primer of every pair of primer adds 1 μ L respectively; The Taq archaeal dna polymerase: 0.4 μ L, the DNA of extraction add 1 μ l as template, use ddH
2O water complements to 30 μ l.The amplification condition of PCR is as follows: pre-sex change: 94 ℃ of 5min; Sex change: 94 ℃ of 30s; Annealing: 60 ℃ of 30s; Extend: 72 ℃ of 30s; Extend at last: 72 ℃ of 7min; Establish 35 circulations altogether.
Table 2 design of primers
As shown in Figure 2, agarose gel electrophoresis detects multiple PCR products, and applied sample amount is 8 μ L.The result shows, detect multiple PCR products with agarose gel electrophoresis, the target DNA fragment size of multiplex PCR amplification is respectively 51bp, 74bp and 88bp, product accumulates in the part below the 100bp of DL 2000DNAMarker with the bulk form, visible traditional agarose gel electrophoresis can't effectively separate it.Wherein DL 2000DNA Marker is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.
Embodiment 3 adopts the magnetic beads for purifying method that lower molecular multiplex PCR-amplified fragments is carried out purifying
1) gets multiple PCR products in 1.5ml Eppendorf centrifuge tube.Add isopyknic magnetic bead in conjunction with liquid (to 100mmol/L Tris-Cl, add 2.0mol/L NaCl, 1.0mol/L MgCl2,15% PEG 6000 and 15% PEG 8000 in the 10mmol/L EDTA solution, the pH value is 6.5) and the DTT solution of 30uL 1mol/L;
2) add the 200uL magnetic bead.Before drawing magnetic bead, the reagent bottle 30s of necessary first vortex vibration dress magnetic bead, even to guarantee the magnetic bead suspended state.Blow and beat repeatedly or, make magnetic bead remain suspended state 5min with liquid getting device with the abundant mixing of whirlpool mixed instrument.
3) centrifuge tube is positioned over leaves standstill 30s on the magnet stand, treat can inhale liquid when magnetic bead is adsorbed in centrifuge tube fully by magnet stand one side, liquid feed is drawn clean.
4) take off centrifuge tube from magnet stand, the ethanol that adds 1mL 70% in the centrifuge tube washs, concussion mixing 1min.Centrifuge tube is positioned on the magnet stand, and magnetic resolution 30s inhales as far as possible and removes liquid.
5) repeating step 4) once.
6) centrifuge tube is placed on the magnet stand all the time, and room temperature was dried 10～15 minutes, every 5min, may be from magnetic bead liquid body exudate, accumulate in the centrifuge tube bottom, with liquid getting device these liquid are in time taken out.
7) (pH8.0) solution shakes mixing 5～10min, makes magnetic bead remain suspended state for 100mmol/L Tris-Cl, 10mmol/L EDTA to add TE to centrifuge tube.
8) magnetic separates, and centrifuge tube is positioned over leaves standstill about 30s on the magnet stand, and careful imbitition is put into new centrifuge tube when making magnetic bead be adsorbed in centrifuge tube by magnet stand one side fully, and gained liquid is the PCR product of purifying.
1) preparation electrophoretic buffer: with 30mL solution is example, takes by weighing the Tris of 323.4mg, the boric acid of 615.9mg, and the EDTA of 22.5mg is settled to 30mL with deionized water, regulates pH value to 7.4 with the boric acid of 1.0mol/L.
2) with 1) in the electrophoretic buffer branch take on 15mL and prepare dissociating buffer.To the HEC that wherein adds 180.0mg.Because the viscosity of HEC is bigger, thus be difficult for dissolving, can adopt the concussion of whirlpool oscillator after, put into water-bath temperature again and bathe and make it to be dissolved in fully electrophoretic buffer, be cooled to 4 ℃ and seal preservation.
3) with 1) and 2) solution cross 45 μ m filter membranes, 4 ℃ of preservations.
4) internal diameter capillaceous is 75 μ m, and inside pipe wall does not have coating, and at 8.2～8.5cm place calcination kapillary, as detection window, capillary pipe length is 48.5cm, and useful length is 40cm.
5) calcination head and tail capillaceous place makes it pass electrode and immerses in the electrophoretic buffer.
6) condition of new pre-treatment capillaceous is as follows: the sodium hydroxide operation 5min of 0.10mol/L; Deionized water operation 10min; Dissociating buffer operation 10min.
7) parameter of capillary electrophoresis apparatus operation is as follows: capillary temperature is 20 ℃, sample introduction 20s under the-5kv, and the operation down of the voltage of-16.5kv detects under Sig.260/8nm and Ref.350/80nm condition.
As shown in Figure 3, the result shows, after the dilution of the process of the PCR product behind the magnetic beads for purifying, sample introduction 20s under the condition of-5kv, capillary electrophoresis can successfully be isolated the PCR product of 3 following different sizes of 100bp, the target DNA fragment size is respectively 51bp, 74bp and 88bp, capillary electrophoresis is good to its isolating resolution, the purified back of sample sample introduction, peak shape is sharp peak, in 10.6min, reach separation, the purity height very of visible sample, the magnetic beads for purifying method helps to carry out separation capillaceous.
The present invention is used for the multi-PRC reaction system of transgenic plant examination, with transgenic corns Bt176 is research object, adopt multiple PCR technique, a PCR reacts CaMV 35S promoter, NOS terminator and the ZSSIIb gene of the corn that increases simultaneously, to improve detection efficiency and detection specificity.And having adopted paramagnetic particle method that multiple PCR products is carried out purifying, this method is quick, effective, easy and simple to handle.A kind of kapillary glue-free sieving system that is used for the above-mentioned multiple PCR products of capillary electrophoresis separation is provided simultaneously, and the electrophoretic buffer of this sieving system is 89mol/L Tris, 332mol/L boric acid, and 2mol/L EDTA, PH are 7.4.Sieving medium is Natvosol (HEC).
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
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Claims (10)
1. an efficient separation method that is used for lower molecular multiplex PCR-amplified fragments is characterized in that, it carries out purifying with the magnetic beads for purifying method to lower molecular multiplex PCR-amplified fragments earlier, and then adopts capillary electrophoresis to separate.
2. separation method according to claim 1 is characterized in that, described magnetic beads for purifying method adopts magnetic bead absorption, washing and elution step successively.
3. separation method according to claim 1 and 2 is characterized in that, the purification step of described magnetic beads for purifying method is:
1) gets multiple PCR products and add the DTT solution of magnetic bead in conjunction with liquid and 30uL 1mol/L;
2) to wherein adding the magnetic bead that keeps even suspended state, make magnetic bead keep suspended state 5min;
3) centrifuge tube is placed carry out magnetic resolution on the magnet stand, inhale and remove liquid;
4) add 70% ethanol then and wash mixing; Carry out magnetic resolution, inhale and remove liquid;
5) repeating step 4) once;
6) under the magnetic condition, room temperature is dried, and inhales and remove liquid;
7) add TE solution, mixing;
8) magnetic resolution, imbitition, gained liquid are the PCR product of purifying.
4. according to any described separation method of claim 1-3, it is characterized in that the used screening solution system of described capillary electrophoresis is made up of sieving medium HEC and matrix damping fluid.
5. separation method according to claim 4 is characterized in that, described matrix damping fluid is made up of 89mol/L Tutofusin tris, 332mol/L boric acid and 2mol/L EDTA, and pH is 7.2～7.5.
6. separation method according to claim 4 is characterized in that, described sieving medium HEC is a water-soluble high-molecular substance, and mass concentration is 1.0～1.5%.
7. according to any described separation method of claim 1-6, it is characterized in that described capillary electrophoresis detailed process is:
1) preparation electrophoretic buffer: the 89mol/L Tutofusin tris, 332mol/L boric acid, 2mol/L EDTA, pH are 7.2～7.5, all the other are deionized water;
2) with 1) in the electrophoretic buffer branch take on part and prepare dissociating buffer, be 1.0～1.5% HEC to wherein adding mass concentration, heating for dissolving is cooled to 4 ℃ of sealings afterwards and preserves;
3) with 1) and 2) solution cross 0.45 μ m filter membrane, 4 ℃ of preservations;
4) internal diameter capillaceous is 75 μ m, and at 8.2～8.5cm place calcination kapillary, as detection window, capillary pipe length is 48.5cm, and useful length is 40cm;
5) calcination head and tail capillaceous place makes it pass electrode and immerses in the electrophoretic buffer;
6) condition of new pre-treatment capillaceous is as follows: the sodium hydroxide operation 5min of 0.10mol/L; Deionized water operation 10min; Dissociating buffer operation 10min;
7) parameter of capillary electrophoresis apparatus operation is as follows: capillary temperature is 20 ℃, sample introduction 20～40s under the-5kv ,-15～-the voltage operation down of 20kv, under Sig.260/8nm and Ref.350/80nm condition, detect.
8. separation method according to claim 7 is characterized in that, the pH value of electrophoretic buffer is 7.4.
9. according to claim 7 or 8 described separation methods, it is characterized in that the mass concentration of HEC is 1.2%.
10. according to claim 7 or 8 described separation methods, it is characterized in that sample injection time is 20s in the step 7), voltage is-16.5kv.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063015A (en) * | 2015-07-28 | 2015-11-18 | 福建师范大学 | Method for rapid purification of specific amplification PCR (polymerase chain reaction) product based on paramagnetic particle method |
| CN108949746A (en) * | 2018-07-23 | 2018-12-07 | 杭州和壹基因科技有限公司 | A kind of method of human faecal mass total DNA sulphite conversion and recovery purifying |
| CN111073886A (en) * | 2020-01-16 | 2020-04-28 | 中国农业科学院农业基因组研究所 | DNA extraction adsorption solution based on superparamagnetic nanoparticles, kit and DNA extraction method |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104419760A (en) * | 2013-09-07 | 2015-03-18 | 上海毕欧桥生物科技有限公司 | Sample purification method applied in analysis of nucleic acid sequence |
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2009
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063015A (en) * | 2015-07-28 | 2015-11-18 | 福建师范大学 | Method for rapid purification of specific amplification PCR (polymerase chain reaction) product based on paramagnetic particle method |
| CN108949746A (en) * | 2018-07-23 | 2018-12-07 | 杭州和壹基因科技有限公司 | A kind of method of human faecal mass total DNA sulphite conversion and recovery purifying |
| CN111073886A (en) * | 2020-01-16 | 2020-04-28 | 中国农业科学院农业基因组研究所 | DNA extraction adsorption solution based on superparamagnetic nanoparticles, kit and DNA extraction method |
| CN111073886B (en) * | 2020-01-16 | 2023-08-29 | 中国农业科学院农业基因组研究所 | DNA extraction adsorption liquid based on superparamagnetism nano-particles, kit and DNA extraction method |
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| CN101705225B (en) | 2012-03-07 |
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