CN101665785A - Method for extracting and purifying nucleic acid from samples by magnetic beads - Google Patents
Method for extracting and purifying nucleic acid from samples by magnetic beads Download PDFInfo
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- CN101665785A CN101665785A CN200910307632A CN200910307632A CN101665785A CN 101665785 A CN101665785 A CN 101665785A CN 200910307632 A CN200910307632 A CN 200910307632A CN 200910307632 A CN200910307632 A CN 200910307632A CN 101665785 A CN101665785 A CN 101665785A
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Abstract
The invention relates to a method for extracting and purifying nucleic acid from samples by magnetic beads, which comprises the following steps: (1) magnetic bead treatment: modifying the magnetic beads by peptide-oligonucleotide; (2) cell lysis: adding the sample containing target nucleic acid and needing to be separated into a centrifuge tube, and then adding lysis buffer to lyse the cells; (3)nucleic acid adsorption: adding the magnetic beads modified by the peptide-oligonucleotide into the solution to lead the nucleic acid to be adsorbed on the surfaces of the magnetic beads; placing thecentrifuge tube into a magnetism separation machine to lead the magnetic beads to be adsorbed on the tube side; (4) impurities removal: adding aqueous phase buffer solution to wash the magnetic bead which are adsorbed with the nucleic acid on the surfaces and adsorbed on the tube side so as to separate other impurities on the magnetic bead; and (5) nucleic acid recovery: directly taking the nucleic acid adsorbed on the surfaces of the magnetic beads as the template for PCR amplification, or adding elution buffer to lead the nucleic acid molecules adsorbed on the surfaces of the magnetic beadsto be released into the buffer for downstream experiments. The magnetic beads used by the method can be reserved at the temperature of 4 DEG C or -20 DEG C, and have convenient transport, high adsorption efficient and lower cost. The nucleic acid extracted and purified by the method has wide application range.
Description
Technical field
The present invention relates to a kind of magnetic bead that adopts and from sample, extract the also method of purification of nucleic acid.
Background technology
The extraction and purification technology of nucleic acid is a basic fundamental of Biochemistry and Molecular Biology.Along with Protocols in Molecular Biology is widely used in biology, medical science and association area thereof, the extraction and purification technology of nucleic acid is also further developed.
Nucleic acid always combines with range protein in cell.The extraction of nucleic acid mainly is meant biomacromolecule separating substances such as nucleic acid and protein, polysaccharide, fat.The extracting method of most of nucleic acid generally all comprises lysis, biomacromolecules such as removal and nucleic acid bonded protein and polysaccharide, lipid, remove other unwanted nucleic acid molecule, precipitate nucleic acids is removed impurity such as salt, organic solvent, steps such as purification of nucleic acid.
1. lysis: lysis can be passed through methods such as mechanical effect, chemical action, enzyme effect and realize.
(1) mechanical effect: comprise physics cleavage methods such as hypotonic cracking, ultrasonic degradation, microwave cracking, freezing-thawing and cracking and grain breakage, these methods make cytoclasis with mechanical force, but mechanical force also can cause the nucleic acid chain break, thereby is not suitable for the separation of high molecular long-chain nucleic acid; (2) chemical action: under certain pH environment and sex change condition, cell rupture, protein denaturation precipitation, nucleic acid is released to water, and the sex change condition can obtain by heating, adding tensio-active agent (SDS, TritonX-100, Tween20, NP-40, CTAB, sar-cosyl, Chelex-100 etc.) or strong ionic agent (guanidinium isothiocyanate, Guanidinium hydrochloride, creatine guanidine etc.); (3) enzyme effect: mainly be by adding N,O-Diacetylmuramidase or proteolytic enzyme (Proteinase K, plant protease or pronase) so that cell rupture, nucleic acid discharges.
2. enzyme is handled: in the nucleic acid extraction process, can make unwanted mass degradation by adding suitable enzyme, be beneficial to the isolation and purification of nucleic acid, can degrade proteins as in lysate, adding proteolytic enzyme (Proteinase K or pronase), deactivation nuclease (DNase and RNase), DNase and RNase also are used to remove unwanted nucleic acid.
3. the separation of nucleic acid and purifying: the high electric charge phosphoric acid skeleton of nucleic acid makes it compare protein, polysaccharide, other biological macromolecular substance such as fat have more wetting ability, difference according to their physico-chemical properties, use selective precipitation, chromatography, methods such as density gradient centrifugation can be with separate nucleic acid, purifying, the ordinary method of existing separation and purification nucleic acid has: (1) phenol extraction/precipitator method: the water that centrifugation after the lysis is contained nucleic acid, add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1 volumes) mixed solution, two-phase is through vortex oscillation mixing (be applicable to separate small molecular weight nucleic acid) or centrifugation after simply putting upside down mixing (being applicable to separation of high molecular weight nucleic acid), again with the nucleinate organic solvent deposit, by the precipitation condensed nucleic acid, change the kind of nucleic acid dissolving damping fluid and remove some impurity molecule; (2) density gradient centrifugation: double-stranded DNA, single stranded DNA, RNA and protein have different density, thereby can form the pure sample area band of different densities through density gradient centrifugation, and this method is applicable to the preparation of a large amount of sample of nucleic acid; (3) chromatography, chromatography is to utilize the difference of some physico-chemical property of different substances and the method for separating and analyzing set up, comprise methods such as adsorption chromatography, affinity chromatography, ion exchange chromatography, under certain ionic environment, nucleic acid can optionally be adsorbed onto tripoli, silica gel or glass surface and with the other biological molecular separation; Other selective adsorption method, as solid phase carrier, magnetic bead can need not centrifugal by magnetic field separation with the magnetic bead of modified or bag quilt, and the nucleic acid that is bonded to solid phase carrier can be with low salt buffer or water elution.
Paramagnetic particle method is the method for extracting nucleic acid that just grew up in recent years, its reagents series does not contain the big organic solvents of toxicity such as chloroform, phenol, the nucleic acid quality of extracting purifying is good, output is high, extraction step is simpler, do not need centrifugal, easily be automated, these advantages and characteristics make its method of extracting nucleic acid that the development trend of all other methods of substituting be arranged.
Magnetic bead is the particle with certain magnetic and special surface structure that is composited by magnetic grain and the various material that contains activity functional groups.In biomagnetism was separated, the magnetic bead surfaces active group was an important factors.Use more magnetic bead surfaces aglucon that monoclonal antibody (Monoclonal antibody), Streptavidin (Streptavidin), oligonucleotide (Oligonucleotides) are arranged at present, and introduce amino (NH on the magnetic microsphere surface with silylating reagent
2), hydroxyl (OH), sulfydryl (SH), carboxyl (COOH), sulfonic group (SO
3-) etc., on this basis, also can introduce polysaccharide compound (as Mierocrystalline cellulose, agarose), vitamin H, antibiotin etc.
The magnetic microsphere of surface bonding antibody, promptly so-called " immunomagnetic beads ", utilization antibody and specificity cell surface antigen bonded principle are used for the separation and concentration of stem cell in the sharp separation, marrow of blood cell.
The magnetic bead that Streptavidin is modified can be used to extraction separation nuclear candy nucleoprotein/ribodesose nucleoprotein (DNP/RNP).Be combined with the magnetic bead that the DNA of vitamin H or RNA sequence and Streptavidin modify and interact, protein identification these sequences, just can be attached on immobilized DNA/RNA, this method is used to separate transcription factor, restriction enzyme, replication protein etc.
Oligo (dT) 25 magnetic microspheres are used to extract mRNA from complicated lysate.Poly (A)-tail is ubiquitous one section base on the mRNA sequence, so magnetic bead surfaces only needs Oligo (dT) 25 sequences, just can be effectively with mRNA on Poly (A)-tail hybridization, and can remove rRNA, tRNA and other RNA.
MagneSil
TMThe superparamagnetism magnetic bead is magnetic particle and SiO
2The surface band silicon hydroxyl that forms and the center is the magnetic bead of magnetic core.DNA and silicon hydroxyl interact and form mixture, and this mixture has certain responsiveness in magnetic field.
These are by the magnetic bead of Streptavidin, vitamin H, antibody or few nucleic acid fragment isoreactivity identification molecular modification, owing in storage and transportation process, be unfavorable for discerning the preservation of molecular activity, use range is narrow, and adsorption efficiency is not high, very former thereby not widespread use such as costliness of price.
Summary of the invention
The purpose of this invention is to provide a kind of magnetic bead and can place 4 ℃ or-20 ℃ of preservations, and convenient transportation, use range is extensive, and adsorption efficiency height, the employing magnetic bead that price is lower extract the also method of purification of nucleic acid from sample.
The present invention's employing magnetic bead extracts from sample and the method for purification of nucleic acid may further comprise the steps:
(1) peptide-oligonucleotide (peptide-oligonucleotide) is modified magnetic bead:
With γ Fe
2O
3And Fe
3O
4Magneticsubstance synthetic homogeneous, super paramagnetic, single diversity poly microballoon are the magnetic source, and each microsphere bag is carried the magnetic macromolecular microsphere of carboxylate salt by one deck poly material by letex polymerization or the preparation of diffuse-aggregate method; Through poly material core particle surface cover one deck magnet that carboxylate salt is modified, oligo (dT)
14Covalently bind on this surface, nucleic acid molecule can be good at being anchored on this oligonucleotide again;
(2) lysis: the sample to be separated that will contain target nucleic acid adds in the centrifuge tube, adds the lysate lysing cell again, to discharge nucleic acid;
(3) nucleic acid absorption: add peptide-oligonucleotides-modified magnetic bead in solution, nucleic acid is adsorbed by magnetic bead surfaces, and protein and other impurity then are not adsorbed; Centrifuge tube is placed magnetic separator, and magnetic bead is adsorbed in test tube wall fully;
(4) remove impurity: add the water damping fluid, the magnetic bead that the surface is adsorbed with nucleic acid and is attached to test tube wall washs, and the flushing of the impurity on the magnetic bead is separated;
(5) nucleic acid reclaims: the nucleic acid that is adsorbed on magnetic bead surfaces can perhaps add elutriant directly as the template of pcr amplification, makes the nucleic acid molecule desorb that is adsorbed on magnetic bead surfaces, enters in the elutriant, is used further to the downstream experiment.
The preferred polystyrene of described poly material.
Described magnetic microsphere (being magnetic bead) modified surface is with different aglucons (carboxylate salt, oligonucleotide etc.), as absorption with in conjunction with the carrier of corresponding molecule.
Described sample to be separated comprises virus, bacterium, fungi, tissue, blood, plant, Stomatocyte, PCR reaction product, medical jurisprudence sample and environmental sample etc.
Described nucleic acid refers to two strands or single stranded DNA, RNA or nucleic acid analog.
Described lysate is the solution that contains tensio-active agent, preferably contains the solution of 0.01%～10% SDS, LLS, Chelex-100, Triton X-100, Tween 20 or NP-40.
Described magnetic bead solution is meant pH6.5 ± 0.2, by the HEPES of 100mmol/L～2M/L and the buffered soln that 100mmol/L～2M/L NaCl constitutes.
Described water damping fluid is by NaCl, the KCl of Triton X-100, the 100mmol/L～2M/L of 0.1% to 2% (volume ratio) or the solution that LiCl forms.
Described elutriant can be 10mM Tris-HCl and 1mM EDTA, the solution of pH8.0.
The nucleic acid of described extraction can be applicable to the diagnosis of pathogenic micro-organism, forensic analysis, tissue and blood grouping, the detection of transgenation, fields such as gene sequencing.
The experiment of described downstream refers to that detection, clone, sequential analysis, pcr amplification, molecular hybridization and cDNA are synthetic etc.
The present invention uses the oligonucleotides-modified magnetic microsphere of peptide (magnetic bead) surface, and the magnetic bead surfaces of modified is adsorbed nucleic acid thereon, and protein and other impurity then are not adsorbed, and with the water damping fluid other impurity are rinsed well; The nucleic acid that is adsorbed on magnetic bead surfaces can perhaps add elution buffer directly as the template of pcr amplification, and the nucleic acid molecule that is adsorbed on magnetic bead surfaces is released in the damping fluid and is used for the downstream experiment.
The magnetic macromolecular microsphere through carboxyl modified among the present invention has unspecific enrichment target cell and the multi-functional character of extracting DNA from biological sample.As in the application of hbv nucleic acid (HBV DNA): the genome of hepatitis B virus is a circular double stranded DNA molecule that part strand district is arranged, and two strand length are different.Long-chain L (3.2kb) is a minus strand, and short chain S is a normal chain, and its length is uncertain, is about about the 50%-80% of minus strand.Because its 3 ' end has different disappearances with the different sources strain, so position changeable has produced the part ring texture.Genome relies on the sticky end of the about 240bp of normal chain 5 ' end and the complementation of minus strand gap portions to keep ring texture.5 ' end of long-chain has a covalently bound terminal protein, may be primase.Short chain 5 ' end then combines with the short rna with cap sequence by covalent linkage.The magnetic bead of carboxyl modified can be good at combining with 5 ' terminal primase, thereby other molecules are not then adsorbed by magnetic bead with impurity hepatitis B virus DNA is well separated and purifying.At magnetic bead surfaces covalent attachment oligo (dT) 14 again, because Poly (A)-tail is ubiquitous one section base on the mRNA sequence, magnetic bead surfaces has Oligo (dT) 14 sequences, just can be effectively with mRNA on Poly (A)-tail hybridization, be that pathogenic agent and other samples of RNA also has good adsorption effect therefore for nucleic acid.
Advantage of the present invention: with the oligonucleotides-modified magnetic bead of peptide, DNA, mRNA can be good at being anchored on this modified magnetic bead, protein, rRNA, tRNA and other impurity then can effectively be removed, thereby make DNA, RNA can access effective separation and purifying.Magnetic bead can place 4 ℃ or-20 ℃ of preservations, and convenient transportation; Utilization this method separation and purification gained purpose nucleic acid output height, the purity height does not need to use simultaneously deleterious organic reagents such as phenol chloroform, and operates quick, easy, saving manpower, easily is automated.
The present invention almost is suitable for separating highly purified DNA of high yield and RNA from any raw material, comprises virus, bacterium, fungi, plasmid, tissue, blood, plant, Stomatocyte, PCR reaction product, and the medical jurisprudence sample etc.The nucleic acid that is obtained can be used for detection of nucleic acids, clone, sequential analysis, pcr amplification, and molecular hybridization and cDNA are synthetic etc., are widely used in the diagnosis of pathogenic micro-organism, forensic analysis, tissue and blood grouping, fields such as the detection of transgenation.
Description of drawings
Fig. 1 (a) is peptide-oligonucleotides-modified magnetic bead schema;
Fig. 1 (b) extracts the DNA operational flowchart for paramagnetic particle method;
Fig. 2 is the fluorescence quantitative PCR detection result of serum HBV DNA;
Fig. 3 is the fluorescence quantitative PCR detection result of serum HBV DNA;
Fig. 4 (a) is the fluorescence quantitative PCR detection result (20IU/ml) of serum HBV DNA;
Fig. 4 (b) is the fluorescence quantitative PCR detection result (10IU/ml) of serum HBV DNA;
Fig. 4 (c) is the fluorescence quantitative PCR detection result (5IU/ml) of serum HBV DNA;
Fig. 5 (a) is fluorescence quantitative PCR detection result's (magnetic bead of the present invention) of serum HBV DNA;
Fig. 5 (b) is fluorescence quantitative PCR detection result's (Dynal magnetic bead) of serum HBV DNA;
Fig. 5 (c) is fluorescence quantitative PCR detection result's (Carboxylate speed magnetic bead) of serum HBV DNA;
Fig. 5 (d) is fluorescence quantitative PCR detection result's (Carboxylate magnetic bead) of serum HBV DNA;
Fig. 6 is the fluorescence quantitative PCR detection result of HCV RNA in the serum.
Among Fig. 2 Fig. 6: X-coordinate is the amplification cycles number, and ordinate zou is a fluorescent signal.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail:
Embodiment 1: HBV DNA in peptide-oligonucleotide magnetic bead separating and purifying serum
Present embodiment may further comprise the steps: (1) peptide-oligonucleotides-modified magnetic bead: with γ Fe
2O
3And Fe
3O
4Magneticsubstance synthetic homogeneous, super paramagnetic, single diversity poly microballoon are the magnetic source, and each microsphere bag is carried the magnetic macromolecular microsphere of carboxylate salt by one deck polystyrene by letex polymerization or the preparation of diffuse-aggregate method; Through polystyrene core particle surface cover one deck magnet that carboxylate salt is modified, oligo (dT)
14Covalently bind on this surface, nucleic acid molecule can be good at being anchored on [referring to Fig. 1 (a)] on this oligonucleotide again; (2) lysis: HBV DNA positive serum 200 μ l are added the 1.5ml centrifuge tube, add 300 μ l lysates subsequently, lysate is made up of the GuScN mixing of Triton X-100, the 1mol/L of the SDS, 2% (V/V) of 0.5% (W/V), and mixing is to discharge nucleic acid; (3) nucleic acid absorption: after simple centrifugal, add 100 μ l through peptide-oligonucleotides-modified magnetic bead solution, adding by HEPES, the 200mmol/L NaCl of 200mmol/LpH6.5 ± 0.2,200 μ g/ml through peptide-oligonucleotides-modified magnetic bead solution, the concussion mixing, nucleic acid is adsorbed by magnetic bead surfaces, protein and other impurity then are not adsorbed, simply centrifugal behind the room temperature placement 10min, centrifuge tube is placed magnetic separator, treat behind the 5min that magnetic bead is adsorbed in centrifugal tube wall fully, from centrifuge tube bottom slowly with the supernatant liquor sucking-off; (4) remove impurity: add 600 μ l water washingss (NaCl of Triton X-100, the 200mmol/L of 0.2% (V/V) forms), the concussion mixing, magnetic bead is suspended fully, after simple centrifugal, sample hose is placed the magnetic bead separator, treat that magnetic bead is adsorbed in test tube wall fully, with the complete sucking-off of liquid, realize that other impurity are rinsed separation from the test tube bottom; (5) nucleic acid reclaims: add 100 μ l 10mM Tris-HCl, and 1mMEDTA, the elutriant that pH8.0 forms is inhaled with liquid-transfering gun and to be beaten magnetic bead, makes DNA and magnetic bead desorb, and enters [described (2)-(5) go on foot referring to Fig. 1 (b)] in the elutriant.
The centrifuge tube that magnetic bead and elutriant are housed is placed magnetic separator again, and magnetic bead is adsorbed on centrifugal tube wall, and 100 μ l elutriants and DNA are wherein all changed in the 100 μ l slit cuvettes, uses ultraviolet spectrophotometer to measure DNA purity.Promptly measure the absorbancy of above-mentioned dna solution at 260nm and 280nm.DNA absorbs the UV-light at 260nm place, and protein adsorption 280nm place.Purer DNA sample should not contain protein substantially, and 260/280 absorbance ratio should be higher, pure DNA OD among water or the TE
260/ OD
280Ratio is generally 1.7～1.9.Its OD of DNA of utilization present method separation and purification
260/ OD
280Ratio is 1.84 (OD
260=0.046, OD
280=0.025).
Embodiment 2: the separation and purification of serum HBV DNA and fluorescence quantitative PCR detection
According to embodiment 1 described method, after absorption, the washing, get the complete wash-out of magnetic bead that 50 μ l PCR reaction solutions will be adsorbed in centrifugal tube wall repeatedly several times with pipettor, be transferred in the PCR reaction tubes, the utilization quantitative real time PCR Instrument carries out detection by quantitative.
1) the HBV DNA positive serum of 3 kinds of concentration of demarcating with Chinese biological goods calibratings institute (middle inspection institute) hepatitis B virus (HBV) nucleic acid quantification standard substance is a sample to be tested, and concentration is respectively 5.0 * 10
6IU/ml, 5.0 * 10
4IU/ml, 5.0 * 10
2IU/ml, each concentration repeated experiments 8 times, the result is as shown in the table.
As shown in Figure 2, use method separation and purification nucleic acid of the present invention, good reproducibility is even different people operation also can obtain consistent result.Thereby guaranteed the stable of subsequent experimental, difference and the mistake of avoiding manual operation to cause.
2) the HBV strong positive serum of demarcating with middle inspection institute hepatitis B virus (HBV) nucleic acid quantification standard substance is initial sample, is diluted to 2.0 * 10
9IU/ml, downward successively then gradient dilution to 2.0 * 10
8IU/ml, 2.0 * 10
7IU/ml, 2.0 * 10
6IU/ml, 2.0 * 10
5IU/ml, 2.0 * 10
4IU/ml, 2.0 * 10
3IU/ml, 200IU/ml, 20IU/ml, each concentration repeats twice, and as Fig. 3, the linear dependence of the typical curve of 9 concentration gradient standard substance formulations is 0.998 thus, and the quantitative scope of setting-out line can reach 2.0 * 10
9IU/ml～2.0 * 10
1IU/ml.
Get low concentration sample 2.0 * 10
3IU/ml is diluted to 20IU/ml, 10IU/ml, and 5IU/ml, each concentration repeats 8 times, as Fig. 4 (a) and (b), (c), 20IU/ml, the amplification of 10IU/ml equal 100%, and only 12.5% the sample of 5IU/ml does not have amplification.
The DNA of utilization separation and purification of the present invention carries out fluorescence quantitative PCR detection wide, the highly sensitive characteristics that have linearity range.Even the amount of sample to be tested amplifying nucleic acid is extremely low, also can obtain separation and purification by method of the present invention.
3) be sample to be tested with the linear sensitivity reference material in the HBV national standard product, (concentration is respectively 5.0 * 10 with the positive criteria product of gradient dilution
6IU/ml, 5.0 * 10
5IU/ml, 5.0 * 10
4IU/ml, 5.0 * 10
3IU/ml) adopt method separation and purification nucleic acid of the present invention and use fluorescence quantitative PCR detection, the accordance of analyzing and testing value and theoretical value.
| Linear sensitivity reference material | Quality standard (IU/m1) theoretical concentration logarithmic value | Detected value | The detectable level logarithmic value |
| ????L0 | ?7.762×10 7～6.165×10 8?8.342 | ?2.36×10 8 | ?8.372 |
| ????L1 | ?1.479×10 7～1.175×10 8?7.619 | ?2.96×10 7 | ?7.471 |
| ????L2 | ?1.585×10 6～1.259×10 7?6.649 | ?3.19×10 6 | ?6.503 |
| ????L3 | ?1.659×10 5～1.318×10 6?5.672 | ?3.63×10 5 | ?5.559 |
| ????L4 | ?1.820×10 4～1.479×10 5?4.715 | ?4.18×10 4 | ?4.621 |
| ????L5 | ?1.514×10 3～1.230×10 4?3.636 | ?3.06×10 3 | ?3.485 |
| ????L6 | ?3.890×10 1～3.090×10 2?2.043 | ?6.28×10 1 | ?1.797 |
The double logarithmic curve linearly dependent coefficient (r) of theoretical concentration and detectable level is 0.9997, as seen uses the nucleic acid free of losses of this method separation and purification, the extraction efficiency height, and it is accurate to be applied to follow-up quantitative experiment result.
Embodiment 3: peptide of the present invention-oligonucleotide magnetic bead HBV DNA extraction and the comparison of existing paramagnetic particle method separation and purification nucleic acid in the market
The magnetic bead of three kinds of different treatment modes of utilization with of the present invention through the acid-treated magnetic bead of peptide-oligonucleoside to serum HBV DNA separation and purification and carry out fluorescence quantitative PCR detection comparison.Three kinds of magnetic beads are respectively: Invetrogen Dynal magnetic bead, Carboxylate (carboxylate salt) speed magnetic bead, Carboxylate magnetic bead.
The HBV positive serum of demarcating with middle inspection institute hepatitis B virus (HBV) nucleic acid quantification standard substance is initial sample, gradient dilution to 10
5IU/ml, 10
4IU/ml, 10
3IU/ml, 10
2IU/ml, 10IU/ml, 5 positive serums and a negative serum adopt above-mentioned three kinds of magnetic beads and peptide oligonucleotide magnetic bead of the present invention according to the method for embodiment 1 HBV DNA to be carried out separation and purification as sample to be tested.Utilization ABI7300 carries out fluorescence quantitative PCR detection to above-mentioned isolating nucleic acid.
Be respectively magnetic bead of the present invention as Fig. 5 (a) and (b), (c), (d), Dynal magnetic bead, Carboxylatespeed magnetic bead, the fluorescence quantitative PCR detection figure of Carboxylate magnetic bead separation and purification DNA.The detected result of Carboxylate speed and Carboxylate magnetic bead is approximate, and the Dynal magnetic bead is 10
3Can't detect Ct below the IU/ml, the DNA of these three kinds of magnetic bead separation and purification all has more loss, and productive rate is not high, thereby the sample of low concentration utilization quantitative fluorescent PCR can't detect.And the DNA productive rate of peptide oligonucleotide magnetic bead of the present invention separation and purification is higher, free of losses, but detectable level is low to moderate the sample of 10IU/ml.
Embodiment 4: the separation and purification of HCV RNA and fluorescence quantitative PCR detection in the serum
With known HCVRNA concentration is 5 * 10
5The serum sample of IU/ml is diluted to 5 * 10
4IU/ml, 5 * 10
3IU/ml, 5 * 10
2IU/ml, 50IU/ml, 25,12.5 and three hepatitis C sera negative are as sample to be tested.1.5ml add hepatitis C ribonucleic (HCVRNA) positive serum 200 μ l in the centrifuge tube, the GuScN, 200 μ g/ml magnetic beads (oligonucleotides-modified with peptide) of Triton X-100,1mol/L that adds the LLS, 2% (V/V) of 600 μ l extracting solutions 1: 0.5% (W/V) subsequently mixes and forms mixing; Simple centrifugal back adds 100 μ l extracting solutions 2, and present embodiment extracting solution 2 is made up of HEPES (pH6.5 ± 0.2), the 200mmol/L NaCl of 200mmol/L; Concussion mixing 10s is simply centrifugal behind the room temperature placement 10min.Sample tube is placed magnetic separator, treats behind the 5min that magnetic bead is adsorbed in test tube wall fully, from test tube bottom slowly with the supernatant liquor sucking-off; Add 600 μ l washingss (TritonX-100 of 0.2% (V/V), the KCl of 200mmol/L form), the concussion mixing suspends magnetic bead fully, simple sample hose is placed the magnetic bead separator after centrifugal, treats that magnetic bead is adsorbed in test tube wall fully, from the test tube bottom with the complete sucking-off of liquid.Get the complete wash-out of magnetic bead that 50 μ lPCR reaction solutions will be adsorbed in tube wall repeatedly several times with pipettor, be transferred in the PCR reaction tubes, the utilization quantitative real time PCR Instrument carries out detection by quantitative.
The PCR response procedures is:
Claims (10)
1. one kind is adopted magnetic bead to extract the also method of purification of nucleic acid from sample, it is characterized in that, may further comprise the steps:
(1) the oligonucleotides-modified magnetic bead of peptide:
With γ Fe2O3 and Fe3O4 magneticsubstance synthetic homogeneous, super paramagnetic, single diversity poly microballoon is the magnetic source, each microsphere bag is carried the magnetic macromolecular microsphere of carboxylate salt by one deck poly material by letex polymerization or the preparation of diffuse-aggregate method; Through poly material core particle surface cover one deck magnet that carboxylate salt is modified, oligo (dT) 14 covalently bind on this surface again, and nucleic acid molecule can be good at being anchored on this oligonucleotide;
(2) lysis: the sample to be separated that will contain target nucleic acid adds in the centrifuge tube, adds the lysate lysing cell again, to discharge nucleic acid;
(3) nucleic acid absorption: add the oligonucleotides-modified magnetic bead of peptide in solution, nucleic acid is adsorbed by magnetic bead surfaces, and protein and other impurity then are not adsorbed; Centrifuge tube is placed magnetic separator, and magnetic bead is adsorbed in test tube wall;
(4) remove impurity: add the water damping fluid, the magnetic bead that the surface is adsorbed with nucleic acid and is attached to test tube wall washs, and the flushing of the impurity on the magnetic bead is separated;
(5) nucleic acid reclaims: the nucleic acid that is adsorbed on magnetic bead surfaces perhaps adds elutriant directly as the template of pcr amplification, makes the nucleic acid molecule desorb that is adsorbed on magnetic bead surfaces, enters in the elutriant, is used further to the downstream experiment.
2. employing magnetic bead according to claim 1 extracts from sample and the method for purification of nucleic acid, it is characterized in that, described magnetic bead surfaces is modified with different aglucons, as absorption with in conjunction with the carrier of corresponding molecule.
3. employing magnetic bead according to claim 2 extracts the also method of purification of nucleic acid from sample, it is characterized in that described aglucon is carboxylate salt and oligonucleotide.
4. employing magnetic bead according to claim 1 extracts the also method of purification of nucleic acid from sample, it is characterized in that described nucleic acid is two strands or single stranded DNA, RNA or nucleic acid analog.
5. employing magnetic bead according to claim 1 and 2 extracts the also method of purification of nucleic acid from sample, it is characterized in that described lysate is the solution that contains tensio-active agent.
6. employing magnetic bead according to claim 5 extracts the also method of purification of nucleic acid from sample, it is characterized in that the described solution that contains tensio-active agent is the solution that contains 0.01%～10% SDS, LLS, Chelex-100, TritonX-100, Tween 20 or NP-40.
7. employing magnetic bead according to claim 1 and 2 extracts the also method of purification of nucleic acid from sample, it is characterized in that, described magnetic bead solution is meant pH6.5 ± 0.2, by the HEPES of 100mmol/L～2M/L and the buffered soln that 100mmol/L～2M/L NaCl constitutes.
8. employing magnetic bead according to claim 1 and 2 extracts the also method of purification of nucleic acid from sample, it is characterized in that described water damping fluid is by NaCl, the KCl of Triton X-100, the 100mmol/L～2M/L of 0.1% to 2% (volume ratio) or the solution that LiCl forms.
9. employing magnetic bead according to claim 1 and 2 extracts the also method of purification of nucleic acid from sample, it is characterized in that described elutriant is 10mM Tris-HCl and 1mM EDTA, the solution of pH8.0.
10. one kind is adopted magnetic bead to extract from sample and the application in diagnosis, forensic analysis, tissue and the blood grouping of pathogenic micro-organism, detection in Gene Mutation, gene sequencing of nucleic acid that the method for purification of nucleic acid is extracted as claimed in claim 1 or 2.
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