Plasmid DNA extraction method based on silicon dioxide particles and kit

Abstract

The invention discloses a method and a kit for extracting plasmid DNA (deoxyribonucleic acid) based on silicon dioxide particles. The kit comprises the silicon dioxide particles, a neutralizing solution and chaotropic salt, the silicon dioxide particles are spherical silicon dioxide particles or irregular silicon dioxide particles with uniform particle sizes of 18 nm to 50 nm; the neutralization solution comprises potassium acetate and guanidine hydrochloride; the chaotropic salt is guanidine hydrochloride. The extraction method comprises the following steps: cracking a bacterial liquid converted with plasmids by adopting an alkali lysis solution; adding potassium acetate and guanidine hydrochloride, upside down and uniformly mixing; centrifuging to remove the precipitate, adding guanidine hydrochloride, upside down and uniformly mixing; adding silicon dioxide particles, and performing rotary incubation; and air-drying the silicon dioxide particles, and eluting with an eluent to obtain the plasmid DNA. According to the method disclosed by the invention, the plasmid DNA with high yield and high purity can be extracted at low cost by utilizing the unmodified silicon dioxide particles, and the extraction can be expanded in equal proportion, so that the method has a relatively good commercial transformation prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nucleic acid extraction, specifically, the present application belongs to the technical field of DNA extraction, more specifically, the present application relates to a method and kit for extracting plasmid DNA using unmodified silica particles (silica beads). BACKGROUND

[0002] Plasmid is a small circular DNA molecule existing in microorganisms such as bacteria and yeast, which can replicate independently of the chromosome. Plasmid DNA (pDNA) is an important tool in molecular cloning, gene expression and genetic engineering technologies. If the extracted pDNA contains impurities such as protein, RNA, genomic DNA and endotoxin, it will seriously inhibit the downstream enzymatic reaction (such as restriction enzyme digestion, ligation, transformation and transfection), leading to experimental failure or result deviation; if the extracted pDNA is not pure enough, it will directly affect the transfection efficiency, sequencing accuracy and product safety in gene expression, sequencing and gene therapy. Therefore, high-quality pDNA extraction is the prerequisite for its successful application, and is the key to ensuring the accuracy and repeatability of subsequent research.

[0003] The extraction of plasmid DNA from E. coli mainly includes three core steps: bacterial culture, lysis and plasmid purification. The most commonly used method for lysis is alkaline lysis, and the purification methods mainly include phenol-chloroform extraction, magnetic bead adsorption and silica gel purification. Phenol-chloroform extraction requires multiple centrifugal precipitation, DNA is easy to lose, the steps are tedious and time-consuming, and phenol-chloroform is a volatile toxic reagent, which also limits its application. The magnetic bead method uses the surface of the silica hydroxyl or carboxyl modification to adsorb DNA, which has good repeatability, simple and fast operation, but is expensive and not suitable for large-scale extraction. The silica gel adsorption method mainly uses the combination of DNA and silica oxide (SiO2) under high salt to purify DNA, which is eluted in a low salt environment. According to the form of silica gel, it can be divided into silica gel membrane centrifugal purification column and silica particles (silica beads). The purification column is the most commonly used method in commercial kits, which involves multiple centrifugation in the operation process, and the yield is relatively low due to the adsorption capacity of the silica membrane, and the cost is also relatively high for large-scale extraction. Since Boom et al. first proposed using silica beads as a solid-phase adsorbent for DNA purification, it has been widely used due to its simple, fast and low-cost operation. Researchers have made various optimizations to this method, mainly focusing on silica bead modification, but it has also significantly increased the cost of plasmid DNA extraction.

[0004] At present, the method for extracting plasmid DNA based on unmodified silica particles usually uses irregular mixed particles with a particle size of 0.5-10 μm (such as Sigma S5631, wherein 80% of the particles have a particle size of 1-5 μm). However, the mixed particles with different particle sizes can result in unstable DNA purification quality, such as insufficient purity or too low DNA concentration. SUMMARY

[0005] Therefore, the present application aims to provide a method and a kit for extracting plasmid DNA. The method of the present application uses unmodified silica particles to extract plasmid DNA at low cost, high yield and high purity.

[0006] The technical solutions for achieving the above-mentioned application objectives include the following.

[0007] In a first aspect, the present application provides the use of silica particles in the extraction of plasmid DNA. The silica particles are spherical silica particles or irregular silica particles with a particle size of 18-50 nm and uniform particle size.

[0008] In a second aspect, the present application provides a method for extracting plasmid DNA, which comprises the following steps:

[0009] (1) obtaining a bacterial solution transformed with plasmid, and lysing the bacterial solution with an alkaline lysis solution;

[0010] (2) adding potassium acetate and guanidine hydrochloride to the lysed bacterial solution and mixing well; the final concentration of the potassium acetate is 0.5-2 M, and the final concentration of the guanidine hydrochloride is 0.5-3 M;

[0011] (3) removing the precipitate by centrifugation, adding guanidine hydrochloride and mixing well to obtain a crude plasmid solution;

[0012] (4) adding 0.125-1 mg of silica particles per mL of the bacterial solution to the crude plasmid solution and rotating for incubation; the silica particles are spherical silica particles or irregular silica particles with a particle size of 18-50 nm and uniform particle size;

[0013] (5) centrifuging, rinsing the silica particles, air-drying, adding an eluent for elution, centrifuging and collecting the supernatant to obtain the plasmid DNA.

[0014] In a third aspect, the present application provides a kit for extracting plasmid DNA, which comprises silica particles, a neutralizing solution and a chaotropic salt; the silica particles are spherical silica particles or irregular silica particles with a particle size of 18-50 nm and uniform particle size; the neutralizing solution comprises potassium acetate and guanidine hydrochloride; and the chaotropic salt is guanidine hydrochloride.

[0015] The inventors of the present application find that, when extracting plasmid DNA, using unmodified silica particles with uniform particle size and particle size of 18-50 nm as adsorbent can greatly improve the quality of the extracted plasmid DNA while ensuring the purity of the extracted plasmid DNA, thus realizing high-quality and high-purity extraction of plasmid DNA even without any modification of the silica particles, greatly saving the cost and being suitable for large-scale industrial plasmid DNA extraction.

[0016] In the method for extracting plasmid DNA of the present application, by using unmodified silica particles with uniform particle size and particle size of 18-50 nm, and by optimizing the amount of silica particles and the composition of the neutralizing solution after alkaline lysis of the bacterial solution, the method of the present application can extract plasmid DNA with high purity and yield, achieving similar effects as conventional centrifugal column kit; and the extraction can be scaled up proportionally, and the yield and purity of the plasmid DNA obtained by scaling up the extraction are not much different from those of the plasmid DNA obtained by using conventional silica gel membrane large-scale adsorption column purification method, which can meet the conventional needs of enzyme digestion and cell transfection. Therefore, the method of the present application greatly reduces the cost as the silica particles do not need to be modified, and the obtained DNA has high purity, high yield and good quality, and has good commercial conversion prospects.

[0017] Further, based on the foregoing improvements, by adjusting the final concentration of the chaotropic salt, the time for adsorbing DNA by the silica particles, the volume of the eluent and the elution time, etc., the comprehensive performance of the finally extracted plasmid DNA can be further optimized. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 4 is an agarose gel electrophoresis diagram of plasmid DNA extracted by silica beads with different shapes and particle sizes in Example 2 of the present application; wherein M lane corresponds to DNA Marker, and lanes 1-11 correspond to the serial numbers in Table 1 in sequence.

[0019] Figure 2 Figure 5 is an agarose gel electrophoresis diagram of plasmid DNA extracted by using neutralizing solutions with different formulations in Example 3 of the present application; wherein lanes 1-12 correspond to the serial numbers in Table 2 in sequence.

[0020] Figure 3 Figure 6 is an agarose gel electrophoresis diagram of plasmid DNA extracted by using silica beads with different amounts in Example 4 of the present application.

[0021] Figure 4 Figure 7 is an agarose gel electrophoresis diagram of plasmid DNA extracted by using different concentrations of guanidine isothiocyanate in Example 5 of the present application.

[0022] Figure 5Agarose gel electrophoresis diagram of plasmid DNA extracted by different concentrations of guanidine hydrochloride in Example 5 of the present application.

[0023] Figure 6 Agarose gel electrophoresis diagram of plasmid DNA extracted by different adsorption times of silica beads in Example 6 of the present application.

[0024] Figure 7 Agarose gel electrophoresis diagram of plasmid DNA extracted by different elution volumes in Example 6 of the present application.

[0025] Figure 8 Agarose gel electrophoresis diagram of plasmid DNA extracted by different elution times in Example 6 of the present application.

[0026] Figure 9 Agarose gel electrophoresis comparison diagram of plasmid DNA extracted by different methods in Example 7 of the present application, wherein lanes 1-3 correspond to the serial numbers in Table 9, respectively.

[0027] Figure 10 Agarose gel electrophoresis comparison diagram of plasmid DNA extracted by different methods in Example 8 of the present application, wherein lanes 1-2 correspond to the serial numbers in Table 10, respectively.

[0028] Figure 11 Agarose gel electrophoresis comparison diagram of plasmid DNA extracted by different methods in Example 8 of the present application, wherein lanes 1-2 correspond to the serial numbers in Table 10, respectively.

[0029] Figure 12 Fluorescence comparison diagram of plasmid DNA extracted by different methods in Example 8 of the present application, wherein the upper diagram is DNA extracted by the silica gel membrane large purification column (PD7) of Axygen Company, and the lower diagram is DNA extracted by the method of Example 1 of the present application. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present application, the present application will be described more fully below. The present application can be realized in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The use of the terms "and / or" includes a combination of one or more of the associated listed items.

[0032] The experimental methods in the following examples, unless otherwise specified, are generally carried out according to conventional conditions, or according to the conditions recommended by the manufacturers. The various common chemical reagents used in the examples are commercially available products.

[0033] In some embodiments of the present application, the use of silica particles in the extraction of plasmid DNA is disclosed, wherein the silica particles are spherical silica particles or irregularly shaped silica particles with a particle size of 18 nm to 50 nm and a uniform particle size.

[0034] In the existing method for extracting plasmid DNA based on unmodified silica particles (hereinafter also referred to as silica beads), irregularly shaped mixed silica particles with a particle size of 0.5 to 10 μm are generally used, most of which have a particle size of 1 to 5 μm. However, such mixed silica particles with a particle size can result in unqualified plasmid DNA purification or insufficient concentration of plasmid DNA. The inventors have found through a large number of experiments that the use of spherical silica particles or irregularly shaped silica particles with a uniform particle size and a particle size of 18 nm to 50 nm as a DNA adsorbent can significantly improve the concentration and yield of plasmid DNA while ensuring the purity of the extracted plasmid DNA. Therefore, spherical silica particles or irregularly shaped silica particles with a uniform particle size and a particle size of 18 nm to 50 nm can be used to extract plasmid DNA.

[0035] In some embodiments of the present application, the use of silica particles in the extraction of plasmid DNA is disclosed, wherein the silica particles are spherical silica particles or irregularly shaped silica particles with a particle size of 18 nm to 50 nm and a uniform particle size.

[0036] (1) obtaining a bacterial solution transformed with a plasmid, and lysing the bacterial solution with an alkaline lysis solution;

[0037] (2) adding potassium acetate and guanidine hydrochloride to the lysed bacterial solution and mixing well; the final concentration of the potassium acetate is 0.5 M to 2 M, and the final concentration of the guanidine hydrochloride is 0.5 M to 3 M;

[0038] (3) after centrifugation to remove the precipitate, adding guanidine hydrochloride and mixing well to obtain a crude plasmid solution;

[0039] (4) adding 0.125 mg to 1 mg of silica particles per mL of bacterial solution to the crude plasmid solution and rotating for incubation; the silica particles are spherical silica particles or irregularly shaped silica particles with a particle size of 18 nm to 50 nm and a uniform particle size;

[0040] (5) centrifuging, rinsing the silica particles, air-drying, adding an elution solution for elution, centrifuging, and collecting the supernatant to obtain the plasmid DNA.

[0041] The method for extracting plasmid DNA of the present application first centrifuges and resuspends the bacteria liquid transformed with plasmid, then denatures and lyses the bacteria with an alkaline lysis solution, and then adds a certain final concentration of potassium acetate and guanidine hydrochloride as a neutralizing solution (in the existing method for extracting plasmid DNA, after the bacteria liquid transformed with plasmid is lysed with an alkaline lysis solution, a pH = 4.2-5.5, 3M final concentration of potassium acetate is usually used as a neutralizing solution. On the one hand, the sodium hydroxide in the alkaline lysis solution is neutralized to neutralize the pH, so that the circular plasmid DNA renatures, while the linear bacterial genomic DNA with a large molecular weight is difficult to renature. On the other hand, potassium acetate can provide potassium ions to form a precipitate with SDS in the alkaline lysis solution to precipitate the genomic DNA, denatured proteins and polysaccharides to form a tangled precipitate complex and precipitate out together. The inventor found that adding a certain amount of guanidine hydrochloride to the potassium acetate as a neutralizing solution can improve the concentration and yield of the plasmid DNA) to renature the plasmid DNA, and the bacterial chromosomal DNA and proteins are precipitated out; then a certain amount of (the use amount of the silica particles also has a great influence on the yield and purity of the extracted plasmid DNA, and too low (less than 0.125 mg) or too high (more than 1 mg) use amount will make the purity of the plasmid DNA not meet the requirements) non-modified silica particles (silica beads) with uniform particle size and a particle size of 18-50 nm in spherical or irregular shape are added to the supernatant (i.e. the crude plasmid extract) rich in plasmid DNA to adsorb the DNA, and finally the silica particles are air-dried and eluted to obtain the plasmid DNA.

[0042] The plasmid herein can be a cloning vector (Cloning Vectors), an expression vector (Expression Vectors), a shuttle vector (Shuttle Vectors), a gene editing vector (CRISPR / Cas9 vector), a reporter vector (Reporter Vectors), and various types of plasmids. In one embodiment, the plasmid is pCDH-EF1-YFP-IRES-Puro. It can also be other various commonly used commercial plasmids, and the method is universal.

[0043] The bacteria herein are Escherichia coli (Escherichia coli), such as common cloning strains: DH5α, TOP10, Stbl2 / 3 / 4; expression strains: BL21 (DE3), JM110, etc.

[0044] In one embodiment, the method for preparing the bacteria liquid of step (1) is: a single colony of bacteria transformed with plasmid is inoculated in LB medium, and cultured at 37±1℃ for 12-16h to OD 600is 0.5 to 0.8, and the precipitate is collected by centrifugation and resuspended. In this context, resuspension is performed using a bacterial suspension known in the art. In one embodiment, the bacterial suspension comprises 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), and 100 μg / mL RNase A.

[0045] In one embodiment, the particle size of the spherical silica particles or irregularly shaped silica particles in step (4) is 18 nm to 22 nm. The yield of the extracted plasmid DNA is significantly higher when the spherical or irregularly shaped silica particles have a particle size of 18 nm to 22 nm. When the silica particles are irregularly shaped silica particles having a particle size of 20 nm, the extracted plasmid DNA band is significantly brighter, and the proportion of supercoiled DNA in the total DNA is the highest.

[0046] In one embodiment, in step (4), 0.5 mg to 1 mg of silica particles are added per mL of bacterial solution. When the mass of the silica particles is within this range, the yield of the extracted plasmid DNA is high, and the purity is high, and the brightness of the DNA and the proportion of supercoiled structure are significantly higher.

[0047] In one embodiment, in step (4), 0.8 mg to 1 mg of silica particles are added per mL of bacterial solution. When the mass of the silica particles is within this range, the yield of the extracted plasmid DNA is the highest.

[0048] In one embodiment, in step (2), the final concentration of potassium acetate is 1.8 M to 2 M, and the final concentration of guanidine hydrochloride is 0.8 M to 1.2 M. When the final concentrations of potassium acetate and guanidine hydrochloride are within this range, the yield of the obtained DNA is significantly increased, and exceeds 15 μg.

[0049] In one embodiment, in step (3), the final concentration of guanidine hydrochloride is 1 M to 4 M.

[0050] In one embodiment, in step (3), the final concentration of guanidine hydrochloride is 1.5 M to 4 M.

[0051] In one embodiment, in step (3), the final concentration of guanidine hydrochloride is 1.8 M to 2.2 M. When the final concentration of guanidine hydrochloride is within this range, the yield of the final plasmid DNA is significantly higher.

[0052] In one embodiment, in step (4), the time for the rotational incubation is 5 min to 30 min.

[0053] In one embodiment, in step (4), the time of the rotating incubation is 10-15 minutes. When the time of the adsorption of the DNA on the silica particles is 10-15 minutes, the combination of the silica particles and the nucleic acid can achieve the best effect.

[0054] In one embodiment, in step (5), the volume of the elution solution is 25-100 μL. Different volumes of the elution solution have different effects on the elution of the DNA adsorbed on the silica particles. When the volume of the elution solution is higher than 100 μL, the purity cannot meet the requirement.

[0055] In one embodiment, in step (5), the volume of the elution solution is 25-50 μL. When the volume of the elution solution is 25-50 μL, the purity of the DNA is high, the yield is high, and the total amount can reach about 16 μg.

[0056] In one embodiment, in step (5), the volume of the elution solution is 45-50 μL. When the volume of the elution solution is in this range, the operability is the strongest.

[0057] In one embodiment, in step (5), the time of the elution of the elution solution is 5-30 minutes.

[0058] In one embodiment, in step (5), the time of the elution of the elution solution is 12-18 minutes.

[0059] In one embodiment, in step (5), the time of the elution of the elution solution is 14-16 minutes. When the elution time is in this range, the adsorbed DNA can be completely eluted.

[0060] In some embodiments of the present application, a method for extracting plasmid DNA is disclosed, comprising the following steps:

[0061] (1) Taking the bacterial solution transformed with the plasmid, centrifuging to discard the supernatant, adding the bacterial suspension (containing 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0) and 100 μg / mL RNase A) to resuspend the bacterial body, to obtain a resuspension;

[0062] (2) Taking the resuspension of step (1), adding the alkaline lysis solution (containing 0.2 M NaOH and 1% SDS), and inverting to mix 4-6 times, so that the bacterial body is fully denatured and lysed;

[0063] (3) Adding potassium acetate with a final concentration of 0.5-2 M and guanidine hydrochloride with a final concentration of 0.5-3 M to the lysed bacterial solution, inverting up and down 4-6 times, and mixing;

[0064] (4) After removing the precipitate by centrifugation, add guanidine hydrochloride with a final concentration of 1M-4M, mix well upside down, and obtain the crude plasmid solution;

[0065] (5) Add 0.125mg-1mg of silica particles per mL of bacterial solution, add spherical or irregular silica particles with a particle size of 18nm-50nm and uniform particle size in the crude plasmid solution of step (4), rotate and incubate for 5min-30min, and centrifuge to discard the supernatant;

[0066] (6) Add PE solution (containing 80% ethanol and 10mM Tris-HCl (pH 7.5)) to resuspend and wash the precipitate, and centrifuge to discard the supernatant;

[0067] (7) Air dry the silica particles for 5-10min;

[0068] (8) Add 25μL-100μL of TE solution (containing 10mM Tris-HCl (pH 8.0) and 1mM EDTA (pH 8.0)) to resuspend the precipitate in the air-dried silica particles, elute at 65℃ for 5min-30min, and mix gently 3-5 times during the period; centrifuge to collect the supernatant, and obtain the product.

[0069] In some other embodiments of the present application, a kit for extracting plasmid DNA is disclosed, comprising: silica particles, neutralizing solution, and chaotropic salt; the silica particles are spherical silica particles or irregular silica particles with a particle size of 18nm-50nm and uniform particle size; the neutralizing solution is potassium acetate and guanidine hydrochloride; and the chaotropic salt is guanidine hydrochloride.

[0070] In the following examples of the present application, the silica particles (silica beads) used are all purchased from Bishui New Material (Suzhou) Co., Ltd. and are not modified. The particle size of the irregular silica particles is determined by a laser particle size analyzer.

[0071] The present application will be described in detail below in combination with the accompanying drawings and specific examples.

[0072] Example 1: A method for extracting plasmid DNA

[0073] comprising the following steps:

[0074] 1. From an E. coli plate transformed with pCDH-EF1-YFP-IRES-Puro plasmid, pick a single colony and inoculate into LB medium containing AMP antibiotic (final concentration 100μg / mL), and incubate at 37℃ for 14h (add 200μL of bacterial solution to a 96-well plate, detect by an enzyme marker, and the OD 600 is about 0.6);

[0075] 2. Take 2 mL of E. coli bacterial solution, centrifuge at 12,000 rpm for 1 min at room temperature, discard the supernatant, and add 250 μL of bacterial suspension (containing 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), and 100 μg / mL RNase A) to resuspend the bacterial cells;

[0076] 3. Add 250 μL of alkaline lysis solution (containing 0.2 M NaOH and 1% SDS) to the resuspension of step 2, invert mix 4-6 times to fully denature and lyse the bacterial cells;

[0077] 4. Add 250 μL of neutralization solution (containing 2 M potassium acetate and 1 M guanidine hydrochloride, and adjust the pH to 4.2-5.5 with acetic acid) to the lysed resuspension, immediately invert mix 4-6 times (renaturation), and observe the white flocculent precipitate (bacterial chromosomal DNA and proteins);

[0078] 5. Centrifuge at 12,000 rpm for 10 min at room temperature, and transfer the supernatant (rich in plasmid DNA) to a new 1.5 mL EP tube;

[0079] 6. Add a chaotropic agent (2 M guanidine hydrochloride) to the supernatant, invert mix, and obtain the crude plasmid extract;

[0080] 7. Add 2 mg of 20 nm irregular SiO2 (SiO2 is prepared in advance as 50 mg / mL with H2O) to the crude plasmid extract, and incubate at room temperature for 10 min with rotation;

[0081] 8. Centrifuge at 12,000 rpm for 1 min at room temperature, and discard the supernatant;

[0082] 9. Add 500 μL of PE solution (containing 80% ethanol and 10 mM Tris-HCl (pH 7.5)) to resuspend and wash the precipitate, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the supernatant; repeat the washing once;

[0083] 10. Air dry for 5-10 min;

[0084] 11. Add 50 μL of TE solution (containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0)) to resuspend the precipitate, elute at 65°C for 15 min, and gently mix 3-5 times during the elution;

[0085] 12. Centrifuge at 12,000 rpm for 1 min at room temperature, and collect the supernatant to obtain the plasmid DNA solution.

[0086] Example 2 Comparison of the yield and quality of plasmid DNA extracted by different particle sizes and regular Si beads

[0087] This embodiment compares the use of seven different particle size spherical silica beads and four irregular silica beads to extract plasmid DNA, and the effects on the yield and quality of the extracted plasmid DNA. The specific method is as follows:

[0088] 1. From the E. coli plate transformed with pCDH-EF1-YFP-IRES-Puro plasmid, a single colony was picked and inoculated into LB medium containing AMP antibiotic (final concentration 100 μg / mL) and cultured at 37°C for 14 h (200 μL of bacterial solution was added to a 96-well plate, and the OD 600 was about 0.6);

[0089] 2. Take 2 mL of E. coli bacterial solution, centrifuge at 12,000 rpm at room temperature for 1 min, discard the supernatant, and add 250 μL of bacterial suspension (containing 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0) and 100 μg / mL RNase A) to resuspend the bacterial cells;

[0090] 3. Add 250 μL of alkaline lysis solution (containing 0.2 M NaOH and 1% SDS) to the resuspension of step 2, mix by inverting 4-6 times to fully denature and lyse the bacterial cells;

[0091] 4. Add 250 μL of neutralization solution (3 M potassium acetate, adjust pH to 4.2-5.5 with acetic acid) to the lysed resuspension, immediately mix by inverting 4-6 times (renaturation), and a white flocculent precipitate (bacterial chromosomal DNA and protein) can be seen;

[0092] 5. Centrifuge at 12,000 rpm for 10 min at room temperature, and transfer the supernatant (rich in plasmid DNA) to a new 1.5 mL EP tube;

[0093] 6. Add a chaotropic agent (final concentration 4 M guanidine hydrochloride) to the supernatant, mix well by inverting, and obtain the crude plasmid extract;

[0094] 7. Add 1 mg of SiO2 particles (silica beads shape and diameter as shown in Table 1, SiO2 is prepared in advance with H2O to 50 mg / mL) to the crude plasmid extract, and incubate at room temperature for 20 min;

[0095] 8. Centrifuge at 12,000 rpm for 1 min at room temperature, and discard the supernatant;

[0096] 9. Add 500 μL of PE solution (containing 80% ethanol and 10 mM Tris-HCl (pH 7.5)) to resuspend and wash the precipitate, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the supernatant; repeat once;

[0097] 10. Air dry for 5-10 min;

[0098] 11. Add 50 μL TE solution (containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0)) to resuspend the precipitate, elute at 65 °C for 10 min, and mix gently 3-5 times during the elution;

[0099] 12. Centrifuge at 12,000 rpm for 1 min at room temperature, collect the supernatant, and obtain the plasmid DNA solution.

[0100] The yield and purity of the plasmid DNA extracted finally were compared, and the results are shown in Table 1. 260 / A 280 between 1.8 and 2.0 and A 260 / A 230 >2.0 indicated that the DNA was of good purity.

[0101] Table 1

[0102] No. Silica bead shape Silica bead diameter Concentration (ng / μL) Total amount (μg) 260 / 280 260 / 230 1 Spherical 20 nm 241.33 12.07 1.91 2.23 2 Spherical 50 nm 172.85 8.64 1.87 2.19 3 Spherical 300 nm 107.53 5.38 1.92 2.00 4 Spherical 500 nm 80.01 4.00 1.89 1.93 5 Spherical 1 μm 98.42 4.92 1.92 2.07 6 Spherical 5 μm 38.16 1.91 1.99 2.19 7 Spherical 10 μm 43.65 2.18 1.98 2.04 8 Irregular 20 nm 201.87 10.09 1.86 2.11 9 Irregular 1 μm 118.50 5.93 1.90 2.20 10 Irregular 5 μm 87.24 4.36 1.88 1.91 11 Irregular 10 μm 52.98 2.65 1.92 1.98

[0103] As can be seen from the results in Table 1, the A 260 / A 280 and A 260 / A 230 ratios of the plasmid DNA extracted by the other 8 kinds of silica beads were within the standard range, but the particle size and shape of the silica beads had a significant influence on the concentration and yield of the plasmid DNA. Among the 11 kinds of silica beads, only the 20 nm spherical silica beads and the 20 nm irregular silica beads were used to extract the plasmid DNA whose concentration exceeded 200 ng / μL and the yield exceeded 10 μg.

[0104] The brightness and band type of the DNA were observed by agarose gel electrophoresis, and the purity and supercoiled content of the DNA were compared, and the results are shown in Table 2. Figure 1 As can be seen from Table 2, the brightness of the plasmid DNA band extracted by the 20 nm irregular silica beads was significantly stronger than that of the other groups, and the proportion of supercoiled DNA in the total DNA was the highest. Figure 1 The results of this example show that the comprehensive performance of the plasmid DNA extracted by the 20 nm irregular silica beads is obviously superior to that of the silica beads of other particle sizes and shapes.

[0105] Example 3 Comparison of the yield and quality of the plasmid DNA extracted by different neutralizing solutions

[0106] In this example, 12 kinds of compound solutions of potassium acetate and guanidine hydrochloride with different concentrations were used as neutralizing solutions for the extraction of plasmid DNA, and the influence of the neutralizing solutions on the yield and quality of the DNA was compared.

[0107]

[0108] ​The extraction procedure was the same as Example 2, except that the neutralization solution in Step 4 was changed to different concentrations (as shown in Table 2) of potassium acetate and guanidine hydrochloride, and 20 nm irregularly shaped silica beads were added in Step 7. The yield and purity of the final extracted plasmid DNA were compared with the neutralization solution in the conventional method as a control, and the results are shown in Table 2 and Figure 2

[0109] Table 2

[0110]

[0111] As can be seen from Table 2 and Figure 2 , compared with the conventional neutralization solution formula, the addition of guanidine hydrochloride to potassium acetate as the neutralization solution improved the concentration and yield of the plasmid DNA to varying degrees, and the use of 2 M potassium acetate and 1 M guanidine hydrochloride as the neutralization solution had the best effect, with the yield of the obtained DNA being more than 15 μg, which was significantly higher than that of the other groups.

[0112] Example 4 Comparison of the yield and quality of plasmid DNA extracted using different amounts of silica beads

[0113] This example compared the effects of using six gradients of the amount of silica beads on the yield and quality of the extracted plasmid DNA.

[0114] The extraction procedure was the same as Example 2, except that the neutralization solution in Step 4 was 2 M potassium acetate and 1 M guanidine hydrochloride, and different amounts (0.125 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 4 mg) of 20 nm irregularly shaped silica beads were added in Step 7. The yield and purity of the final extracted plasmid DNA were compared, and the results are shown in Table 3 and Figure 3

[0115] Table 3

[0116] Silica bead amount (mg) Concentration (ng / μL) Total amount (μg) 260 / 280 260 / 230 0.125 41.01 2.05 1.89 1.77 0.25 105.97 5.30 1.89 2.05 0.5 211.50 10.57 1.87 2.12 1 292.56 14.63 1.87 2.14 2 303.07 15.15 1.86 2.12 4 316.17 15.81 1.90 1.96

[0117] As can be seen from Table 3 and Figure 3 , under the same conditions for extracting plasmid DNA, the concentration and total amount of the obtained plasmid DNA increased with the increase in the amount of silica beads, but when 0.125 mg and 4 mg of silica beads were added in 2 mL of bacterial solution, the A 260 / A 230 ratio of the extracted plasmid DNA was less than 2, and the purity was not high enough, and when the amount was 0.25 mg, 0.5 mg, 1 mg, and 2 mg, the A 260 / A 280 and A 260 / A 230 ratios of the extracted DNA were all within the standard range, and the brightness and supercoiled structure of the DNA were significantly higher, and the use of 2 mg of silica beads had the best effect. ​​

[0118] Example 5 Comparison of the yield and quality of plasmid DNA extracted by different chaotropic salts

[0119] Chaotropic salts promote the specific binding of nucleic acids to silica beads through electrostatic adsorption, hydrophobic interaction, etc., thereby separating plasmid DNA. This embodiment compares the effects of using different chaotropic salts (guanidine hydrochloride or guanidine isothiocyanate) to extract plasmid DNA on the yield and quality of the extracted plasmid DNA.

[0120] The extraction steps are the same as in Example 2, except that in Step 4, the neutralization solution is 2M potassium acetate and 1M guanidine hydrochloride, and in Step 6, different final concentrations (0M, 0.5M, 1M, 2M, 4M) of guanidine hydrochloride or guanidine isothiocyanate are added, and in Step 7, 2mg of 20nm irregular silica beads are added. The yield and purity of the plasmid DNA obtained after the final extraction are compared, and the results are shown in Tables 4-5 and Figure 4-5 .

[0121] Table 4

[0122] Guanidinium isothiocyanate (M) Concentration (ng / μL) Total amount (μg) 260 / 280 260 / 230 0 67.08 3.35 1.87 1.70 0.5 100.56 5.03 1.89 0.60 1 297.10 14.86 1.89 0.74 2 307.48 15.37 1.96 0.24 4 219.85 10.99 1.90 0.20

[0123] Table 5

[0124] Guanidinium hydrochloride (M) Concentration (ng / μL) Total amount (μg) 260 / 280 260 / 230 0 78.71 3.94 1.91 1.51 0.5 87.19 4.36 1.91 1.58 1 258.24 12.91 1.89 2.00 2 302.88 15.14 1.88 2.07 4 278.37 13.92 1.89 2.22

[0125] From Tables 4-5 and Figure 4-5 the results, the type of chaotropic salt has a very large effect on the purity of the extracted plasmid DNA. Using guanidine isothiocyanate as the chaotropic salt, whether at high or low concentration, the ratio of A 260 / A 230 is very small, and the purity of the DNA cannot meet the requirements; using guanidine hydrochloride as the chaotropic salt, when the concentration is more than 1M, high-purity plasmid DNA can be obtained, and among them, the yield with 2M guanidine hydrochloride is significantly higher than that of other concentration groups.

[0126] Example 6 Comparison of the yield and quality of plasmid DNA extracted under different process conditions

[0127] This embodiment compares the effects of different adsorption times of silica beads, different elution volumes of elution solution, and different elution times of elution solution on the extraction of plasmid DNA on the yield and quality of the extracted plasmid DNA.

[0128] 1. Adsorption time

[0129] The extraction steps are the same as in Example 2, except that in Step 4, the neutralization solution is 2M potassium acetate and 1M guanidine hydrochloride, in Step 6, 2M guanidine hydrochloride is added, and in Step 7, 2mg of 20nm irregular silica beads are added and incubated at room temperature for different times (5min, 10min, 15min, 20min, 30min). The yield and purity of the plasmid DNA obtained after the final extraction are compared, and the results are shown in Tables 6 andFigure 6 as shown.

[0130] Table 6

[0131] Adsorption time (min) Concentration (ng / μL) Total amount (μg) 260 / 280 260 / 230 5 273.86 13.69 1.87 2.06 10 313.99 15.70 1.87 2.07 15 315.21 15.76 1.86 2.01 20 307.35 15.37 1.88 2.12 30 308.94 15.45 1.88 2.18

[0132] Table 6 and Figure 6 The results show that the combination of the silica beads and nucleic acids can reach the best effect after 10 minutes of adsorption, and the obtained plasmid DNA has high purity and high yield.

[0133] 2, Elution volume

[0134] The extraction steps are the same as those in Example 2, wherein the neutralizing solution in Step 4 is 2M potassium acetate and 1M guanidine hydrochloride, 2M guanidine hydrochloride is added in Step 6, 2mg of 20nm irregular silica beads are added in Step 7 for rotation incubation at room temperature for 10 minutes, and different volumes (25μL, 50μL, 100μL, 150μL and 200μL) of elution solution are used for elution in Step 11. The yield and purity of the finally extracted plasmid DNA are compared, and the results are shown in Table 7 and Figure 7 as shown.

[0135] Table 7

[0136] Elution volume (μL) Concentration (ng / μL) Total amount (μg) 260 / 280 260 / 230 25 648.96 16.22 1.86 2.05 50 319.05 15.95 1.87 2.10 100 144.47 14.45 1.88 2.17 150 95.25 14.29 1.87 1.95 200 71.93 14.39 1.87 1.87

[0137] From Table 7 and Figure 7 it can be seen that different volumes of elution solution have great differences in the elution effect of DNA adsorbed on the silica beads. After elution with 150μL and 200μL of elution solution, the A 260 / A 230 value of the DNA is less than 2; after elution with 100μL of elution solution, the purity of the DNA can meet the requirements, but the yield is relatively low; after elution with 25μL and 50μL of elution solution, the purity of the DNA is high, and the total amount is about 16μg. Considering the operability, 50μL of elution volume is the most suitable.

[0138] 3, Elution time

[0139] The extraction steps are the same as those in Example 2, wherein the neutralizing solution in Step 4 is 2M potassium acetate and 1M guanidine hydrochloride, 2M guanidine hydrochloride is added in Step 6, 2mg of 20nm irregular silica beads are added in Step 7 for rotation incubation at room temperature for 10 minutes, and 50μL of elution solution is used for elution at different times (5min, 10min, 15min, 20min and 30min) in Step 11. The yield and purity of the finally extracted plasmid DNA are compared, and the results are shown in Table 8 and Figure 8 as shown.

[0140] Table 8

[0141] Elution time (min) Concentration (ng / μL) Total amount (μg) 260 / 280 260 / 230 5 282.95 14.15 1.88 2.20 10 307.29 15.36 1.87 2.18 15 325.54 16.28 1.87 2.13 20 325.47 16.27 1.88 2.15 30 321.79 16.09 1.88 2.15

[0142] From Table 8 and Figure 8 It can be found that the yield of the final DNA increases with the increase of the elution time, and the yield of the DNA does not change when the elution time is more than 15 min, which indicates that the adsorbed DNA can be completely eluted in 15 min. The improved plasmid DNA extraction method based on the above is the extraction method described in Example 1.

[0143] Example 7 Comparison of the yield and purity of the plasmid DNA extracted by the method of the application and the conventional centrifugal column method

[0144] This example compares the yield and purity of the plasmid DNA extracted by the extraction method of the application (i.e. the method of Example 1) and the conventional centrifugal column extraction method (two commercially available plasmid small-scale extraction kits are used).

[0145] The results are shown in Table 9 and Figure 9 .

[0146] Table 9

[0147] No. Method Concentration (ng / μL) Total amount (μg) 260 / 280 260 / 230 1 Centrifugal column (Shark Kit, A) 292.07 14.60 1.80 2.36 2 Centrifugal column (Tiangen Kit, B) 383.83 19.19 1.93 1.95 3 Silica bead method (Example 1) 317.44 15.87 1.87 2.08

[0148] The results show that the yield of the plasmid DNA extracted by the method of Example 1 of the application is higher than that of the plasmid DNA extracted by the A kit, and the purity is qualified; while the yield of the plasmid DNA extracted by the B kit is much higher, but the A 260 / A 230 The ratio is slightly lower than the normal range. In addition, the plasmid DNA extracted by the three methods has a high supercoiled structure content, and no visible RNA or other contaminants.

[0149] The results of this example prove that the yield and purity of the plasmid DNA extracted by the method of the application can achieve a similar effect to that of the commercially available kit.

[0150] Example 8 Comparison of the yield and purity of the plasmid DNA extracted by the method of the application and the conventional silica gel membrane large-scale extraction and adsorption column purification method

[0151] This example compares the yield and purity of the plasmid DNA extracted by the extraction method of the application (i.e. the method of Example 1) and the conventional commercially available silica gel membrane large-scale extraction and adsorption column purification method (PD7, Axygen Company) (1 mL of eluent is finally added for elution).

[0152] The results are shown in Table 10 and Figure 10 .

[0153] Table 10

[0154] No. Method Concentration (ng / μL) Total amount (μg) 260 / 280 260 / 230 1 Silica gel membrane macro column 337.08 337.08 1.87 2.30 2 Silica bead method (Example 1) 417.82 417.82 1.88 2.22

[0155] The results show that there is no significant difference in the supercoiling proportion and DNA purity between the two extraction methods, but the yield of the plasmid DNA extracted by the method of the application is higher, more than 400 μg.

[0156] The plasmid DNA purified by the two methods was added to the restriction enzyme EcoRI and BamHI for enzyme digestion, and the results are shown in Figure 11 The results show that the plasmid DNA purified by the two methods of large extraction has clear enzyme digestion fragments, and the sizes are consistent, indicating that the method of the application has no significant effect on the restriction enzyme digestion reaction of the plasmid DNA.

[0157] Then the transfection efficiency of the plasmids extracted by the two methods was detected at the cell level. The trypsin-digested HEK 293T cells were counted, and 8x10 5 cells / well were inoculated into a 12-well plate at 37℃, 5% CO2, and cultured until the cell confluence reached 70%-80%. 2 μg of the plasmid DNA purified by the two methods was added to 100 μL of serum-free medium, mixed thoroughly, and then 4 μL of PEI40,000 transfection reagent (CYTOCH Company, CT0009) was added, and the mixture was incubated at room temperature for 20 min. The transfection complex was added to the cells, shaken gently, and cultured at 37℃, 5% CO2. After 48 h, fluorescence observation and photographing were performed, and the results are shown in Figure 12 The fluorescence shows that there is no significant difference in the expression level of the fluorescent protein after transfection of the two groups of plasmid DNA.

[0158] The results of the present embodiment show that the plasmid DNA obtained by the plasmid DNA extraction method of the application can meet the conventional needs of enzyme digestion, cell transfection, etc., and the purity is not much different from that of the plasmid DNA extracted by the commercially available product, but the method of the application can improve the DNA yield, and is more simple and low in cost, and has better comprehensive advantages.

[0159] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not exist, they should be considered as the scope of the description.

[0160] The above-described embodiments only express several embodiments of the application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are within the scope of the application. Therefore, the protection scope of the patent of the application should be subject to the appended claims.

Claims

1. Use of silica particles for the extraction of plasmid DNA, characterized in that, The silica particles are spherical silica particles or irregular silica particles with a particle size of 18-50 nm and uniform particle size; preferably, the spherical silica particles or irregular silica particles have a particle size of 18-22 nm.

2. A method for extracting plasmid DNA, characterized by, The method comprises the following steps: (1) obtaining bacteria liquid transformed with a plasmid, and lysing the bacteria liquid with an alkaline lysis solution; (2) adding potassium acetate and guanidine hydrochloride to the lysed bacteria liquid and mixing uniformly; the final concentration of the potassium acetate is 0.5-2 M, and the final concentration of the guanidine hydrochloride is 0.5-3 M; (3) after removing the precipitate by centrifugation, adding guanidine hydrochloride and mixing uniformly to obtain a crude plasmid solution; (4) adding 0.125-1 mg of silica particles per mL of the bacteria liquid, adding the silica particles to the crude plasmid solution and rotating for incubation; the silica particles are spherical silica particles or irregular silica particles with a particle size of 18-50 nm and uniform particle size; (5) centrifuging, rinsing the silica particles, air-drying, adding an eluent for elution, centrifuging, and collecting the supernatant to obtain plasmid DNA.

3. The method for extracting plasmid DNA according to claim 2, characterized in that, The spherical silica particles or irregular silica particles in step (4) have a particle size of 18-22 nm; Preferably, the silica particles in step (4) are irregular silica particles with a particle size of 20 nm.

4. The method for extracting plasmid DNA according to claim 2, characterized in that, In step (4), 0.8-1 mg of silica particles are added per mL of the bacteria liquid; And / or, the final concentration of the guanidine hydrochloride in step (3) is 1-4 M.

5. The method for extracting plasmid DNA according to claim 2, characterized in that, In step (2), the final concentration of the potassium acetate is 1.8-2 M, and the final concentration of the guanidine hydrochloride is 0.8-1.2 M; And / or, the final concentration of the guanidine hydrochloride in step (3) is 1.8-2.2 M.

6. The method for extracting plasmid DNA according to any one of claims 2 to 5, characterized in that, In step (4), the rotating incubation time is 5-30 min.

7. The method for extracting plasmid DNA according to claim 6, characterized in that, The rotating incubation time is 10-15 min.

8. The method for extracting plasmid DNA according to any one of claims 2 to 5, characterized by, In step (5), the volume of the eluent is 25-100 μL; And / or, the elution time of the eluent is 5-30 min.

9. The method for extracting plasmid DNA according to claim 8, characterized in that, In step (5), the volume of the eluent is 45-50 μL; And / or, the elution time of the eluent is 14-16 min.

10. A kit for extracting plasmid DNA, characterized by comprising: The method comprises: Silica particles, a neutralizing solution, and a chaotropic salt; The silica particles are spherical silica particles or irregular silica particles with a particle size of 18-50 nm and uniform particle size; The neutralizing solution comprises potassium acetate and guanidine hydrochloride; The chaotropic salt is guanidine hydrochloride.

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