Method for purifying single-stranded DNA and application thereof
By using primers with 5'-terminal acrylamide groups and alkaline agarose gel electrophoresis for separation and purification in single-stranded DNA preparation, the problem of balancing yield, purity and cost in existing technologies has been solved, achieving efficient and low-cost single-stranded DNA purification.
Patent Information
- Application Number
- CN202510981804.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-10-28
AI Technical Summary
Among existing single-stranded DNA preparation technologies, it is difficult to balance yield, purity and cost. Chemical synthesis methods are costly and have limited length, enzymatic synthesis methods have unstable yield and quality, magnetic bead separation methods have low purity, Lambda exonuclease methods have incomplete or excessive digestion, and methanol copolymer precipitation methods are cumbersome and prone to loss.
Polymerization was performed using primers with acrylamide groups modified at the 5' end to generate linear polyacrylamide-modified primers. Polyacrylamide-crosslinked double-stranded DNA was obtained by PCR amplification. The target single-stranded DNA was separated by agarose gel electrophoresis under alkaline conditions and purified by gel extraction.
It effectively reduces costs, improves the purity and yield of single-stranded DNA, and is suitable for large-scale production in laboratories or enterprises, achieving efficient single-stranded DNA purification.
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Figure CN120843645A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for purifying single-stranded DNA and its application. Background Technology
[0002] Single-stranded DNA (ssDNA), as an important genetic material and biomaterial, plays a crucial role in gene editing, molecular markers, and nanotechnology. In gene editing, ssDNA can serve as a template for homologous recombination repair, precisely introducing gene modifications and bringing new hope to gene therapy. In the field of molecular markers, ssDNA probes, with their specific binding ability, can be used for disease diagnosis and pathogen detection. In nanotechnology, ssDNA is a crucial building block for constructing complex nanostructures, contributing to the development of cutting-edge technologies such as nanorobots and drug delivery vectors.
[0003] However, existing ssDNA preparation technologies face a dilemma in balancing yield, purity, and cost. Chemical synthesis methods, which involve cyclically adding oligonucleotides, not only limit the length and quality of synthesized oligonucleotides but also incur high costs. Typically, chemically synthesized ssDNA is no longer than 200 nt; as chain length increases, the error incorporation rate rises, and synthesis efficiency drops sharply. Each base extension requires significant reagent and time costs. Enzymatic synthesis methods, due to variations in the enzymes used, produce products with inconsistent length, yield, and quality. While preparing single-stranded DNA from double-stranded templates is relatively efficient, it has limitations in terms of the concentration and purity of the recovered strand. For example, magnetic bead separation utilizes biotin-streptavidin interactions to separate ssDNA; however, alkaline denaturation weakens the binding ability of magnetic beads, leading to increased non-specific recovery rates. In practice, the chemical groups on the surface of magnetic beads undergo partial hydrolysis under alkaline conditions, reducing the binding affinity between biotin and streptavidin. Impurity DNA that shouldn't bind is also recovered, affecting the purity of the final product. The Lambda exonuclease method selectively digests phosphorylated single strands of dsDNA to obtain ssDNA, but often suffers from incomplete digestion leading to double-strand contamination, affecting the reliability of downstream experiments. Over-digestion can also reduce product yield or shorten the target strand. This method is extremely sensitive to reaction time and temperature; high enzyme activity in the early stages of the reaction easily leads to over-digestion, while decreased enzyme activity in the later stages results in incomplete digestion. The methanol copolymer precipitation method relies on polymer selective precipitation, requiring multiple precipitation and resuspension cycles, which is cumbersome and prone to non-specific precipitation. Each precipitation and resuspension process can result in the loss of the target ssDNA, and the polymer's precipitation effect varies for different sequences of single-stranded DNA, making it difficult to guarantee product homogeneity.
[0004] Therefore, there is an urgent need for a preparation method that can effectively solve the imbalance between yield, purity and cost in existing single-stranded DNA preparation technologies. Summary of the Invention
[0005] The purpose of this invention is to provide a method for purifying ssDNA, which not only effectively reduces costs but also improves the purity of ssDNA.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] The purpose of this invention is to provide a method for purifying single-stranded DNA, the method comprising the following steps:
[0008] S1: Polymerization reaction was carried out using primers with acrylamide groups modified at the 5' end to generate linear polyacrylamide-modified primers LPA-primer;
[0009] S2: Using the LPA-primer described in step S1 as a primer, polyacrylamide-crosslinked double-stranded DNA LPA-dsDNA was obtained by PCR amplification.
[0010] S3: The LPA-dsDNA described in step S2 is subjected to denaturing agarose gel electrophoresis under alkaline conditions to separate the target ssDNA from the non-target ssDNA by utilizing the difference in migration rate caused by the polyacrylamide modification group.
[0011] S4: The target ssDNA band described in step S3 is cut and recovered to obtain purified ssDNA.
[0012] Preferably, the polymerization reaction in step S1 is a reaction between a primer with an acrylamide group modified at the 5' end and a mixed reaction solution; the mixed reaction solution includes acrylamide, sodium acrylate, TBE and water; the mass percentage of acrylamide to sodium acrylate is 99:1.
[0013] Preferably, the polymerization reaction described in step S1 is catalyzed by ammonium persulfate and tetramethylethylenediamine under anaerobic conditions.
[0014] Preferably, the PCR amplification reaction conditions in step S2 are: 96℃ pre-denaturation for 3 min, 96℃ denaturation for 30 s, 55-60℃ annealing for 45 s, and 72℃ final extension for 5 min, for 28 cycles; the PCR amplification system in step S2 consists of 100 nM primers, 200 μM dNTPs, 1×Pfu buffer, and 2.5 μL KOD polymerase.
[0015] Preferably, the alkaline condition in step S3 is an electrophoresis buffer containing 30-50 mM NaOH.
[0016] Preferably, the length of the target purified ssDNA in step S4 is 100-2000 nt.
[0017] The present invention also provides an application of ssDNA prepared according to the method described above in the preparation of DNA origami structures.
[0018] Preferably, the DNA origami structure is a drug-loaded nanodevice.
[0019] The present invention also provides an application of ssDNA prepared according to the above-described method in the preparation of fluorescent in situ hybridization probes.
[0020] Preferably, the fluorescent in situ hybridization probe is a ring-shaped signal amplification probe.
[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0022] (1) This invention modifies the non-target strand in double-stranded DNA with linear polyacrylamide (LPA) to create a huge molecular weight difference between the non-target strand and the target strand, and then separates and purifies the target ssDNA by alkaline agarose gel electrophoresis.
[0023] (2) The method for purifying ssDNA provided by the present invention effectively reduces costs, improves ssDNA purity, and increases yield, and is suitable for large-scale laboratory or enterprise use. Attached Figure Description
[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0025] Figure 1 Figure A shows a schematic diagram of the process for preparing ssDNA; Figure B shows a schematic diagram of the process for producing polyacrylamide-modified primers through polymerization; Figure B shows a schematic diagram of the process for ssDNA isolation and purification.
[0026] Figure 2Figure 1 shows the evaluation and comparative analysis of ssDNA recovery efficiency; Figure A is a schematic diagram of the separation effect of different NaOH concentrations; Figure B is the agarose gel electrophoresis characterization of 100-800nt ssDNA; Figure C is the agarose gel electrophoresis characterization of 1500nt and 2000nt ssDNA; Figure D is a statistical analysis of the recovery rate (%) of 100-2000nt ssDNA; Figure E is the electrophoresis diagram of ssDNA separated and purified by different methods; Lane 1: λ exonuclease digestion method (λ-Exo); Lane 2: T7 exonuclease digestion method (T7 Exo); Lane 3: Biotin-streptavidin magnetic bead method (Magnetic... Beads); Lane 4: Purification method based on linear polyacrylamide modification and denaturing agarose gel electrophoresis (LAP-DAGE); Figure F shows the relative yield analysis of ssDNA separated and purified by different methods; Figure G shows the double-stranded DNA residual rate analysis; Double-stranded DNA residual rate (%) = gray value of dsDNA band / (gray value of target ssDNA band + gray value of dsDNA band) × 100; Data are expressed as Mean ± SEM, n = 3.
[0027] Figure 3Figure A shows the RNA-FISH workflow for preparing ssDNA probes for RNA-FISH analysis. The workflow includes: i. Hybridizing "split" locus probes onto processed cell or tissue samples to target RNA molecules, with 10 probe pairs designed for each RNA molecule. ii. Hybridizing primary and secondary amplification probes to amplify the signal. iv. Hybridizing fluorescent probes to visualize the signal. Figure B shows four types of "split" locus probes: Circle C, Double Z, Double C, and Cruciform, targeting the inverted confocal imaging signal of the Actb gene in NIH3T3 cells. Figure C shows the quantitative comparison of the signal-to-noise ratio (SNR) of the four types of "split" locus probes for the same gene. For each experiment, 30 cells were selected for SNR analysis (n=30). Data are expressed as Mean±SEM, ***P<0.001. Scale bar: 50 μm; Figure D is a schematic diagram of multicolor imaging of endogenous Actb mRNA FISH, exogenous EGFP mRNA FISH, and EGFP protein signals in NIH3T3 cells. From left to right, they are: nuclear DAPI staining signal (represented by blue), Actb FISH signal (Tamra dye, represented by red), green fluorescent protein signal (represented by green), EGFP FISH signal (Alexa647 dye, represented by magenta), and the signals of the four channels superimposed (merged). Scale bar: 50 μm; Figure E is a schematic diagram comparing the detection effects of RNAFISH, smFISH, and π-FISH based on the preparation of ssDNA amplification probes. Blue represents nuclear DAPI staining signal, and red represents Actb FISH signal. Scale bar: 20 μm. Figure F shows the signal-to-noise ratio of FISH signals under the three strategies. In each experiment, 30 cells were selected to calculate the signal-to-noise ratio, n=30. The data are expressed as Mean±SEM. ***P<0.001, *P<0.05.
[0028] Figure 4Figure A shows the preparation process of the DNAFISH probe library for ploidy detection and analysis in HeLa cells. Figure B shows the electrophoretic characterization of the DNAFISH probes, where lane 1 contains dsDNA template (approximately 100 bp) and lane 2 contains purified ssDNA probes (approximately 100 nt). Figure C shows the in situ validation of the generated ssDNA probe library with FAM labeling on cultured HeLa cells. The library contains 48 probe sequences, each targeting the repetitive region of the centriole region of chromosome 17 in HeLa cells. The green dots are the DNAFISH labeling signals, indicating that chromosome 17 in HeLa cells is mostly triploid. The scale bar is 10 μm. Figure D shows the statistical results of DNAFISH detection of the proportion of triploid, diploid, and other polyploids on chromosome 17 in HeLa cells. The data are expressed as Mean ± SEM, n = 3.
[0029] Figure 5 Figure A shows a schematic diagram of preparing long ssDNA for DNA origami, and the electrophoretic characterization of circular DNA origami is shown in Figure A. Lane 1: 1500nt ssDNA; Lane 2: annealing reaction solution, including a folded 38nm diameter ring origami structure and excess staple sequence; Figure B shows the AFM characterization of the ring DNA origami, with scale bars of 100nm and 40nm; Figure C shows the electrophoretic characterization of the quadrilateral DNA origami; Lane 1: 2000nt ssDNA; Lane 2: annealing reaction solution, including a 50nm wide quadrilateral origami structure and excess staple sequence; Figure D shows the AFM characterization of the quadrilateral DNA origami, with scale bars of 100nm and 40nm; Figure E shows the width and height measurement of the ring DNA origami; Figure F shows the width and height measurement of the quadrilateral DNA origami; Figure G shows the folding efficiency statistics of the ring and quadrilateral DNA nanostructures, folding rate = number of fully folded origami / (number of fully folded origami + number of incompletely folded origami) × 100, data are expressed as Mean ± SEM, n = 3. Detailed Implementation
[0030] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.
[0031] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.
[0032] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.
[0033] Example 1 Synthesis of polyacrylamide-modified primers
[0034] The polymerization reaction produces polyacrylamide-modified primers, the steps of which are as follows: Figure 1 As shown in Figure A.
[0035] The reaction was carried out in 15 mL centrifuge tubes with a total reaction volume of 500 μL, containing 5% acrylamide, 0.05% sodium acrylate, 100 μM acrylamide-modified primers, 1×TBE, 0.005% TEMED, and 0.005% APS.
[0036] Before the reaction begins, acrylamide, sodium acrylate, TBE, primers and water are mixed to obtain a mixed reaction solution. Then, nitrogen gas is blown into the mixed reaction solution for 10 minutes to remove oxygen. After that, TEMED and APS are added, and nitrogen gas is blown continuously for 10 minutes until a viscous gel-like mixture is formed. Finally, the tube cap is tightened, sealed and placed in the dark at room temperature overnight for reaction.
[0037] The next day, purify the primers using the following steps:
[0038] (1) Add 4.5 mL of 1×TE buffer to 500 μL of reaction solution and vortex at high speed to disperse it completely;
[0039] (2) Add 25 μL of 5M NaCl and vortex for 15 seconds;
[0040] (3) Slowly add 5 mL of ice-cold methanol while vortexing to prevent the formation of a large amount of local precipitation. After adding, incubate on ice for 2 min to allow the polymer to precipitate fully.
[0041] (4) Centrifuge at 150g for 5 minutes, discard the supernatant, and a milky white precipitate will be visible at the bottom of the tube;
[0042] (5) Resuspend the precipitate with 5 mL of 1×TE buffer, then add 25 μL of 5M NaCl and vortex for 15 seconds;
[0043] (6) Then repeat steps (3) and (4) to precipitate again;
[0044] (7) The collected precipitate was resuspended in 2.5 mL of 1×TE buffer, aliquoted and stored at -20℃ for later use.
[0045] Example 2: Isolation and purification of ssDNA
[0046] The steps for isolating and purifying ssDNA are as follows: Figure 1 As shown in B
[0047] 1. PCR reaction generates linear polyacrylamide-modified double-stranded DNA (LPA-primer)
[0048] The PCR reaction was carried out in a total reaction volume of 100 μL, and the reaction mixture contained 100 nM primers, 200 μM dNTPs, 1× Pfu buffer, and 2.5 μL of 147D polymerase.
[0049] The PCR reaction was performed by heating at 96°C for 3 minutes to completely denature the template, followed by 28 PCR cycles, including denaturation at 96°C for 30 seconds, annealing at 55-60°C for 30 seconds, and extension at 72°C, with a final extension at 72°C for 5 minutes. Typically, the annealing temperature was 5°C lower than that of the primers with the lowest Tm value. For all smaller amplification products (<500 nt), the extension temperature was 15 seconds; while for larger amplification products, an extension of 1 minute per kb of amplification product was generally used.
[0050] 2. Denaturing agarose gel electrophoresis
[0051] Denaturing agarose gel electrophoresis was performed under alkaline conditions. A basic agarose gel containing 1.5% agarose, 50 mM NaOH, and 1 mM EDTA was prepared.
[0052] First, weigh 1.5g of agarose into 50mL of deionized water, heat to dissolve, and wait for the temperature to drop to about 60℃. Then add 1mL of 5M NaOH, 1mL of 100mM EDTA and 10μL of GelRed nucleic acid dye. After adding deionized water to 100mL, mix thoroughly and pour into a mold to solidify for later use.
[0053] Before electrophoresis, add 1× alkaline denaturation buffer to the sample and heat at 75℃ for 5-10 min (the heating time varies depending on the DNA length; generally, heat for 1 min for DNA less than 500bp, 5 min for DNA between 500-1000bp, and 10 min for DNA greater than 1000bp). Immediately after heating, place the sample on ice for 2 min, then add the sample to the gel well for electrophoresis. The electrophoresis running voltage is 100V, and the duration is 60 min, using 1× alkaline electrophoresis buffer.
[0054] After electrophoresis, the gel was soaked in 1×Tris-HCl buffer (pH 7.6) for 0.5 h to neutralize. The target band was then observed and cut using a blue light gel cutter. The ssDNA was recovered using a gel recovery kit. For ssDNA smaller than 500 nt, 0.5 volumes of isopropanol and 2 volumes of anhydrous ethanol were added to the sol to improve the recovery efficiency.
[0055] Example 3: Comparison and Validation of the Effects of Different ssDNA Isolation and Purification Methods
[0056] In this invention, the separation effects of denaturing buffer solutions containing different concentrations of NaOH were systematically compared, and the results are as follows: Figure 2 As shown in Figure A, when the NaOH concentration is too low, the double-stranded DNA cannot be sufficiently denatured, thus limiting the separation of ssDNA; when the NaOH concentration is too high, it will damage the chemical structure and stability of DNA, causing DNA hydrolysis and breakage. Experiments have verified that a NaOH concentration of 50 mM can fully separate ssDNA and maintain structural stability.
[0057] To comprehensively evaluate the purified ssDNA, this invention employs multiple analytical methods. Agarose gel electrophoresis analysis yielded the following results: Figure 2 As shown in B and C, ssDNA samples of different lengths all exhibited a single migration band. This invention also determined, using ultraviolet spectrophotometry, that approximately 8-13 pmol of ssDNA product could be stably obtained per 100 μL PCR reaction system, with the recovery rate relative to the initial PCR amplification product maintained between 65% and 85%. Figure 2 As shown in D.
[0058] The data above show that the preparation method provided by this invention is not only applicable to the preparation of ssDNA of different lengths, but also can maintain stable high purity and recovery rate.
[0059] In this invention, the ssDNA preparation method provided by this invention is compared and analyzed with other ssDNA production methods based on double-stranded DNA. Specifically, the overall yield and purity of ssDNA are systematically evaluated to verify the technical effectiveness of the method of this invention.
[0060] First, double-stranded DNA of approximately 500 bp in length was amplified, and the target ssDNA was isolated and purified using different methods. The quality of the ssDNA obtained from the PCR products was then assessed using agarose gel electrophoresis. The agarose gel electrophoresis results are shown below. Figure 2As shown in E, the electrophoretic bands of ssDNA prepared by the λ-Exo and T7-Exo digestion methods are weak and contain incompletely digested dsDNA. The ssDNA bands obtained by the biotin-streptavidin magnetic bead method are brighter, but there is still a problem of dsDNA contamination, requiring an additional agarose gel purification step to remove any uneluted double-stranded DNA. In contrast, the ssDNA prepared by the separation and purification method provided in this invention has bright and uniform electrophoretic bands.
[0061] Quantitative analysis data of electrophoresis results, such as Figure 2 As shown in F and G, the relative yield of ssDNA obtained by the separation and purification method provided by the present invention (87.50±5.79%) is significantly higher than that of the λ exonuclease digestion method (16.30±4.17%) and the T7 exonuclease digestion method (31.53±8.06%). Meanwhile, the residual rate of dsDNA generated by the method of the present invention (8.26±1.67%) is significantly lower than that of the magnetic bead method (35.93±6.22%) and the T7 exonuclease method (36.97±1.81%).
[0062] The above experimental data results demonstrate that the separation and purification method provided by this invention has excellent ssDNA separation and purification capabilities.
[0063] Example 4: ssDNA probe for RNA-FISH
[0064] 1) Prepare NIH3T3 cells for overnight adherent culture, aspirate the DMEM medium from the cells, wash once with 1× phosphate-buffered saline (PBS) at room temperature, then immediately fix in 4% PFA solution for 10 min, and then wash three times with 1× PBS for 5 min each time.
[0065] 2) To permeabilize the cell samples, wash the cells three times with PBST buffer (1×PBS, containing 0.5% Triton X-100) for 5 min each time before hybridization.
[0066] 3) Prepare the primary probe hybridization solution. Add each primary probe to the hybridization solution (6×SSC, 1% Triton X-100, 25% formamide, 10% dextran sulfate, 0.1% DEPC) to a final concentration of 10 nM. Immerse the cells in the primary probe hybridization solution and incubate overnight at 37°C on a shaker. Then wash three times at 37°C for 10 min each time with washing buffer containing 25% formamide (2×SSC, 1% Triton X-100, 25% formamide, 0.1% DEPC).
[0067] 4) Prepare the secondary probe hybridization solution (25% formamide, 2×SSC, 1% Triton X-100, 10% dextran sulfate, DEPC, 5nM secondary probe). Immerse the cells in the secondary probe hybridization solution and incubate on a shaker at 37°C for 5 h. Then wash the cells three times at 37°C for 10 min each time with washing buffer containing 25% formamide (2×SSC, 1% Triton X-100, 25% formamide, 0.1% DEPC).
[0068] 5) Prepare a tertiary probe hybridization solution (25% formamide, 2×SSC, 1% Triton X-100, 10% dextran sulfate, 0.1% DEPC, 5 nM tertiary probe). Immerse the cells in the tertiary probe hybridization solution and incubate at 37°C on a shaker for 3 h. Then wash the cells three times at 37°C for 10 min each time with a washing buffer containing 25% formamide (2×SSC, 1% Triton X-100, 25% formamide, 0.1% DEPC).
[0069] 6) Prepare a fluorescent hybridization solution (20% formamide, 2×SSC, 1% Triton X-100, 10% dextran sulfate, 0.1% DEPC, 40 nM fluorescent probe). Immerse the cells in the fluorescent probe hybridization solution and incubate at 37°C on a shaker for 0.5 h. Then wash the cells three times at 37°C for 10 min each time with a washing buffer containing 20% formamide (2×SSC, 1% Triton X-100, 20% formamide, 0.1% DEPC).
[0070] 7) Finally, stain with DAPI for 15 min and rinse the sample 3 times with 1×PBS, then place it at 4℃ for imaging. Next, place a circular coverslip with cell samples attached into a magnetic confocal imaging dish for imaging.
[0071] This invention utilizes ssDNA probes synthesized using the LPA-DAGE method for RNA FISH experiments, such as... Figure 3 As shown in Figure A, the bottom targeting region of the pedestal probe binds complementary to the target RNA, while the upper region binds to the non-repetitive sequence of the primary amplification probe. The 40nt repetitive sequence region of the primary amplification probe can bind to the non-repetitive sequence of the secondary amplification probe; similarly, the 20nt repetitive sequence region of the secondary amplification probe can bind to the fluorescent probe. This branched amplification strategy achieves multi-stage signal amplification while reducing hybridization background signal.
[0072] To achieve optimal hybridization results, this invention tested four "split" type base probes targeting the Actb gene in NIH3T3 cells: Circle C, Double Z, Double C, and Cruciform. The experimental results are as follows... Figure 3 As shown in B and C, under the same laser excitation parameters, the cells targeted by the "C-shaped" probe exhibit a high-density signal distribution. Quantitative analysis shows that, compared with the other three base probes, the "C-shaped" probe has a higher signal-to-noise ratio (SNR = 27.17 ± 5.65, Mean ± SEM, n = 30).
[0073] To assess the specificity of hybridization, EYFP protein and FISH targeting EYFP mRNA were simultaneously detected in NIH3T3 cells transiently transfected with the pCAG-EYFP plasmid. Imaging analysis showed... Figure 3 As shown in Figure D, the Actb probe exhibited a high-density signal distribution in all detected cells, while the EGFP mRNAFISH signal and the green fluorescent protein signal were distributed in the same cell.
[0074] To further analyze the signal amplification effect of the prepared ssDNA probe, this invention selected Actb from NIH3T3 cells as the target molecule. Under the condition of having the same number of base probes, its signal detection effect was compared with that of smFISH and π-FISH. Schematic diagrams and imaging results of the ssDNA probe system established in this invention, along with those of smFISH and π-FISH probe systems, are shown below. Figure 3 As shown in E, the fluorescence imaging results allow for a direct observation of significant differences in signal distribution characteristics. For example... Figure 3 Quantitative analysis results showed that the single-stranded probe system based on the branched DNA strategy had a significant advantage in signal-to-noise ratio (SNR = 27.17 ± 5.65). Combined with fluorescence signal intensity profiles detected by different methods, it can be found that under the same exposure conditions, its fluorescence intensity distribution curve exhibits a steeper gradient characteristic.
[0075] Example 5: Application of DNAFISH probes in ploidy detection in HeLa cells
[0076] 1. After HeLa cells adhered to the plate overnight, the DMEM medium was aspirated, and the cells were washed twice with PBS. Then, the cells were fixed with 4% PFA for 10 min, and washed three times with 1X PBS for 5 min each time.
[0077] 2. After fixation, permeabilize by sequentially treating with 0.5% PBST for 10 min, 0.1% PBSTw for 2 min, 0.1M HCl for 5 min, and washing twice with 2×SSCT for 5 min each time.
[0078] 3. Next, denature the sample with a mixture of 50% (v / v) formamide-2XSSCT (50% formamide, 2×SSC, 0.1% Triton X-100) at room temperature for 10 min, then denature it at 60°C for 1 h, and then denature it at 75°C for 10 min with a mixture of 70% (v / v) formamide-2XSSCT (70% formamide, 2×SSC, 0.1% Triton X-100) preheated to 75°C. Immediately transfer the sample to 70% ethanol precooled at -20°C for 5 min, and then treat it with 90% and 100% ice-cold ethanol for 5 min each.
[0079] 4. After air drying, add hybridization buffer containing 40% formamide, which includes 2×SSCT, 40% (v / v) formamide, 10% (w / w) dextran sulfate, 40 ng / μL RNase A, and 1 μg / μL single-stranded DNA probe. Place the sample in a water bath and incubate overnight at 37°C.
[0080] 5. After hybridization, the samples were washed twice in 2×SSCT at 60℃ for 15 minutes each time, and then washed twice in 2×SSCT at room temperature for 5 minutes each time.
[0081] 6. Finally, after incubating with DAPI at room temperature for 15 min, rinse three times with PBS and soak in PBS for imaging.
[0082] like Figure 4 As shown in Figure A, 48 targeting sequences were designed based on different centrioles of HeLa cells chromosome 17. Using these sequences as templates, dsDNA was amplified by PCR. Then, polyacrylamide-modified ssDNA and FAM-labeled ssDNA were separated by alkaline agarose gel electrophoresis. The recovered FAM probes were as follows: Figure 4 As shown in B, it can be used for chromosome DNA FISH detection. Figure 4 As shown in C and D, based on the signal labeling and quantitative analysis results, it can be found that chromosome 17 in the tested HeLa cells exhibits significant ploidy heterogeneity: triploids account for the highest proportion (56.77±10.17%), followed by diploids (28.37±0.75%), and a small number of other polyploids also exist (14.86±10.91%).
[0083] Example 6: Long ssDNA used for DNA origami
[0084] This invention uses the following steps to prepare DNA origami structures: First, a DNA origami reaction mixture is prepared in a PCR tube: a 5 nM long ssDNA scaffold and a 100 nM excess of oligonucleotide sequences (staple strands) are added to a 1×TAE / Mg solution. 2+Mix the buffer solution to a final reaction volume of 100 μL. Then, place the mixture in a PCR thermal cycler for annealing, following the procedure as follows: reduce the temperature from 80°C to 75°C at a rate of 1°C per minute; reduce the temperature from 75°C to 30°C at a rate of 0.5°C per minute; reduce the temperature from 30°C to 20°C at a rate of 1°C per minute, and finally maintain the temperature at 4°C. After annealing, the DNA origami product for electrophoresis and imaging is obtained.
[0085] The assembly effect of the annealed products was evaluated using 1.5% agarose gel electrophoresis, and the results are as follows: Figure 5 As shown in A and C, from left to right, lane 1 represents the long ssDNA scaffold, and lane 2 represents the thermally cycled DNA origami composite system. Electrophoresis results show that lane 1 contains only a single ssDNA scaffold band, while lane 2 contains no other impurity bands besides the target DNA origami product and excess short nucleotide sequences (staple chains). This result indicates that the ssDNA recovered in this invention has high purity and high yield, and the DNA origami structure obtained through annealing is stable and uniform.
[0086] Further morphological analysis of the two DNA nano-origami structures was performed using atomic force microscopy, such as... Figure 5 As shown in Figures B and D, regular ring and quadrilateral structures can be observed in both the two-dimensional and three-dimensional topological images, along with the periodic arrangement of surface trenches. These characteristics are consistent with the preset two-dimensional lattice design parameters. Measurements show that the diameter of the ring structure is 38.06 ± 0.55 nm, and the width of the quadrilateral structure is 50.76 ± 0.87 nm, which highly match the predicted values from the computer-aided design model (ring: theoretical size 38 nm; quadrilateral: theoretical size 50 nm). Figure 5 As shown in E and F. Statistical results show that both the formed ring-shaped and quadrilateral origami structures were fully folded with high yields. Specifically, the folding yield of the ring-shaped structure was 92.14 ± 2.56%, and the folding yield of the quadrilateral structure was 79.82 ± 4.69%. Figure 5 As shown in G.
[0087] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A method for purifying single-stranded DNA, characterized in that, The method includes the following steps: S1: Polymerization reaction was carried out using primers with acrylamide groups modified at the 5' end to generate linear polyacrylamide-modified primers LPA-primer; S2: Using the LPA-primer described in step S1 as a primer, polyacrylamide-crosslinked double-stranded DNA LPA-dsDNA was obtained by PCR amplification. S3: The LPA-dsDNA described in step S2 is subjected to denaturing agarose gel electrophoresis under alkaline conditions to separate the target ssDNA from the non-target ssDNA by utilizing the difference in migration rate caused by the polyacrylamide modification group. S4: The target ssDNA band described in step S3 is cut and recovered to obtain purified ssDNA.
2. The method according to claim 1, characterized in that, The polymerization reaction in step S1 is a reaction between a primer with an acrylamide group modified at the 5' end and a mixed reaction solution; the mixed reaction solution includes acrylamide, sodium acrylate, TBE and water; the mass percentage of acrylamide to sodium acrylate is 99:
1.
3. The method according to claim 1, characterized in that, The polymerization reaction described in step S1 is catalyzed by ammonium persulfate and tetramethylethylenediamine under anaerobic conditions.
4. The method according to claim 1, characterized in that, The PCR amplification reaction conditions described in step S2 are: 96℃ pre-denaturation for 3 min, 96℃ denaturation for 30 s, 55-60℃ annealing for 45 s, and 72℃ final extension for 5 min, for 28 cycles; the PCR amplification system described in step S2 consists of 100 nM primers, 200 μM dNTPs, 1×Pfu buffer, and 2.5 μL of KOD polymerase.
5. The method according to claim 1, characterized in that, The alkaline condition for step S3 is an electrophoresis buffer containing 30-50 mM NaOH.
6. The method according to claim 1, characterized in that, The target purified ssDNA in step S4 has a length of 100-2000 nt.
7. The application of ssDNA prepared by the method according to any one of claims 1-6 in the preparation of DNA origami structures.
8. The application according to claim 7, characterized in that, The DNA origami structure is a drug-loaded nanodevice.
9. The application of the ssDNA prepared by the method according to any one of claims 1-6 in the preparation of fluorescent in situ hybridization probes.
10. The application according to claim 9, characterized in that, The fluorescent in situ hybridization probe is a ring-shaped signal amplification probe.