An industrialized method for improving the efficiency of gene amplification of trace cells

Abstract

The present invention belongs to the field of gene amplification technology, and specifically relates to a low-cost batch processing method for improving the efficiency of micro-cell gene amplification. The processing method for improving the efficiency of micro-cell gene amplification includes the following steps: step 1. material preparation; step 2. PBS cleaning; step 3. cell pipetting; step 4. sample blowing; step 5. terahertz wave irradiation; step 6. PCR reaction configuration; step 7. gene amplification; step 8. agarose gel electrophoresis, and the material preparation is to prepare a magnetron, a power supply, a rectangular waveguide excitation cavity, a microwave leak detector, an ice maker, a Taq enzyme, and a centrifuge. The invention uses the terahertz band to cause resonance of nucleic acids, proteins, polysaccharides, and lipid molecules, which destroys the molecular spatial structure and removes the inhibitory effect of active biological macromolecules on DNA synthase, thereby improving the reaction yield. The core step of the process realizes the chemical change of biological macromolecules through physical methods, avoids the complex effect of adding additional chemical reagents and the lengthy processing flow, is low-cost, fast, repeatable, easy to use, can be enlarged, and can be applied in large-scale industry.

Description

Technical Field

[0001] The invention belongs to the technical field of gene amplification, and in particular relates to an industrialized processing method for improving the efficiency of trace cell gene amplification. Background Art

[0002] Micro-cell gene amplification and even single-cell genome amplification technology are the basis of single-cell gene sequencing analysis, which is the technology to amplify the DNA in a cell completely and accurately. In 2009, Tang Fuchou et al. (2009) invented the first single-cell RNA sequencing technology by improving the transcriptome amplification method of microarray analysis. In 2011, Navin et al. (2011) invented the first single-cell DNA sequencing technology. In 2013, "Science" magazine ranked single-cell sequencing as the top of the six most noteworthy fields of the year, and "Nature Methods" rated the technology as the most important methodological progress in 2013. Since 2015, single-cell sequencing technology has been widely used in basic scientific research and clinical research, and the corresponding results have also been favored by CNS. Articles have also frequently appeared in high-scoring journals, and single-cell gene amplification technology related to single-cell sequencing has also developed accordingly. Existing whole genome amplification methods can be divided into three categories according to their principles:

[0003] 1 PCR-based WGA method

[0004] 1.1 Degenerate Oligonucleotide Primer PCR

[0005] Primed PCR DOP-PCR, ):

[0006] 1.2 Ligation-mediated PCR

[0007] polymerase chain reaction LM-PCRR,)

[0008] 1.3 Pre-amplification primer extension reaction (PEP-PCR) primers are completely random primers composed of 15 nucleotides. PCR consists of 50 cycles, and the annealing temperature of each cycle is continuously increased from 37°C to 55°C, thereby ensuring that primers of different compositions anneal to as many genomic sequences as possible to amplify the entire genome.

[0009] 2MDA technology (Multiple displacement amplification): Primer hexamer (6 random nucleotides) anneals with template DNA at multiple sites, phi29 DNA polymerase initiates replication at multiple sites of DNA at the same time, synthesizes DNA along the DNA template, and replaces the complementary chain of the template at the same time. The displaced complementary chain becomes a new template for amplification.

[0010] 3ddMDA and eMDA technologies: These two technologies have the same principle, which is to place the MDA reaction into tens of millions of droplets. This can greatly improve the uniformity of genome amplification and greatly reduce the difference in amplification multiples between different regions.

[0011] 4TruePrime technology: The random hexamer primers of the original MDA technology are removed, and a primer synthesizer with DNA as a template is used to synthesize the primer directly, which increases the dissociation rate of the primer synthesizer, so that phi29 can continue to synthesize on the already synthesized primers. In theory, it ensures that each region is amplified with equal opportunities.

[0012] 5 Multiple Annealing and Looping

[0013] Based Amplification Cycles MALBAC, ):Combining the advantages of MDA amplification technology and PCR amplification technology, by using specially designed primers, the end of the amplicon is cleverly made to form a loop through complementarity, so that 93% of the genome in a single cell can be sequenced.

[0014] 6 A method based on Tn5 transposase insertion to achieve single-cell genome amplification. The basic principle is the same as the Tn5 library construction principle.

[0015] Among the above six technologies, the first four are related to gene chain amplification, and DNA polymerase activity is the key to improving the success rate of chain amplification. There are many reasons for amplification failure, and the inhibition of the activity of key enzymes in gene chain amplification is the key to failure. Common amplification enzyme inhibitors are SDS, phenol, ethanol, xylidine, isopropanol, sodium acetate, sodium chloride, EDTA, heme, heme chloride, tannic acid, bromophenol blue, heparin, urea, humus, complex polysaccharides, polystyrene, polypropylene, iron ions, bilirubin, bile salts, collagen, melanin, and proteases.

[0016] When gene amplification is performed on cells, the cell metabolites are different due to the differences in cells. For example, melanoma cells (B16F10, B16BL6, B78D4, SK-MEL-5, SK-MEL-28, SK-MEL-31, MeWo, WM3734) will produce a large amount of melanin, which inhibits gene amplification. Fibroblasts, chondrocytes, osteoblasts, epithelial or endothelial cells can synthesize procollagen, bone collagen and collagen in the basement membrane in connective tissue, respectively, to inhibit gene amplification. Some cells produce more polysaccharides and proteases, which will inhibit gene amplification.

[0017] Therefore, many companies have launched amplification kits that can counteract amplification inhibition. Its main mechanism of action is to add antagonists to the inhibitor. The main antagonists are listed below. Betaine, dimethyl sulfoxide (DMSO), formamide, glycerol, BSA, gelatin, tween-20, NP-40, Triton X-100, tetramethylammonium chloride, PEG, spermidine. However, these kits have different formulas from different companies, and the price is high. For industrial production, the cost is unbearable, and new technologies need to be developed.

[0018] Terahertz waves, also known as T waves, are electromagnetic waves located between microwaves and infrared rays in the electromagnetic spectrum. Terahertz radiation is generally understood as electromagnetic radiation with a frequency range from about 0.1THz to 10THz, corresponding to a wavelength of 3mm to 30μm in a vacuum. Such frequencies are higher than those of radio waves and microwaves, but lower than those of infrared light. Since the wavelength is in the range of 0.03mm to 3mm and is usually less than 1mm, terahertz radiation is sometimes also called submillimeter radiation. We propose an industrialized method for improving the efficiency of gene amplification in trace cells to solve the above problems. Summary of the invention

[0019] The purpose of the present invention is to provide an industrialized treatment method for improving the efficiency of trace cell gene amplification, which can use the terahertz band to cause resonance of nucleic acids, proteins, polysaccharides, and lipid molecules, thereby destroying the molecular spatial structure and inactivating biological macromolecules. It does not use chemical reagents, avoids the complex effects of chemical reagents and lengthy development processes, is low-cost, fast, repeatable, easy to use, scalable, and can be applied industrially on a large scale.

[0020] The technical solution adopted by the present invention is as follows:

[0021] An industrialized treatment method for improving the efficiency of gene amplification of trace cells, using terahertz waves with a wavelength of 1-5THz to instantly destroy DNA synthase inhibitors naturally present in cells, using terahertz waves with a power of 300-1500W to instantly destroy DNA synthase inhibitors naturally present in cells, and using terahertz waves to destroy DNA synthase inhibitors naturally present in cells within 1-3 minutes;

[0022] The processing method for improving the efficiency of micro-cell gene amplification comprises the following steps:

[0023] Step 1. Material preparation;

[0024] Step 2. PBS washing;

[0025] Step 3. Cell pipetting;

[0026] Step 4. Sample blowing;

[0027] Step 5. Terahertz wave irradiation;

[0028] Step 6. PCR reaction configuration;

[0029] Step 7. Gene amplification;

[0030] Step 8. Agarose gel electrophoresis.

[0031] In a preferred embodiment, the material preparation includes a magnetron, a power supply, a rectangular waveguide excitation cavity, a microwave leak detector, an ice maker, Taq enzyme, a centrifuge, a gene amplifier, an incubator, a culture bottle, a cell scraper, a pipette, a centrifuge tube, and a terahertz wave generator.

[0032] In a preferred embodiment, the PBS washing is to take out the cells that need to be gene amplified from the incubator, discard the liquid in the culture bottle by suction, wash them once with room temperature PBS, and discard the PBS by suction.

[0033] In a preferred embodiment, the cell pipetting is performed by scraping the cells with a cell scraper, and transferring the scraped cell suspension to a 1.5 mL centrifuge tube with a pipette.

[0034] In a preferred embodiment, the sample is blown by repeatedly blowing the cells 30-60 times, ice is taken from an ice maker, and the sample centrifuge tube is inserted on the ice.

[0035] In a preferred embodiment, the terahertz wave irradiation is to transfer the centrifuge tube to a terahertz wave generator and irradiate for 1-10 minutes using 0THz, 3THz, 6THz, 9THz wavelengths and 300W power, respectively.

[0036] In a preferred embodiment, the PCR reaction is configured as 10ul of 10× amplification buffer, 200umol / L of each of the four dNTP mixtures, 10-100pmol of each primer, 0.1-2ug of template DNA, 2.5u of TaqDNA polymerase, 1.5mmol / L of Mg2+, and double distilled water is added to 100ul.

[0037] In a preferred embodiment, the gene amplification is DNA pre-denaturation: 96°C for 3 min, followed by DNA pre-denaturation: 96°C for 30 s, annealing: 55°C for 30 s, extension: 72°C for 60 s, and 72°C for 300 s, and the above conditions are used for 30 sets of amplification cycles.

[0038] In a preferred embodiment, the agarose gel electrophoresis includes the preparation of agarose gel, gel casting, sample loading, electrophoresis, staining and observation. The preparation of agarose gel is to weigh agarose and dissolve it in electrophoresis buffer, according to the agarose content of 0.3-1.5%, heat it in a microwave oven or a boiling water bath until it is completely dissolved, take it out and shake it well. The gel casting is to gently pour the agarose solution cooled to 80°C into the horizontal plate of the electrophoresis tank, add electrophoresis buffer into the electrophoresis tank after the agarose gel solidifies, and then pull out the comb. The sample loading is to mix the DNA sample with the sample loading buffer according to After mixing at a ratio of 4:1, add the mixed solution to the sample tank with a micropipette, add 10-20ul to each tank, record the sample spotting order and sample addition amount, the electrophoresis is to install the electrode wire, connect one end of the spotting hole to the negative electrode and the other end to the positive electrode, turn on the power, adjust the voltage to 3-5V / cm, electrophoresis for 0.5-3hr, when bromophenol blue moves to 1-2cm away from the front of the gel, stop the electrophoresis, stain and observe the gel, put it in a staining solution containing ethidium bromide for 30min, observe it under a 254nm ultraviolet lamp, and record the electrophoresis spectrum by photography.

[0039] In a preferred embodiment, the magnetron is a Samsung magnetron produced in Malaysia, the power supply is an adjustable power supply of the terahertz quantum generator of Shanghai Lvqing Industry, the assembly is according to "Terahertz Solid-State Electronic Devices and Circuit Assembly" written by Feng Zhihong et al., the rectangular waveguide excitation cavity is purchased from Zhejiang Jiaxing Xingda Accessories Factory, the microwave leak detector is purchased from Shenzhen Huashengchang Technology Industry Co., Ltd., the ice maker is purchased from Shanghai Biaolong, the Taq enzyme is purchased from Beijing Boosen Biotechnology Co., Ltd., the centrifuge is purchased from Wuhan Seville Biotechnology Co., Ltd., and the gene amplifier is purchased from Beijing Liuyi Factory.

[0040] The technical effects achieved by the present invention are:

[0041] The terahertz band is used to induce resonance of nucleic acids, proteins, polysaccharides, and lipid molecules, thereby destroying the molecular spatial structure and inactivating biological macromolecules. Chemical reagents are not used, thus avoiding the complex effects of chemical reagents and lengthy development processes. The method is low-cost, fast, repeatable, easy to use, and can be enlarged and applied in large-scale industries.

[0042] Depending on the required sample size and objectives, different concentrations of agarose gel can be selected to obtain the best electrophoresis results. After staining with ethidium bromide, the electrophoresis pattern can be observed and recorded under ultraviolet light to facilitate analysis and interpretation of the results.

[0043] The configuration and operation steps of the PCR reaction conform to the requirements of conventional PCR experiments and are easy to implement. The processing method is applicable to different types of samples and can be used to detect the destruction and amplification of proteins, polysaccharides and large segments of DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a staining result diagram of EB of the present invention;

[0045] Figure 2 The electrophoresis pattern was recorded photographically under ultraviolet light;

[0046] Figure 3 Promote amplification efficiency electrophoresis profile. DETAILED DESCRIPTION

[0047] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0048] See also Figure 1 As shown, the present invention provides an industrialized processing method for improving the efficiency of trace cell gene amplification, using terahertz waves with a wavelength of 1-5THz to instantly destroy the DNA synthase inhibitors naturally present in the cells, using terahertz waves with a power of 300-1500W to instantly destroy the DNA synthase inhibitors naturally present in the cells, and using terahertz waves to destroy the DNA synthase inhibitors naturally present in the cells within 1-3 minutes;

[0049] The processing method for improving the efficiency of gene amplification of trace cells includes the following steps:

[0050] Step 1. Material preparation;

[0051] Step 2. PBS washing;

[0052] Step 3. Cell pipetting;

[0053] Step 4. Sample blowing;

[0054] Step 5. Terahertz wave irradiation;

[0055] Step 6. Gene amplification;

[0056] Step 7. Agarose gel electrophoresis.

[0057] Materials preparation includes magnetron, power supply, rectangular waveguide excitation cavity, microwave leak detector, ice maker, Taq enzyme, centrifuge, gene amplifier, incubator, culture bottle, cell scraper, pipette, centrifuge tube, terahertz wave generator;

[0058] PBS washing: Take out the cells that need to be gene amplified from the incubator, discard the liquid in the culture bottle, wash them with room temperature PBS, and discard the PBS;

[0059] Cell pipetting is to scrape the cells with a cell scraper and transfer the scraped cell suspension to a 1.5 mL centrifuge tube with a pipette;

[0060] The sample was pipetted by repeatedly pipetting the cells 30-60 times, ice was taken from the ice maker, and the sample centrifuge tube was inserted on ice;

[0061] Terahertz wave irradiation is to transfer the centrifuge tube to a terahertz wave generator and irradiate for 1-10 minutes using 0THz, 3THz, 6THz, 9THz wavelengths and 300W power respectively;

[0062] The PCR reaction was configured as follows: 10ul of 10× amplification buffer, 200umol / L of each of the four dNTP mixtures, 10-100pmol of each primer, 0.1-2ug of template DNA, 2.5u of TaqDNA polymerase, 1.5mmol / L of Mg2+, and double distilled water to 100ul;

[0063] Gene amplification was performed as follows: DNA pre-denaturation: 96°C for 3 min, followed by DNA pre-denaturation: 96°C for 30 s, annealing: 55°C for 30 s, extension: 72°C for 60 s, and 72°C for 300 s. The above conditions were used for 30 amplification cycles.

[0064] Agarose gel electrophoresis includes the preparation of agarose gel, gel filling, sample loading, electrophoresis, staining and observation. The preparation of agarose gel is to weigh agarose and dissolve it in electrophoresis buffer. According to the agarose content of 0.3-1.5%, it is placed in a microwave oven or a boiling water bath and heated until it is completely dissolved. Take it out and shake it well. Gel filling is to gently pour the agarose solution cooled to 80°C into the horizontal plate of the electrophoresis tank. After the agarose gel solidifies, add electrophoresis buffer into the electrophoresis tank, and then pull out the comb. Sample loading is to mix the DNA sample with the sample loading buffer at a ratio of 4:1. Use a micropipette to add the mixed solution to the sample slot, add 10-20ul to each slot, record the order and amount of sample spotting, install the electrode wire for electrophoresis, connect one end of the spotting hole to the negative electrode and the other end to the positive electrode, turn on the power, adjust the voltage to 3-5V / cm, and perform electrophoresis for 0.5-3hr. When bromophenol blue moves to 1-2cm from the front of the gel, stop the electrophoresis. For staining and observation, take out the gel, put it in the staining solution containing ethidium bromide for staining for 30min, and then observe it under a 254nm ultraviolet lamp, and record the electrophoresis pattern by photography;

[0065] The magnetron used was Samsung magnetron produced in Malaysia, the power supply used was adjustable power supply of terahertz quantum generator produced by Shanghai Lvqing Industry, and the assembly was based on the book "Terahertz Solid-State Electronic Devices and Circuit Assembly" written by Feng Zhihong et al. The rectangular waveguide excitation cavity was purchased from Xingda Parts Factory in Jiaxing, Zhejiang, the microwave leak detector was purchased from Shenzhen Huashengchang Technology Industry Co., Ltd., the ice maker was purchased from Shanghai Biaolong, the Taq enzyme was purchased from Beijing Bioson Biotechnology Co., Ltd., the centrifuge was purchased from Wuhan Sewell Biotechnology Co., Ltd., and the gene amplifier was purchased from Beijing Liuyi Factory.

[0066] Embodiment 2

[0067] 1. Terahertz wave damage to protein structure

[0068] 1.1 Materials:

[0069] 1.1.130% acrylamide / N,N'-methylbisacrylamide (Acr / Bis): 29g acrylamide, 1g N,N'-methylbisacrylamide, add distilled water to 100mL. 2.10% ammonium persulfate (Aps): 0.4g ammonium persulfate, 4mL distilled water, TEMED (N,N,N',N'-tetramethylethylenediamine).

[0070] 1.1.2 1.5 mol / L Tris-HCl (pH 8.8): Take 50 mL of 3 mol / L Tris mother solution, adjust the pH to 8.8 with hydrochloric acid, and dilute to 100 mL with redistilled water (store at 4°C);

[0071] 1.1.30.5mol / L Tris-HCl (pH 6.8): Take 50mL of 1mol / L Tris solution, adjust the pH to 6.8 with hydrochloric acid, and make up to 100mL with redistilled water (store at 4℃).

[0072] 1.1.4 10% sodium dodecyl sulfate (SDS) (stored at room temperature).

[0073] 1.1.5 1×TBE buffer: 89 mol / L Tris-borate, 2 mol / L EDTA (pH 8.0).

[0074] 1.1.6 Type I loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% sucrose aqueous solution (W / V) stored at 4°C.

[0075] 1.1.7 Sealing glue: 1% agarose solution (prepared with distilled water).

[0076] 1.1.8 Staining solution: 0.2g Coomassie Brilliant Blue R-250, 84mL 95% ethanol, 20mL glacial acetic acid, dilute to 200mL, filter and set aside.

[0077] 1.1.9 Decolorizing solution: medical alcohol: glacial acetic acid: water = 4.5:0.5:5 (V:V:V).

[0078] 1.1.10 Preservative solution: 7% glacial acetic acid.

[0079] 1.1.11 Glass plates, spacers, electrophoresis tanks, beakers, magnetic stirring bars, microwave ovens or heaters, balances, combs, UV analyzers

[0080] 1.2 Steps:

[0081] 1.2.1 Prepare 100mL gel solution: 37.5mL distilled water. Heat in microwave for 30s, stir with stirring rod 50g urea 10×TBE buffer 8mL 30% acrylamide 10mL before pouring 10% ammonium persulfate 0.8mL TEMED 23.5μL

[0082] 1.2.2 Clean the two glass plates, one long and one short, and the spacer between the two plates with detergent, and then wipe them dry with ethanol.

[0083] 1.2.3 Align one side of the long and short plates, insert a spacer about 1.5 cm wide, and clamp the three sides of the glass plate with a clamp. Seal the three sides of the rubber plate with a sealing glue (1% agarose solution).

[0084] 1.2.4 Add 10% ammonium persulfate and TEMED to the glue solution and mix well. Immediately pour the glue. To avoid bubbles, pour the glue slowly and carefully. If bubbles are generated, use a rubber hammer or pencil tip to tap the glue board from bottom to top to drive out the bubbles.

[0085] 1.2.5 When the glue is about 0.5cm from the upper edge of the short glass plate, stop adding glue. Gently insert a comb of appropriate size, do not allow bubbles to form under the comb, clamp the comb and the glue plate, and then add the remaining glue to both sides of the comb.

[0086] 1.2.6 The coagulation time at room temperature is about 0.5 to 1 hour.

[0087] 1.2.7 Carefully remove the comb, clean the sample wells with water, fix the gel on the electrophoresis tank, and add 1×TBE buffer.

[0088] 1.2.8 Take out the cells from the incubator, discard the liquid in the culture bottle, wash them with room temperature PBS, and discard the PBS.

[0089] 1.2.9 Use a cell scraper to scrape off the cells. Use a pipette to transfer the scraped cell suspension to a 1.5 mL centrifuge tube.

[0090] 1.2.10 Repeatedly pipette and pump for 30 to 60 times and place the sample centrifuge tube on ice.

[0091] 1.2.11 Transfer the centrifuge tube to the terahertz wave generator and irradiate for 1-10 minutes using 0THz, 3THz, 6THz, 9THz wavelengths and 300W power, respectively.

[0092] 1.2.12 Take the cell sample and mix with 6 times type I loading buffer. Add 20 μL of sample to each well (it is recommended to add sample first and then working solution to ensure complete reaction). The voltage gradient is 1V / cm to 8V / cm.

[0093] 1.2.13 After electrophoresis, remove the gel and stain it in EB for 30 minutes. The concentration of EB is (0.5 μg / mL 1x TBE buffer).

[0094] Analyze and take photos under UV light. Figure 1 .

[0095] 2. Terahertz wave damage to polysaccharide structure

[0096] 2.1 Materials:

[0097] 2.1.1 Electrode buffer (borax-sodium hydroxide buffer, pH 9.4): Dissolve 4.77 g borax and 0.445 g NaOH, adjust the pH to 9.4, and make up to 1000 mL.

[0098] 2.1.2 Schiff reagent: Weigh 1g of basic fuchsin, dissolve it in 200mL of boiling water for 5min, cool to 40-50℃, add 1.37g of sodium metabisulfite and 20mL of 1mol·L-1HCl, cool to room temperature, add 2 spoons of activated carbon and stir until the solution becomes colorless and transparent, filter, and store in a refrigerator at 4℃ away from light (Zhang Weijie 1999).

[0099] 2.1.31% periodic acid solution: weigh 1g periodic acid and dissolve it to make up to 100mL.

[0100] 2.1.4 0.5% sodium metabisulfite: weigh 1g sodium metabisulfite and dissolve it to make up to 200mL.

[0101] 2.2 Steps:

[0102] 2.2.1 Prepare 100mL gel solution: 37.5mL distilled water. Heat in microwave for 30s, stir with stirring rod 50g urea 10×TBE buffer 8mL 30% acrylamide 10mL before pouring 10% ammonium persulfate 0.8mL TEMED 23.5μL

[0103] 2.2.2 Clean the two glass plates, one long and one short, and the spacer between the two plates with detergent, and then wipe them dry with ethanol.

[0104] 2.2.3 Align one side of the long and short plates, insert a spacer about 1.5 cm wide, and clamp the three sides of the glass plate with a clamp. Seal the three sides of the rubber plate with a sealing glue (1% agarose solution).

[0105] 2.2.4 Add 10% ammonium persulfate and TEMED to the glue solution and mix well. Immediately pour the glue. To avoid bubbles, pour the glue slowly and carefully. If bubbles are generated, use a rubber hammer or pencil tip to tap the glue board from bottom to top to drive out the bubbles.

[0106] 2.2.5 When the glue is about 0.5cm from the upper edge of the short glass plate, stop adding glue. Gently insert a comb of appropriate size, do not allow bubbles to form under the comb, clamp the comb and the glue plate, and then add the remaining glue to both sides of the comb.

[0107] 2.2.6 The coagulation time at room temperature is about 0.5 to 1 hour.

[0108] 2.2.7 Carefully remove the comb, clean the sample wells with water, fix the gel on the electrophoresis tank, and add 1×TBE buffer.

[0109] 2.2.8 Take out the cells from the incubator, discard the liquid in the culture bottle, wash them with room temperature PBS, and discard the PBS.

[0110] 2.2.9 Use a cell scraper to scrape off the cells. Use a pipette to transfer the scraped cell suspension to

[0111] 1.5mL centrifuge tube.

[0112] 2.2.10 Repeatedly pipette and pump for 30 to 60 times and place the sample centrifuge tube on ice.

[0113] 2.2.11 Transfer the centrifuge tube to the terahertz wave generator and irradiate for 1-10 minutes using 0THz, 3THz, 6THz, 9THz wavelengths and 300W power respectively.

[0114] 2.2.12 Take the cell sample and mix with 6 times type I loading buffer. Add 20 μL of sample to each well (it is recommended to add sample first and then working solution to ensure complete reaction). The voltage gradient is 1V / cm to 8V / cm.

[0115] 2.2.13 Borax-NaOH buffer system with pH 9.4 was used as electrode buffer for electrophoresis.

[0116] 2.2.14 Perform PAS staining under different treatment conditions on a shaker (speed of 85 r·min-1). First, fix with 10% trichloroacetic acid (TCA) for different times of 5 to 30 minutes, and wash the gel with distilled water 2 to 3 times. Add 1% periodic acid for 15 to 30 minutes of oxidation reaction; then wash with distilled water 3 times, 5 minutes each time, to thoroughly wash out the periodic acid; add Schiff reagent, stain at room temperature in the dark for 30 minutes; pour out the staining solution, wash the gel 3 times with 0.5% newly prepared sodium metabisulfite, change every 5 minutes (can be tested by adding formaldehyde, if there is no color change, it means it is clean), and store in 7% acetic acid solution.

[0117] 3. Terahertz wave fragmentation of large segments of DNA

[0118] 3.1 Materials: electrophoresis apparatus, electrophoresis tank, UV transflectometer, constant temperature water bath, microwave oven, microinjector, tris(hydroxymethyl)aminomethane, hydrochloric acid, sodium acetate, EDTA, agarose, bromophenol blue, ethidium bromide.

[0119] 3.2 Steps:

[0120] 3.2.1 Install the electrophoresis tank: Wash the organic glass electrophoresis gel bed, dry it, seal the openings at both ends with tape, place it on a horizontal workbench, and insert the sample comb.

[0121] 3.2.2 Preparation of agarose gel: Weigh agarose and dissolve it in electrophoresis buffer (according to 0.3-1.5% agarose content, use 1% gel for DNA of 1-25kb, 0.5% gel for DNA of 20-100kb, and 1.5% gel for DNA of 200-2000bp). Heat in a microwave oven or boiling water bath until completely dissolved (do not heat to boiling), take out and shake well.

[0122] 3.2.3 Gel pouring: Gently pour the agarose solution cooled to 60°C onto the horizontal plate of the electrophoresis tank.

[0123] 3.2.4 After the agarose gel solidifies, add electrophoresis buffer into the electrophoresis tank and then remove the comb.

[0124] 3.2.5 Take out the cells from the incubator, discard the liquid in the culture bottle, wash them with room temperature PBS, and discard the PBS.

[0125] 3.2.6 Use a cell scraper to scrape off the cells and transfer the scraped cell suspension to a 1.5 mL centrifuge tube using a pipette.

[0126] 3.2.7 Repeat the pipetting for 30 to 60 times and place the sample centrifuge tube on ice.

[0127] 3.2.8 Transfer the centrifuge tube to the terahertz wave generator and irradiate for 1-10 minutes using 0THz, 3THz, 6THz, 9THz wavelengths and 300W power respectively.

[0128] 3.2.9 Sampling: Take cell samples and

[0129] 3.2.10 After mixing the sample buffer at a ratio of 4:1, use a micropipette to add the mixed solution to the sample slot, adding 10-20 μl to each slot, and record the sample spotting order and sample volume.

[0130] 3.2.11 Electrophoresis: Install the electrode wires, connect one end of the sample well to the negative electrode and the other end to the positive electrode, turn on the power, adjust the voltage to 3-5V / cm, and perform electrophoresis for 1-3 hours. Stop the electrophoresis when bromophenol blue moves to 1-2cm away from the front of the gel.

[0131] Staining and observation: Take out the gel and stain it in a staining solution containing ethidium bromide for 30 minutes. Then observe it under a 254nm ultraviolet lamp, or take a photo under a ultraviolet lamp to record the electrophoresis pattern. Figure 2 .

[0132] 4. The promotion of terahertz waves on gene amplification

[0133] 1. Take out the cells from the incubator, discard the liquid in the culture bottle, wash them with room temperature PBS, and discard the PBS.

[0134] 2. Use a cell scraper to scrape off the cells. Use a pipette to transfer the scraped cell suspension to a 1.5 mL centrifuge tube.

[0135] 3. Repeatedly pipette and beat for 30 to 60 times and place the sample centrifuge tube on ice.

[0136] 4. Transfer the centrifuge tube to the terahertz wave generator and irradiate for 1-10 minutes using 0THz, 3THz, 6THz, 9THz wavelengths and 300W power respectively.

[0137] 5. Configure the PCR reaction system according to the table below.

[0138]

[0139] 6. Perform gene amplification reaction according to the table below.

[0140] a) DNA pre-denaturation: 96℃3min

[0141] b) Perform 30 amplification cycles under the following conditions

[0142] i. DNA pre-denaturation: 96℃30S

[0143] ii. Annealing: 55℃30S

[0144] iii. Extension: 72℃ 60S

[0145] c)72℃300S

[0146] d) 4℃∞

[0147] 7. Agarose Gel Electrophoresis

[0148] a) Preparation of agarose gel: Weigh agarose and dissolve it in electrophoresis buffer (according to 0.3-1.5% agarose content, use 1% gel for DNA of 1-25 kb, 0.5% gel for DNA of 20-100 kb, and 1.5% gel for DNA of 200-2000 bp). Heat in a microwave oven or boiling water bath until completely dissolved (do not heat to boiling), remove and shake well.

[0149] b) Gel pouring: Gently pour the agarose solution cooled to 60℃ into the horizontal plate of the electrophoresis tank. After the agarose gel solidifies, add electrophoresis buffer into the electrophoresis tank and then remove the comb.

[0150] c) Sample loading: Mix the DNA sample and loading buffer at a ratio of 4:1, and add the mixture to the sample wells using a micropipette. Add 10-20 μl to each well. Record the order of sample spotting and the amount of sample loaded.

[0151] d) Electrophoresis: Install the electrode wires, connect one end of the spot hole to the negative electrode and the other end to the positive electrode, turn on the power, adjust the voltage to 3-5V / cm, and perform electrophoresis for 1-3 hours. Stop the electrophoresis when the bromophenol blue moves to 1-2cm from the front of the gel.

[0152] Staining and observation: Take out the gel and stain it in a staining solution containing ethidium bromide for 30 minutes, then observe it under a 254nm ultraviolet lamp and record the electrophoresis pattern by photographing. Figure 3 .

[0153] In the present invention, the terahertz band is used to cause resonance of nucleic acids, proteins, polysaccharides, and lipid molecules, so as to destroy the molecular spatial structure and inactivate biological macromolecules. No chemical reagents are used, thus avoiding the complicated effects of chemical reagents and the lengthy development process. The invention has low cost, high speed, repeatability, convenience in use, scalability, and large-scale industrial application. Agarose gels of different concentrations can be selected according to the required sample size and target to obtain the best electrophoresis result. After staining with ethidium bromide, the electrophoresis pattern can be observed and recorded under ultraviolet light, which is convenient for analysis and interpretation of the results. The configuration and operation steps of the PCR reaction meet the requirements of conventional PCR experiments and are easy to implement. The treatment method is suitable for different types of samples and can be used to detect the destruction and amplification of proteins, polysaccharides, and large segments of DNA.

[0154] The above is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention shall be implemented according to the conventional means in the art unless otherwise specified and limited.

Claims

1. An industrialized method for improving the efficiency of gene amplification of trace cells, characterized in that: Terahertz waves are used to instantly destroy DNA synthase inhibitors naturally present in cells; the wavelength of the terahertz waves is 1-5 THz, and the power of the terahertz waves is 300-1500 W; the terahertz waves destroy DNA synthase inhibitors naturally present in cells within 1-3 minutes.

2. The industrialized method for improving the efficiency of gene amplification of trace cells according to claim 1, characterized in that: Step 1. Material preparation; Step 2. PBS washing; Step 3. Cell pipetting; Step 4. Sample blowing; Step 5. Terahertz wave irradiation; Step 6. PCR reaction configuration; Step 7. Gene amplification; Step 8. Agarose gel electrophoresis.

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