CN109735532A - A kind of bead suspension and its application for nucleic acid purification and screening - Google Patents
A kind of bead suspension and its application for nucleic acid purification and screening Download PDFInfo
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- CN109735532A CN109735532A CN201811643990.5A CN201811643990A CN109735532A CN 109735532 A CN109735532 A CN 109735532A CN 201811643990 A CN201811643990 A CN 201811643990A CN 109735532 A CN109735532 A CN 109735532A
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Abstract
The invention belongs to magnetic bead applied technical fields, disclose a kind of bead suspension and its application for nucleic acid purification and screening, bead suspension of the invention contains the nanometer magnetic bead of carboxylated modification, PEG polyethylene glycol, sodium chloride and/or magnesium chloride, ethylenediamine tetra-acetic acid, Tris-HCl, N- sodium lauroyl sarcosine, polysorbas20, solvent is water, and wherein the concentration of nanometer magnetic bead is 0.1-5mg/mL.Bead suspension of the invention can purification of nucleic acid, the rate of recovery is up to 96.42%, and has good DNA fragmentation screening function.Bead suspension performance of the invention is stablized, and high throughput sample operations are suitble to, and at low cost, flexibility is high, and each work station is suitble to use.
Description
Technical field
The invention belongs to magnetic bead applied technical fields, suspend more particularly to a kind of for nucleic acid purification and the magnetic bead of screening
Liquid and its application.
Background technique
As the fast development of genomics detection technique, especially new-generation sequencing (NGS) technology reach its maturity, application
To after clinical diagnosis or medical research, high-throughput, quick, highly sensitive and standardized experimental implementation demand is more more and more urgent.
NGS sequencing technologies library construction step is a quite time-consuming laborious job, needs to carry out sample between enzymatic reaction step
Purification process is tested in next step to can be carried out.
In bench-scale testing or scientific research project work requirements, silicon matrix Filter column or filter membrane is can be used in nucleic acid purification,
Sample to be purified is filtered by centrifugation or negative pressure, washs and then dries using cleaning solution, dissolve, to reach purifying core
The purpose of acid.The purifying of column method is crossed, manual operation degree of participation height is needed, is difficult to realize automate, and general purification column carries
750 μ L of volume <, it is difficult to purify big system sample；It crosses column purification mode and also relies on the equipment such as centrifuge and reach purification process,
Cumbersome, flexibility ratio is limited, and the rate of recovery is low.Purifying plate batch operation may be implemented in filter membrane purifying, but there is still a need for by true
The heavy instrument such as sky pump；The finite volume of single purifying, less than 200 μ L；Purification and recovery is expensive, is not suitable for mass, rule
Modelling enterprise produces and uses.The Agencourt AMPure XP magnetic beads for purifying recovering effect of import brand BeckMan company compared with
It is good but expensive, price Pu Bianwei $50/mL-$70/mL.
The building of two generation sequencing libraries, most of library type need DNA points to genome or length greater than 1000bp
Son carries out random fragmentation processing.Physics ultrasound interrupts or chemical enzymatic cleavage methods fragmentation, and fragmentation products are still in a certain fixed length
There is diffusing phenomenon in degree range.It carries out carrying out piece in library preparation or library preparation process again after needing to carry out segment screening
Section screening, to adapt to optimal sequencing strategy (related with sequencing length).Traditional segment screening, can take Ago-Gel electric
Swimming carries out segment separation, carries out cutting glue, recycling to purpose nucleic acid；This segment screening mode list batch operation time time-consuming is at 1
Hour or more, if sample size increases, the time cost for needing to put into can be more.Although this segment screening mode screening length essence
Standard, but sample of nucleic acid loss greatly, need to put into electrophoresis apparatus, gel imager, glue recycling etc. relevant devices, DNA input amount compared with
Height, generally higher than 1 μ g, and it is difficult to realize mass, scale, automatic operation.Two generations represented sequencing is detected as with NIPT
Technical application has further pushed the development of NGS technology to clinical detection service, and requires experimental implementation towards simple, automatic
Change direction to develop.
Super-paramagnetism nano microballoon (super paramagnetics nano spheres/particles/beads) letter
Claim magnetic bead, it, can the biomolecule progress such as specificity and cell, protein, nucleic acid by the active group that external sheath is distributed
Coupling, to can make the separation of biomolecule Yu primary liquid environment under the influence of a magnetic field, realizes the purification and recovery of biomolecule.
For magnetic microsphere since birth, it just receives the concern of researcher, and obtains in biochemical analysis field
Successfully application, nucleic acid purification recycling and segment screening are one of them application directions, just meet two generations sequencing batch
The requirement of scale library construction.The action principle of magnetic beads for purifying is point based on the reversible fixation of a kind of solid phase carrier (SPRI)
From purification process.It is generally comprised in magnetic bead system: magnetic bead, nucleic acid, polyethylene glycol (PEG) and salt ion etc., in a certain concentration
PEG and salt ion environment in, DNA can be adsorbed onto the Polymer Magnetic bead surface (i.e. solid phase carrier) of carboxyl modified, which is
Reversible, the DNA molecular combined under proper condition can be eluted recycling.Nanometer magnetic bead surface nature is different, separation principle
Also it is not quite similar, but substantially solid spheroidal material composition has no too many differences, foundation structure is generally divided into 3 layers, innermost layer
Core be polystyrene, second layer coated magnetic substance --- ferroso-ferric oxide (Fe3O4), outermost surface be carboxyl (-
COOH) high molecular material modified is constituted, and wherein carboxyl exercises the work in conjunction with nucleic acid.In whole system, PEG is shadow
Ring the deciding factor of DNA recycling (other factors further include DNA size and concentration, salt ionic concentration, incubation time etc.).?
In certain density PEG environment, NaCl or MgCl is adjusted2Equal salt ionic concentrations, make DNA that dehydration, molecular conformation meeting occur
Change dramatically occurs, curling is compressed to form by threadiness spherical, then aggregate and precipitate, while with PEG molecular weight, concentration and salt
The DNA of the difference of concentration, different length being precipitated out by selectivity.In PEG and salt ion work dis environment
DNA after molecular conformation change occurs because of dehydration, can expose a large amount of negatively charged phosphate in phosphoric acid backbone
, in conjunction with the carboxyl magnetic bead negatively charged with surface, but how to explain the effect between negative negative electrical charge, it is also unknown at present.
But it is believed that this is because the effect (such as Na+) of positively charged salt ion.Electronegative phosphate group by dissociation salt
Ion (such as Na+) and carboxyl form ionic bridge, make DNA by specific adsorption to carboxyl magnetic bead surfaces.When PEG and salt are removed
Later, aqueous molecule is added, understands quickly abundant aquation DNA, the ionic interaction between its three is released, so that being adsorbed onto magnetic
The DNA of pearl is purified out.
The Agencourt AMPure XP product of German BeckMan company is that one kind can obtain high quality DNA product
Magnetic bead Nucleic acid purification kits.Purified product purity is high, salt-free ion residues, overall process is without centrifugation or filtering；It both can be manual
Automatically working platform can also be used in operation, and the purifying of batch sample is completed in the form of 96 holes or 384 orifice plates.This product is opened up
Magnetic bead is used for the prelude of nucleic acid purification on a large scale, has healthy and free from worry Axygen to be proposed AxyPrep Mag purifying magnetic bead in succession.In view of
The application for purifying magnetic bead is convenient, flexible, even if expensive, is still used by more and more enterprises.Bake Mann
Agencourt AMPure XP magnetic bead occupies the main market share with its superior performance, and use is more and more extensive.It is domestic
Enterprise has also emerged in large numbers numerous persons of imitating, and magnetic bead types of brand are various at present, and practical purification and recovery rate quality is irregular, has good
The magnetic bead of good segment screening capacity is less.
Summary of the invention
The strong magnetic of fragment ability is verified in and a kind of screening good the purpose of the present invention is to provide purification and recovery effect at low cost
Pearl suspension.
Specifically, the present invention provides a kind of bead suspension, the nanometer magnetic bead containing carboxylated modification, polyethylene glycol
PEG, sodium chloride and/or magnesium chloride, ethylenediamine tetra-acetic acid, Tris-HCl, N- sodium lauroyl sarcosine, polysorbas20, solvent are water,
Wherein concentration of the nanometer magnetic bead in bead suspension is 0.1-5mg/mL.
N- sodium lauroyl sarcosine, plays stable magnetic bead suspension effect in polysorbas20 the application formula, auxiliary improve magnetic bead and
The adsorption efficiency of nucleic acid increases the effect of recovery efficiency.
In bead suspension provided by the invention, the nanometer magnetic bead magnetic core composition of the carboxylated modification is four oxidations three
Iron, magnetic content > 70wt%；Magnetic bead particles diameter is 0.5 μm -1.0 μm, or meets the sizes magnetic bead particles of aforementioned condition
It is used in mixed way.
In above-mentioned bead suspension, the polyethylene glycol PEG is one or more within the scope of PEG6000-PEG10000
It is used in mixed way；Mass percent (g/100mL) of the PEG in bead suspension is 8%-20%.Preferably, PEG is outstanding in magnetic bead
Mass percent in supernatant liquid is 10-20%, it is highly preferred that mass percent of the PEG in bead suspension is 15%.
Molar concentration of the sodium chloride in bead suspension is 0.5mol/L-5mol/L；It is preferred that 1mol/L-3mol/
L；
Molar concentration 0.3mol/L-1mol/L of the magnesium chloride in bead suspension；It is preferred that 0.5mol/L-1mol/L
The molar concentration of the ethylenediamine tetra-acetic acid is 0.1mmol/L to 10mmol/L；-
The molar concentration of the Tris-HCl is 0.1mmol/L to 10mmol/L, pH value 7.5-8.5.
The mass volume ratio of the N- sodium lauroyl sarcosine is 0-0.1% (g/100mL)；
The percent by volume of the polysorbas20 is 0-5%.
The present invention provides the kits for being used for nucleic acid purification or screening containing above-mentioned bead suspension.
The present invention provides application of the above-mentioned bead suspension in high-throughput sequencing library building.
The present invention provides application of the above-mentioned bead suspension in nucleic acid purification or screening nucleic acid fragment.
Specifically, the present invention provides a kind of methods of purification of nucleic acid, comprising the following steps:
(1) bead suspension of the present invention is added in sample of nucleic acid to be purified and be mixed, the two volume ratio be (0.4:
1)-(2.5:1)；It is statically placed in magnetic frame and is all adsorbed on magnetic frame to magnetic bead；
(2) supernatant for abandoning step (1) is inhaled, is inhaled again after addition 80% ethyl alcohol standing 1.5-3min and abandons supernatant, be added
80% ethyl alcohol stands 1.5-3min, inhales and abandons supernatant, it is ensured that without obvious drop；
(3) supernatant for abandoning step (2) is inhaled, is inhaled again after addition 80% ethyl alcohol standing 1.5-3min and abandons supernatant, be added
80% ethyl alcohol stands 1.5-3min, inhales and abandons supernatant, it is ensured that without obvious drop；
(4) magnetic bead for combining nucleic acid is dried, pure water elutes magnetic bead, and eluent brief centrifugation is statically placed in magnetic frame, until magnetic
Pearl is all adsorbed by magnetic frame, draws supernatant liquid body to get nucleic acid solution after purification.
The present invention also provides a kind of detection methods for screening nucleic acid fragment ability, comprising the following steps:
(1) several 10 μ L of the ladder of 50bp, moisturizing to 50 μ L are taken.(in the case where 50 μ L, supplement pure water to volume
50μL)；It is added any bead suspension of claim 1-5 into mixture, the volume ratio of nucleic acid and magnetic bead is
(0.4:1)-(2.5:1) is mixed；It is statically placed in magnetic frame and is all adsorbed on magnetic frame to magnetic bead
(2) supernatant for abandoning step (1) is inhaled, is inhaled again after addition 80% ethyl alcohol standing 1.5-3min and abandons supernatant, be added
80% ethyl alcohol stands 1.5-3min, inhales and abandons supernatant, it is ensured that without obvious drop；
(3) supernatant for abandoning step (2) is inhaled, is inhaled again after addition 80% ethyl alcohol standing 1.5-3min and abandons supernatant, be added
80% ethyl alcohol stands 1.5-3min, inhales and abandons supernatant, it is ensured that without obvious drop；
(4) magnetic bead for combining nucleic acid is dried, pure water elutes magnetic bead, and eluent brief centrifugation is statically placed in magnetic frame, until magnetic
Pearl is all adsorbed by magnetic frame, and supernatant liquid body is drawn, and loadingbuffer is added and carries out detected through gel electrophoresis.
The beneficial effects of the present invention are: bead suspension provided by the invention can purification of nucleic acid, the rate of recovery is up to
96.42%, reach 97% or more of commercialization Agencourt AMPure XP product recovery rate, closely and is equal to this
The purification effect of magnetic bead is commercialized；And bead suspension of the invention has good DNA fragmentation screening function；Use 1 times
Volume can screen the DNA fragmentation for being 200bp greater than length in the bead suspension dosage of nucleic acid to be purified.Magnetic of the invention
Pearl suspension performance is stablized, cheap, in the case where completing a mass purification or screening, with 50 μ L systems to be purified, and 1
For times magnetic bead dosage: bead suspension dosage is 50 μ L, and about 0.5 yuan of cost, the Ampure XP beads of identical function is general
2.5 yuan -3.5 yuan are needed, high-throughput operation is suitble to, at low cost, flexibility is high, and each work station is suitble to use.Using magnetic beads for purifying,
By taking work station purifies as an example, every batch of purifies 96 samples, used time about 35min.
Detailed description of the invention
Fig. 1 is the screening capacity electrophoresis detection figure of different bead suspensions in the embodiment of the present invention 5.Using 0.7 ×-
The screening of nucleic acid fragment different length may be implemented in the bead suspension of 1.5 × multiple proportions.
Fig. 2 is that embodiment 6 uses bead suspension of the present invention to carry out segment sorting nucleic acid fragment as the sample result of 450bp
1, graphical results are shown, detection segment main peak concentrates near 450bp.
Fig. 3 is that embodiment 6 uses bead suspension of the present invention to carry out segment sorting nucleic acid fragment as the sample result of 450bp
2, graphical results are shown, detection segment main peak concentrates near 450bp.
Fig. 4 is that embodiment 6 uses bead suspension of the present invention to carry out segment sorting nucleic acid fragment as the sample result of 450bp
3, graphical results are shown, detection segment main peak concentrates near 450bp.
Specific embodiment
Following embodiment is merely to illustrate the present invention, and is not intended to limit the scope of the invention.
Raw material used in the following embodiment is commercially available, and can also usually conventional method in that art be made.The present invention
Raw material nano magnetic bead in embodiment is purchased from U.S. GE life science.
The preparation (1) of 1 bead suspension of embodiment
Present embodiments provide a kind of preparation method of bead suspension, specific formula are as follows: particle diameter is at 0.5-1.0 μm
Between carboxylated modification nanometer magnetic bead, the final concentration of 1.0mg/mL in bead suspension, final concentration of 15% (quality
Percentage, g/100mL) polyethylene glycol PEG10000, the sodium chloride of final concentration of 1mol/L, the ethylenediamine tetrem of 5mmol/L
Acid, the Tris-HCl of 2mmol/L, the N- sodium lauroyl sarcosine of mass volume ratio (g/100mL) 0.01%, mass volume ratio are
0.5% polysorbas20, solvent are water.After preparation, 2 DEG C of -8 DEG C of preservations.Bead suspension process for preparation of the invention is preferential
Water, PEG, the high concentrations such as sodium chloride or the big composition of dosage is added, the elder generation of reagent is added in the subsequent composition that other low concentrations are added
Sequence is without particular/special requirement afterwards.Following embodiment addition sequence principle thus.
The preparation (2) of 2 bead suspension of embodiment
Present embodiments provide a kind of bead suspension, specific formula are as follows: solvent is water, the carboxylic that particle diameter is 0.5 μm
The nanometer magnetic bead of baseization modification, the final concentration of 5mg/mL in bead suspension, final concentration of 15% (mass percent, g/
Polyethylene glycol PEG6000 100mL), the magnesium chloride of the sodium chloride of final concentration of 0.5mol/L and final concentration of 1mol/L,
The ethylenediamine tetra-acetic acid of 1mmol/L, the Tris-HCl of 6mmol/L, the N- lauroyl flesh of mass volume ratio 0.05% (g/100mL)
Propylhomoserin sodium, the polysorbas20 that mass volume ratio is 0.5%1%.After preparation, 2 DEG C of -8 DEG C of preservations.
The preparation (3) of 3 bead suspension of embodiment
Present embodiments provide a kind of bead suspension, specific formula are as follows: solvent is water, the carboxyl that particle diameter is 1 μm
Change the nanometer magnetic bead of modification, the final concentration of 1mg/mL in bead suspension, final concentration of 20% (mass percent) is gathered
Ethylene glycol PEG8000, the magnesium chloride of the sodium chloride of final concentration of 2.5mol/L and final concentration of 1.0mol/L, the second of 5mmol/L
Ethylenediamine tetraacetic acid (EDTA), the Tris-HCl of 2mmol/L, the N- sodium lauroyl sarcosine of mass volume ratio 0.01%, mass volume ratio (g/
100mL) the polysorbas20 for being 0.5%.After preparation, 2 DEG C of -8 DEG C of preservations.
1 bead suspension A of comparative example
Compared with the formula of embodiment 1, other than being not added with N- sodium lauroyl sarcosine and polysorbas20, other formulas are homogeneous
Together.Specifically: particle diameter 1.0 μm carboxylated modify nanometer magnetic bead, it is final concentration of in bead suspension
1.0mg/mL, the polyethylene glycol PEG10000 of final concentration of 15% (mass percent, g/100mL), final concentration of 1mol/L's
Sodium chloride, the ethylenediamine tetra-acetic acid of 5mmol/L, the Tris-HCl of 2mmol/L.After preparation, 2 DEG C of -8 DEG C of preservations.
2 bead suspension B of comparative example
The formula and embodiment 1 of bead suspension B compares, and using 7% PEG10000, other formulas are all the same.Implement
Example provides a kind of bead suspension preparation method, specific formula are as follows: carboxylated modification of the particle diameter between 1.0 μm is received
Rice magnetic bead, the final concentration of 1.0mg/mL in bead suspension, the poly- second of final concentration of 7% (mass percent, g/100mL)
Glycol PEG10000, the sodium chloride of final concentration of 1mol/L, the ethylenediamine tetra-acetic acid of 5mmol/L, the Tris-HCl of 2mmol/L,
The N- sodium lauroyl sarcosine of mass volume ratio 0.01%, the polysorbas20 that mass volume ratio is 0.5%, solvent is water.It has prepared
Bi Hou, 2 DEG C of -8 DEG C of preservations.
The compliance test result of 4 bead suspension purification and recovery nucleic acid of embodiment
Embodiment 1-3 is respectively adopted, the magnetic bead of comparative example 1-2 configuration carries out the survey of nucleic acid recyclability by the following method
Examination, and using AMpure XP magnetic bead as control.
The 50 μ L of PCR product of 14.0ng/ μ L is selected, segment is concentrated mainly on 320bp, using purifying magnetic bead of the invention,
1.5 × (75 μ L) purifying is carried out, the sample rate of recovery is measured.50 μ L PCR products are taken, are placed in 1.5ml centrifuge tube, 75 μ of magnetic bead is taken
To sample to be purified, piping and druming mixes to be not less than 6 times L；It is stored at room temperature 7min；The magnetic bead & sample mixtures of previous step are placed in magnetic
2min is stood on power frame, until magnetic bead is all adsorbed on magnetic frame；The supernatant for abandoning previous step is inhaled, 500 μ L preparation is then added
80% ethyl alcohol, stand 2min；The supernatant for abandoning previous step is inhaled, 80% ethyl alcohol of 500 μ L preparation is then added, stands 2min；
It is inhaled again using small-range pipettor and abandons previous step residual alcohol, it is ensured that no obvious drop alcohol exists；Previous step is combined
The magnetic bead of DNA carries out room temperature and dries, the about 3min time, until remaining without obvious washmarking；DNA elution is carried out using 52 μ L of pure water；It will
Eluent and step magnetic bead carry out piping and druming mixing, are stored at room temperature 7min；The magnetic bead eluent of previous step is subjected to brief centrifugation, is placed in
2min is stood on magnetic frame, until magnetic bead is all adsorbed by magnetic frame, eluent is in clear state；Draw supernatant liquid body
50 μ L carry out concentration quantitative, detection using Qubit.Each processing group 3 repeated experiments take result average value, each processing group group
Interior 3 data differences are not significant.Measurement rate of recovery data are shown in Table 1.
1 recovery of nucleic acid test data of table
Compared by detection data it is found that embodiment 1-3, PEG10000, PEG6000 and PEG8000 are to nucleic acid recovery efficiency
Influence unobvious, average recovery rate is respectively 97.14%, 96.42% and 94.28%.
For embodiment 1 compared with comparative example 1, not adding N- sodium lauroyl sarcosine and polysorbas20 has slight shadow to the rate of recovery
It rings, is reduced to 93.57 by 97.14%, reduces about 2 percentage points.
Embodiment 1 is compared with comparative example 2, and using 7% PEG10000, the rate of recovery is substantially reduced, by being reduced by 96.42%
To 40%.The influence importance that PEG concentration recycles nucleic acid purification is absolutely proved.
5 bead suspension of embodiment screens nucleic acid fragment ability measurement (1)
50bp DNA ladder is taken, the bead suspension prepared using the present invention, progress different amounts are gradient-purified, if
Setting value is that (0.7X, 0.8X, 0.9X, 1.0X, 1.2X, 1.5X) measures the sample rate of recovery, each gradient 50bp ladder dosage 10
μ L, 40 μ L of moisturizing take corresponding magnetic bead according to the system of 50 μ L again (each gradient is shown in Table 2).
2 magnetic bead screening capacity magnetic bead dosage gradient of table
The mixture for taking 50 μ LLadder and water, is placed in 1.5ml centrifuge tube, and 1-3 of the embodiment of the present invention is taken to prepare respectively
Bead suspension takes volume and is shown in Table 2 " magnetic bead dosages ", piping and druming, which mixes, to be not less than to 50ml to ladder and in the mixture of water
6 times；It is stored at room temperature 7min；The magnetic bead sample mixtures of previous step are placed on magnetic frame and stand 2min, until magnetic bead all adsorbs
On magnetic frame；The supernatant for abandoning previous step is inhaled, 80% ethyl alcohol of 500 μ L preparation is then added, stands 2min.It inhales and abandons previous step
Supernatant, be then added 500 μ L preparation 80% ethyl alcohol, stand 2min；It is inhaled again using small-range pipettor and abandons previous step
Remain alcohol, it is ensured that no obvious drop alcohol exists；The magnetic bead that previous step combines DNA is carried out room temperature to dry, when about 3min
Between, until being remained without obvious washmarking；DNA elution is carried out using pure water；Add 10 μ L of water volume, eluent and previous step magnetic bead are carried out
Piping and druming mixes, and is stored at room temperature 7min；The magnetic bead eluent of previous step is subjected to brief centrifugation, is placed on magnetic frame and stands 2min,
It is all adsorbed to magnetic bead by magnetic frame, eluent is in clear state；10 μ L of supernatant liquid body is drawn, Loading is added
Buffer carries out agarose gel electrophoresis detection, sees Fig. 1.As shown in Figure 1:
0.7X system bead suspension dosage, nucleic acid fragment screen range >=300bp；
0.8X system bead suspension dosage, nucleic acid fragment screen range >=250bp；
0.9-1.0X system bead suspension dosage, nucleic acid fragment screen range >=200bp；
1.2-1.5X system bead suspension dosage, nucleic acid fragment screen range >=150bp.
6 bead suspension of embodiment screens nucleic acid fragment ability measurement (2)
It takes mankind gDNA 200ng to carry out the processing of physics ultrasound fragmentation, then carries out library construction；Library construction process
It is middle that length screening is carried out to library using two-step purifying screening technique, PCR amplification then is carried out to screening product, library fragments are long
Degree is demonstrated by outstanding segment screening capacity through detecting Stable distritation range about 450bp.
This test splice coupled reaction system is 50 μ L, and purification step operation example is as follows:
Prepare 0.6 × sorting Buffer: taking 600 μ L magnetic beads, 1000 μ L ultrapure waters are added, mixes well, is placed in magnetic frame
Upper standing 2min；Taking supernatant is 0.6X sorting buffer, for use.
It takes 50 μ library construction connection products to be placed in 1.5ml centrifuge tube, 1.2X respective volume bead suspension is added, blows
It beats to mix and be not less than 6 times；It is stored at room temperature 7min；The mixture of previous step is placed on magnetic frame and stands 2min, until magnetic bead is whole
It is adsorbed on magnetic frame；The supernatant for abandoning previous step is inhaled, 80% ethyl alcohol of 500 μ L preparation is then added, stands 2min.It inhales on abandoning
Then 80% ethyl alcohol of 500 μ L preparation is added in the supernatant of one step, stand 2min；It is inhaled in abandoning again using small-range pipettor
One step remains alcohol, it is ensured that no obvious drop alcohol exists；160 μ L 0.6 × sorting buffer are added and previous step magnetic bead is abundant
It mixes, stands 8min；Previous step supernatant is taken, a new 1.5mL centrifuge tube is placed in, 20 μ L magnetic beads are added, and mix well, it is quiet
Set 5min；The magnetic bead sample mixtures of previous step are placed on magnetic frame and stand 2min, until magnetic bead is all adsorbed on magnetic frame；
The supernatant for abandoning previous step is inhaled, 80% ethyl alcohol of 500 μ L preparation is then added, stands 2min.The supernatant for abandoning previous step is inhaled, so
80% ethyl alcohol of 500 μ L preparation is added afterwards, stands 2min；It is inhaled again using small-range pipettor and abandons previous step residual alcohol, really
It protects and exists without obvious drop alcohol；The magnetic bead that previous step combines DNA is carried out room temperature to dry, the about 3min time, until without obvious
Washmarking residual；DNA elution is carried out using pure water；Add 30 μ L of water volume, eluent and previous step magnetic bead are subjected to piping and druming mixing, room
Temperature stands 7min；The magnetic bead eluent of previous step is subjected to brief centrifugation, is placed on magnetic frame and stands 2min, until magnetic bead whole quilt
Magnetic frame absorption, eluent are in clear state；Portion thereof library is taken to carry out micro flow control chip capillary electrophoresis system detection, inspection
It surveys result and sees Fig. 2-Fig. 4.
The above is only the description to general explanation and specific embodiment of the invention, is not to this
The limitation of other forms is made in invention.Anyone skilled in the art can be added based on technology contents disclosed in the present application
To change or be modified to its equivalent integers.Under the premise of without departing substantially from design and spirit of the invention, the present invention is carried out
Any modification or modification belong to the protection scope of technical solution of the present invention.
Claims (10)
1. a kind of bead suspension, which is characterized in that the nanometer magnetic bead containing carboxylated modification, PEG polyethylene glycol, sodium chloride
And/or magnesium chloride, ethylenediamine tetra-acetic acid, Tris-HCl, N- sodium lauroyl sarcosine, polysorbas20, solvent are water, wherein nano magnetic
Concentration of the pearl in bead suspension is 0.1-5mg/mL.
2. bead suspension according to claim 1, which is characterized in that the nanometer magnetic bead magnetic core of carboxylated modification at
Part is ferroso-ferric oxide, magnetic content > 70wt%；Magnetic bead particles diameter is 0.5 μm -1.0 μm, or meets a variety of of aforementioned condition
Size magnetic bead particles are used in mixed way.
3. bead suspension according to claim 1, which is characterized in that the polyethylene glycol PEG is PEG6000-
One or more within the scope of PEG10000 are used in mixed way；Mass percent of the PEG in bead suspension is 8%-20%, institute
State mass volume ratio are as follows: g/100mL.
4. bead suspension according to claim 1, which is characterized in that mole of the sodium chloride in bead suspension
Concentration is 0.5mol/L-5mol/L；Molar concentration 0.3mol/L-1mol/L of the magnesium chloride in bead suspension；It is described
The molar concentration of ethylenediamine tetra-acetic acid is 0.1mmol/L-10mmol/L；The molar concentration of the Tris-HCl is 0.1mmol/L-
10mmol/L, pH value 7.5-8.5.
5. bead suspension according to claim 1 to 4, which is characterized in that the matter of the N- sodium lauroyl sarcosine
Amount volume ratio is 0-0.1%, the mass volume ratio are as follows: g/100mL；The polysorbas20 is to be diluted to volume basis with pure water
Than for 0-5%.
6. the kit for being used for nucleic acid purification or screening containing any bead suspension of claim 1-5.
7. application of any bead suspension of claim 1-5 in high-throughput sequencing library building.
8. application of any bead suspension of claim 1-5 in nucleic acid purification or screening nucleic acid fragment.
9. a kind of method of purification of nucleic acid, which comprises the following steps:
(1) any bead suspension of claim 1-5 is added in sample of nucleic acid to be purified and is mixed, the two volume ratio is
(0.4:1)-(2.5:1)；It is statically placed in magnetic frame and is all adsorbed on magnetic frame to magnetic bead；
(2) supernatant for abandoning step (1) is inhaled, is inhaled again after addition 80% ethyl alcohol standing 1.5-3min and abandons supernatant, be added 80%
Ethyl alcohol stands 1.5-3min, inhales and abandons supernatant, it is ensured that without obvious drop；
(3) supernatant for abandoning step (2) is inhaled, is inhaled again after addition 80% ethyl alcohol standing 1.5-3min and abandons supernatant, be added 80%
Ethyl alcohol stands 1.5-3min, inhales and abandons supernatant, it is ensured that without obvious drop；
(4) magnetic bead for combining nucleic acid is dried, pure water elutes magnetic bead, and eluent brief centrifugation is statically placed in magnetic frame, until magnetic bead is complete
Portion is adsorbed by magnetic frame, draws supernatant liquid body to get nucleic acid solution after purification.
10. a kind of detection method for screening nucleic acid fragment ability, which comprises the following steps:
(1) take the ladder of 50bp several, if sample volume supplements pure water to 50 μ L of volume less than 50 μ L；Into mixture
The volume ratio of any bead suspension of addition claim 1-5, nucleic acid and magnetic bead is (0.4:1)-(2.5:1), is mixed
It is even；It is statically placed in magnetic frame and is all adsorbed on magnetic frame to magnetic bead；
(2) supernatant for abandoning step (1) is inhaled, is inhaled again after addition 80% ethyl alcohol standing 1.5-3min and abandons supernatant, be added 80%
Ethyl alcohol stands 1.5-3min, inhales and abandons supernatant, it is ensured that without obvious drop；
(3) supernatant for abandoning step (2) is inhaled, is inhaled again after addition 80% ethyl alcohol standing 1.5-3min and abandons supernatant, be added 80%
Ethyl alcohol stands 1.5-3min, inhales and abandons supernatant, it is ensured that without obvious drop；
(4) magnetic bead for combining nucleic acid is dried, pure water elutes magnetic bead, and eluent brief centrifugation is statically placed in magnetic frame, until magnetic bead is complete
Portion is adsorbed by magnetic frame, draws supernatant liquid body, and loading buffer is added and carries out detected through gel electrophoresis.
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