CN111073885A - Purification method applied to double-stranded DNA fragment - Google Patents
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Abstract
The invention provides a purification method applied to double-stranded DNA fragments, belonging to the technical field of biochemistry and molecular biology. The invention aims to establish a set of method for separating and purifying double-stranded linear DNA molecules with the difference of 3000bp, so as to be beneficial to the development of the later gene therapy field. According to the characteristics that nucleic acid molecules have negative charges under neutral and large pH conditions, the method adopts anion exchange chromatography to adsorb nucleic acid on a filler, and a target fragment can be obtained by elution; the invention separates and purifies the double-stranded DNA fragments after enzyme digestion by optimizing the process route, can carry out process amplification, can be produced under the GMP condition, and is beneficial to promoting the rapid and standard development of the gene therapy industry. The invention provides a new thought, a simple and effective method for the current situation that few methods for separating and purifying double-stranded DNA fragments after enzyme digestion exist at present, and particularly provides a scheme for separating and purifying double-stranded DNA fragments within 3000bp of each other after enzyme digestion.
Description
Technical Field
The invention relates to the technical field of biochemistry and molecular biology, in particular to a purification method applied to double-stranded DNA fragments.
Background
Plasmids (plasmids) are widely present in the kingdom of life, ranging from bacteria, actinomycetes, filamentous fungi, macrofungi, yeasts to plants and even in the human body. The bacterial plasmid is a closed circular double-stranded DNA molecule except chromosome (or karyoplast) in organisms such as bacteria, microzyme, actinomycetes and the like, has the size of 1kb to 200kb, has the autonomous replication capability, can stably express carried genetic information, and is the most commonly used vector in genetic engineering.
At present, the technology for separating the plasmid with larger molecular weight difference is mature, but the process for separating and purifying the double-stranded DNA fragment after enzyme digestion is rarely explored and optimized, and particularly the plasmid with the difference of two fragments within 3000bp after enzyme digestion is separated and purified, so that the technology has good exploration and application space and is beneficial to the development of the later gene therapy field.
Disclosure of Invention
The invention aims to establish a method for simply separating and purifying double-stranded linear DNA molecules with molecular weight difference within 3000bp after enzyme digestion so as to be beneficial to the development of the later gene therapy field.
In order to achieve the purpose, the invention adopts the following technical scheme:
a purification method applied to double-stranded DNA fragments is completed by one-step chromatography, and the used raw materials and the operation parameters are as follows:
a chromatography system: AKTA Pure 150;
size of the chromatographic column: 21.2 x 150 mm;
flow rate: 3-6 mL/min;
mobile phase A-balance buffer solution and mobile phase B-sample loading eluent;
balancing: until the baseline is stable;
loading: 80mL of enzyme digestion product;
wash: 2-4 CVs;
linear elution: 28% -83% (mobile phase B) 25CV wash.
The technical scheme disclosed by the invention can separate and purify the fragments after enzyme digestion by a process route method, can complete process amplification and simultaneously achieves GMP conditions.
Furthermore, the filler used by the chromatographic column is GE SOURCE 30Q, the framework adopts polystyrene, the flow rate is high in the application process, the stability is good, and the active groups are as follows: R-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3Can effectively combine the groups with negative charges, has smaller average grain diameter of 30 mu m and better resolution.
Further, the baseline is stable until the ultraviolet value is fixedly changed at 0.5-1 mAu.
Further, the equilibration buffer: pH6.5, 50mM Tris-HCl, 1mM EDTA; the loading eluent comprises: pH6.5, 50mM Tris-HCl, 1mM EDTA, 2M NaCl.
Further, the buffer has a high pH and forms an environment with a pH higher than the isoelectric point of the nucleic acid, so that the nucleic acid has a net negative charge and can be adsorbed by the filler. The nucleic acids with different lengths have different charge and different adsorption strengths, and are sequentially dissociated, purified and separated.
Further, the enzyme digestion product is a double-stranded linear DNA molecule with the molecular weight difference within 3000 bp.
Furthermore, the enzyme digestion product is a double-stranded linear DNA molecule with the molecular weight difference of 500-3000 bp.
Further, the enzyme digestion product is subjected to ultrafiltration, concentration and sterilization treatment, and endotoxin is removed by a PVDF filter membrane.
Further, the chromatography is anion exchange chromatography, nucleic acid is adsorbed on a filler, and a target fragment is obtained through elution.
Furthermore, the error linear curve is drawn after the chromatography process, and the method comprises the following steps:
the concentration of the double-stranded DNA fragment is determined by using nanodrop2000, then the double-stranded DNA fragment is diluted to 10 mu g/mL, 20 mu g/mL, 50 mu g/mL, 200 mu g/mL, 500 mu g/mL and 1000 mu g/mL by using TEbuffer, and peak areas are measured to make a linear curve, wherein the used raw materials and reagents are as follows:
a chromatographic column: TSKgel DNA-NPR chromatography column;
reagent: mobile phase a-equilibration buffer (20mM Tris-HCl, ph 9.0);
mobile phase B-elution buffer (20mM Tris-HCl, 1M NaCl, ph 9.0);
0-100% elution;
flow rate: 1 mL/min;
linear elution: 0 → 55% B, 22CV, 55% -65% B, 20 CV.
Further, the elution process is to cause the shedding of nucleic acids with smaller length first by the difference of the adsorption strength of nucleic acids.
Further, the Wash: 4 CV of; the linear elution is as follows: 32% -78% (mobile phase B) of 26 CV.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
according to the characteristics that nucleic acid molecules have negative charges under neutral and large pH conditions, the method adopts anion exchange chromatography to adsorb nucleic acid on a filler, and a target fragment can be obtained by elution; the invention can separate and purify the fragments after enzyme digestion by a process route method, can carry out process amplification, can reach GMP conditions, has the isoelectric point of nucleic acid of about 2-4, has net negative charges on the nucleic acid in an environment with higher pH, can be adsorbed by the filler, has different nucleic acid charge amounts on the nucleic acids with different lengths, has different adsorption capacities on the filler, has firm combination with large charge amount, can be eluted only by higher charge displacement amount, can be sequentially eluted on the nucleic acid fragments with different lengths, achieves the separation effect, is beneficial to the development of the whole industry, has simple and effective process, and can achieve the effect by one-step chromatography.
Drawings
FIG. 1 is a graph showing the linear curve of plasmid concentration versus peak area error in example 1;
FIG. 2 is a chromatogram of a conventional method for separating plasmids having widely different molecular weights;
FIG. 3 is a gel map of the isolated plasmid of FIG. 2;
FIG. 4 is a spectrum obtained by the prior art after the plasmid is digested and purified;
FIG. 5 is a photograph of the gel electrophoresis of FIG. 4;
FIG. 6 is a chromatogram of a plasmid separation experiment after enzyme digestion according to the present invention;
FIG. 7 is a chromatogram of the replicate of FIG. 6;
FIG. 8 is a photograph of the gel electrophoresis of FIG. 6.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The error linear curve is drawn after the chromatographic process of the purification method, and the method comprises the following steps:
the concentration of the double-stranded DNA fragment is determined by using nanodrop2000, then the double-stranded DNA fragment is diluted to 10 mu g/mL, 20 mu g/mL, 50 mu g/mL, 200 mu g/mL, 500 mu g/mL and 1000 mu g/mL by using TEbuffer, and peak areas are measured to make a linear curve, wherein the used raw materials and reagents are as follows:
a chromatographic column: TSKgel DNA-NPR chromatography column;
reagent: mobile phase a-equilibration buffer (20mM Tris-HCl, ph 9.0);
mobile phase B-elution buffer (20mM Tris-HCl, 1M NaCl, ph 9.0);
0-100% elution;
flow rate: 1 mL/min;
linear elution: 0 → 55% B, 22CV, 55% -65% B, 20 CV.
A method for purification of double-stranded DNA fragments, comprising the steps of:
anion exchange chromatography is carried out on double-stranded linear DNA molecules with molecular weight difference within 3000bp, and the used materials and the operation parameters are as follows:
a chromatography system: AKTA Pure 150;
a chromatographic column: GE SOURCE 30Q (21.2 × 150 mm);
flow rate: 3-6 mL/min;
mobile phase A-equilibration buffer (pH 6.550 mM Tris-HCl, 1mM EDTA);
mobile phase B-loading eluent (PH 6.550 mM Tris-HCl, 1mM EDTA, 2M NaCl);
balancing: to baseline plateau (to a fixed change in uv value of 0.5-1 mAu);
loading: 80mL of enzyme digestion product;
wash: 3 CV of;
linear elution: 28% -83% (mobile phase B) 25CV wash.
The utensils are baked to remove heat source and sterilized.
Example 1
The concentration of the double-stranded DNA fragment is determined by using nanodrop2000, then the double-stranded DNA fragment is diluted to 10 mu g/mL, 20 mu g/mL, 50 mu g/mL, 200 mu g/mL, 500 mu g/mL and 1000 mu g/mL by using TEbuffer, and peak areas are measured to make a linear curve, wherein the used raw materials and reagents are as follows:
a chromatographic column: TSKgel DNA-NPR chromatography column;
reagent: mobile phase a-equilibration buffer (20mM Tris-HCl, ph 9.0);
mobile phase B-elution buffer (20mM Tris-HCl, 1M NaCl, ph 9.0);
0-100% elution;
flow rate: 1 mL/min;
linear elution: 0 → 55% B, 22CV, 55% -65% B, 20 CV.
The resulting linear curve is shown in FIG. 1.
A purification method applied to double-stranded DNA fragments is completed by one-step chromatography, and the used raw materials and the operation parameters are as follows:
specifically, the chromatography system: AKTA Pure 150;
specifically, the size of the column: 21.2 x 150 mm;
specifically, the flow rate: 3-6 mL/min;
specifically, mobile phase A-equilibrium buffer solution and mobile phase B-loading eluent;
specifically, balancing: until the baseline is stable;
specifically, loading: 80mL of enzyme digestion product;
specifically, Wash: 2-4 CVs;
specifically, linear elution: 28% -83% (mobile phase B) of 26 CV.
Wherein, the filler used by the chromatographic column is GE SOURCE 30Q, the flow rate is high in the application process, the stability is good, and the active groups are as follows: R-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3Can effectively combine the groups with negative charges, has smaller average grain diameter of 30 mu m and better resolution.
Wherein the baseline is stable until the ultraviolet value is fixedly changed at 0.5-1 mAu.
Wherein the equilibration buffer: pH6.5, 50mM Tris-HCl, 1mM EDTA; the loading eluent comprises: pH6.5, 50mM Tris-HCl, 1mM EDTA, 2M NaCl.
Wherein the buffer has a high pH value and forms an environment with a pH higher than the isoelectric point of the nucleic acid, so that the nucleic acid has a net negative charge and can be adsorbed by the filler. The nucleic acids with different lengths have different charge and different adsorption strengths, and are sequentially dissociated, purified and separated.
Wherein the enzyme digestion product is a double-stranded linear DNA molecule with the molecular weight difference within 3000 bp.
Wherein the enzyme digestion product is subjected to ultrafiltration, concentration and sterilization treatment, and endotoxin is removed by a PVDF filter membrane.
Wherein, the chromatography is anion exchange chromatography, nucleic acid is adsorbed on a filler, and a target fragment is obtained by elution.
Wherein, the elution process is that nucleic acid with smaller length is firstly shed due to the difference of nucleic acid adsorption strength.
Wherein, the Wash: 4 CV of; the linear elution is as follows: 32% -78% (mobile phase B) of 26 CV.
The utensils are baked to remove heat source and sterilized.
The checking process of the result obtained by the purification method comprises the following steps:
(1) pre-experiment: the enzyme digestion system verifies the enzyme digestion effect of the enzyme digestion system with 1 mu g of DNA and 10units, and if the effect is expected, the subsequent steps are carried out:
s1, preliminarily carrying out enzyme digestion on 1mg of plasmid;
s2 balance: equilibrating the chromatographic column GE SOURCE 30Q with equilibration buffer;
s3, sample loading: loading 1mg of plasmid;
s4, Wash: equilibration buffer equilibrates 2CV of column volume;
s5, elution: the gradient elution of 51%, 52%, 53%, 54%, 55% was verified;
s6, collecting samples: collecting two elution peaks;
s7, detection: gel detection is carried out to determine whether the size is combined with the expectation or not and whether the purity is combined with the expectation or not;
equilibration buffer used: pH 8.010 mM Tris-HCl, 1mM EDTA, 0.4M NaCl;
elution buffer used: pH 8.010 mM Tris-HCl, 1mM EDTA, 1M NaCl;
and (3) sterilization: and (5) drying the reagent bottle to remove a heat source.
The solution used subsequently and the pre-experimental solution are in the same preparation batch.
(2) Amplifying and loading:
s1, enzyme digestion of 5mg plasmid loading:
verifying the enzyme digestion effect by gradient elution of 51%, 52%, 53%, 54% and 55%, if the effect is in accordance with the expectation, carrying out the subsequent step test, and if the effect is not in accordance with the expectation, collecting samples by adopting a linear gradient elution mode of 50-60% and 5 CV;
s2, enzyme digestion of 10mg plasmid loading:
the enzyme digestion effect is verified by gradient elution of 51%, 52%, 53%, 54% and 55%, and if the effect is in accordance with the expectation, the subsequent step test is carried out;
s3, enzyme digestion of 20mg plasmid loading:
the enzyme digestion effect is verified by gradient elution of 51%, 52%, 53%, 54% and 55%, and if the effect is in accordance with the expectation, the subsequent step test is carried out;
determining whether to continue purifying according to the amount of the received nutrient, and then carrying out the following steps;
(3) and (3) ultrafiltration concentration:
centrifuging the sample after multiple purification for 10min with a 50mL centrifuge tube 8000prm/min with 10KD for ultrafiltration to change the liquid;
the method comprises the following steps: removing endotoxin (Kejing), and sterilizing with 0.22 μm filter (Kejing).
(3) And (3) detecting the purity, wherein the raw materials and parameters are as follows:
a chromatographic column: TSKgel DNA-NPR;
flow rate: 1 mL/min;
and (3) detection: a wavelength of 254 nm;
mobile phase a-equilibration buffer (20mM Tris-HCl, ph 9.0);
mobile phase B-elution buffer (20mM Tris-HCl, 1M NaCl, ph 9.0);
loading: 100 μ L, (enzyme cleavage product: a ═ 1: 1);
linearity: 0-55%, 22CV, 55 → 65% B, 20 CV;
(4) and (3) detecting the concentration, wherein the raw materials and parameters are as follows:
a chromatographic column: TSKgel DNA-NPR;
flow rate: 1 mL/min;
and (3) detection: a wavelength of 254 nm;
mobile phase a-equilibration buffer (20mM Tris-HCl, ph 9.0);
mobile phase B-elution buffer (20mM Tris-HCl, 1M NaCl, ph 9.0);
loading: 50 μ L, (enzyme digestion product: a ═ 1: 1);
linearity: 50 → 65% B, 10 CV;
(5) and (3) gel detection: preparing 1% gel to detect the purity;
(6) and (3) detecting endotoxin: transferring QC detection;
(7) and (3) sterility detection: and transferring QC detection.
The obtained result meets the requirements of separation and purification.
The existing purification method and the purification method provided by the invention are used for carrying out related experiments, and the obtained spectrogram and the result are as follows:
referring to FIG. 1, an error curve is determined, the concentration quantitative relationship of nucleic acid fragments is labeled, according to the spectrum, the peak area is calculated after loading, and the concentration of nucleic acid can be calculated by substituting into a formula;
referring to FIG. 2, the separation effect of plasmids with different sizes under the prior art can separate plasmids with different molecular weights;
please refer to fig. 3, which is a gel map of the plasmid separation of fig. 2, wherein sample No. 1 is two mixed plasmids to be purified, sample No. 2 is a plasmid control gel electrophoresis band with a larger molecular weight, sample No. 3 is a plasmid with a larger molecular weight after purification, sample No. 4 is a plasmid with a smaller molecular weight after purification, and sample No. 5 is a plasmid control gel result with a smaller molecular weight. The results show that plasmid purification and separation with different molecular weights can be carried out;
referring to fig. 4, a map obtained by performing purification and separation after plasmid digestion in the prior art shows that an absorption peak appears, which indicates that effective separation of fragments is not completed and the obtained effect is poor;
please refer to fig. 5, which is a gel result of fig. 4, wherein the first band from the left of Maker is a plasmid control, the second band is a gel result of the collected peak after purification, and the bands can be visually judged not to be separated according to the band position;
from fig. 2 to 5, it can be seen that in the prior art, separation is mainly performed on a larger plasmid, the obtained effect is more remarkable, a better effect can be achieved, but the effect on a smaller plasmid such as a plasmid after enzyme digestion is separated is poor.
Referring to fig. 6, according to the technical scheme provided by the present invention, after the plasmid is digested, the plasmid is purified, and the two purification peaks are divided into two fragments with smaller molecular weight and two fragments with larger molecular weight, so that the separation effect is good;
referring to fig. 7, the same scheme as that used in fig. 6 is repeated, so that the separation effect is good, and the reproducibility is also good, which indicates that the purification method provided by the present invention is stable and effective;
referring to FIG. 8, the two target products collected after purification in FIG. six were subjected to gel electrophoresis, the first is the electrophoresis result after plasmid digestion, and the second two are the fragments purified after digestion, and are divided into two fragments.
In conclusion, the nucleic acid fragment of less than 2000bp can be well separated by purifying according to the method disclosed by the invention.
Is different from the prior art: according to the characteristics that nucleic acid molecules have negative charges under neutral and large pH conditions, the method adopts anion exchange chromatography to adsorb nucleic acid on a filler, and a target fragment can be obtained by elution; the invention can separate and purify the fragments after enzyme digestion by a process route method, can carry out process amplification, can reach GMP conditions, is beneficial to the development of the whole industry, has simple and effective process, and can achieve the effect by one-step chromatography.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A purification method applied to double-stranded DNA fragments is characterized in that the method is completed by one-step chromatography, and the used raw materials and the operation parameters are as follows:
a chromatography system: AKTA Pure 150;
size of the chromatographic column: 21.2 x 150 mm;
flow rate: 3-6 mL/min;
mobile phase A-balance buffer solution and mobile phase B-sample loading eluent;
balancing: until the baseline is stable;
loading: 80mL of enzyme digestion product;
wash: 2-4 CVs;
linear elution: washing with 28% -83% (mobile phase B) for 23-28 CV.
2. The method of claim 1, wherein the packing material used in the column is GE SOURCE 30Q, and the column has high flow rate, good stability, and active group R-O-CH2-CHOH-CH2-O-CH2-CHOH-CH2-N+(CH3)3Can effectively combine the groups with negative charges, has the average particle size of 30 mu m and ensures good resolution.
3. The purification method applied to double-stranded DNA fragments as claimed in claim 1, wherein the baseline is stabilized to a constant change of UV value of 0.5-1 mAU.
4. The method of claim 1, wherein the equilibration buffer: p H6.5.5, 50mM Tris-HCl, 1mM EDTA; the loading eluent comprises: pH6.5, 50mM Tris-HCl, 1mM EDTA, 2M NaCl.
5. The method as claimed in claim 4, wherein the buffer solution has a higher pH value, and forms an environment with a pH higher than the isoelectric point of the nucleic acid, so that the nucleic acid has a net negative charge and can be adsorbed by the filler, and the nucleic acids with different lengths have different charge and different adsorption strengths, and are sequentially dissociated, purified and separated.
6. The method for purifying double-stranded DNA fragments according to claim 1, wherein the enzyme-cleaved product is a double-stranded linear DNA molecule having a molecular weight difference of 3000 bp.
7. The purification method applied to the double-stranded DNA fragments as claimed in claim 1, wherein the enzyme digestion product is subjected to ultrafiltration concentration sterilization treatment, and endotoxin is removed by a PVDF filter membrane; the chromatography is anion exchange chromatography, nucleic acid is adsorbed on a filler, and a target fragment is obtained by elution.
8. The method for purifying double-stranded DNA fragments as claimed in claim 1, wherein the error linear curve is drawn after the chromatography process, comprising the following steps:
the concentration of the double-stranded DNA fragment is determined by using nanodrop2000, then the double-stranded DNA fragment is diluted to 10 mu g/mL, 20 mu g/mL, 50 mu g/mL, 200 mu g/mL, 500 mu g/mL and 1000 mu g/mL by using TEbuffer, and peak areas are measured to make a linear curve, wherein the used raw materials and reagents are as follows:
a chromatographic column: TSKgel DNA-NPR chromatography column;
reagent: mobile phase a-equilibration buffer (20mM Tris-HCl, ph 9.0);
mobile phase B-elution buffer (20mM Tris-HCl, 1M NaCl, ph 9.0);
0-100% elution;
flow rate: 1 mL/min;
linear elution: 0 → 55% B, 22CV, 55% -65% B, 20 CV.
9. The method according to claim 7, wherein the elution is caused by the difference in nucleic acid adsorption strength such that smaller length nucleic acids are shed first.
10. The purification method applied to double-stranded DNA fragments according to claim 1, wherein the Wash: 4 CV of; the linear elution is as follows: 32% -78% (mobile phase B) of 26 CV.
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Citations (5)
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| CN1243542A (en) * | 1997-01-10 | 2000-02-02 | 罗切诊断学有限公司 | Method for purifying DNA in cross-flow centrifuge |
| US20030165876A1 (en) * | 2001-03-23 | 2003-09-04 | Francis Blanche | Processes for purifying and for detecting target double-stranded DNA sequences by triple helix interaction |
| US20040038278A1 (en) * | 2002-08-12 | 2004-02-26 | George Tzertzinis | Methods and compositions relating to gene silencing |
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| CN1243542A (en) * | 1997-01-10 | 2000-02-02 | 罗切诊断学有限公司 | Method for purifying DNA in cross-flow centrifuge |
| US20030165876A1 (en) * | 2001-03-23 | 2003-09-04 | Francis Blanche | Processes for purifying and for detecting target double-stranded DNA sequences by triple helix interaction |
| US20040038278A1 (en) * | 2002-08-12 | 2004-02-26 | George Tzertzinis | Methods and compositions relating to gene silencing |
| US20050014245A1 (en) * | 2003-05-30 | 2005-01-20 | Advisys, Inc. | Devices and methods for biomaterial production |
| US20130237699A1 (en) * | 2010-11-09 | 2013-09-12 | Qiagen Gmbh | Method and device for isolating and purifying double-stranded nucleic acids |
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| GENERAL ELECTRIC COMPANY: "SOURCE 30Q and 30S", 《GE HEALTHCARE》 * |
| RANJIT R. DESHMUKH ET AL.: "A CASE STUDY: OLIGONUCLEOTIDE PURIFICATION FROM GRAM TO HUNDRED GRAM SCALE", 《NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS》 * |
| 廖杰 等: "高效液相色谱非多孔型阴离子交换柱分析DNA片段", 《色谱》 * |
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