High-resolution DNA topoisomerization analysis method

Abstract

The invention provides a high-resolution DNA topoisomerism analysis method, and particularly belongs to the technical field of gene detection. The high-resolution DNA topoisomerization analysis method comprises the following steps: extracting DNA, and detecting the concentration of the DNA; preparing agarose gel; loading the DNA sample into agarose gel holes according to the same total amount; after the voltage intensity, the electrophoresis time and the environment temperature are set, agarose gel electrophoresis is carried out; after electrophoresis, cutting the gel, and transferring the film; and after transferring the membrane, taking out the nylon membrane, carrying out radiation hybridization and scanning imaging, and analyzing and interpreting DNA topoisomerism information. The method provided by the invention can realize high-resolution DNA topological structure analysis, can be widely applied to the fields of cytobiology, molecular biology, pathology, immunology, endocrinology and the like, and has important significance in the development of the fields of genetic disease diagnosis, gene therapy, DNA map analysis, detection of DNA in a sample and the content thereof, PCR product analysis and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection, and in particular relates to a high-resolution DNA topoisomerism analysis method. Background Art

[0002] The detection of supercoil topological structure of DNA is widely used in the fields of genetic disease diagnosis, DNA map analysis, PCR product analysis, gene copy number change detection, mutation detection, linkage analysis, gene genetic diversity analysis, gene rearrangement and structural variation detection, viral DNA detection, and characteristic analysis of specific regions of the genome. DNA topological isomerism analysis mainly includes gel preparation; electrophoresis; membrane transfer (i.e., transferring and binding the nucleic acid molecules to be determined to a certain solid support (nitrocellulose membrane or nylon membrane) by a certain method, i.e., imprinting); molecular hybridization (i.e., hybridizing the nucleic acid fixed on the membrane in the previous step with the isotope-labeled probe at a certain temperature and ionic strength); and development. Each step requires refined processing. If there is a problem in any step of operation, the result will be unsatisfactory and high-resolution result images cannot be obtained, such as the appearance of many non-specific bands, low band resolution, band deformation, band blur, impurities, too dark or too light background, etc., which will lead to poor final image quality. Therefore, obtaining clearly visible bands is of great significance to the development of DNA map analysis, detection of DNA and its content in samples, PCR product analysis, etc. Summary of the invention

[0003] The purpose of the present invention is to provide a high-resolution DNA topoisomerism analysis method. The method of the present invention can realize high-resolution DNA topoisomerism analysis.

[0004] The present invention provides a high-resolution DNA topoisomerism analysis method, comprising the following steps:

[0005] Extract DNA and detect DNA concentration;

[0006] Prepare agarose gel;

[0007] Load the DNA into the wells of the agarose gel in the same total amount;

[0008] After setting the voltage intensity, electrophoresis time and ambient temperature, perform agarose gel electrophoresis;

[0009] After electrophoresis, the gel is cut and transferred to a membrane; the transfer device is filled with a transfer buffer, a lower glass plate for bridging is placed parallel to the short side of the transfer tank and through the center of the transfer tank, and a first filter paper is placed on the lower glass plate to form a bridge to obtain a thick filter paper bridge; a second filter paper of the same size as the gel block is laid flat on the thick filter paper bridge; the cut gel block is placed on the second filter paper and laid flat; a nylon membrane of the same size as the gel block is placed on top of the gel block and laid flat; a third filter paper of the same size as the gel block is placed on top of the nylon membrane and laid flat; an absorbent paper of a size greater than or equal to the size of the gel block is placed on top of the third filter paper and laid flat; an upper glass plate is placed on top of the absorbent paper, and then a weight is placed;

[0010] After transfer, the nylon membrane is removed for radiation hybridization, scanning imaging, and analysis and interpretation of DNA topological isomerism information.

[0011] Preferably, DNA concentration is dually measured using a Nanodrop instrument and a Qubit4 instrument.

[0012] Preferably, the voltage intensity is 50V; the electrophoresis time is 48h; and the ambient temperature is 4°C.

[0013] Preferably, the gel cutting position includes: cutting the gel from 1.5 cm below the observed genomic DNA band until a length of 10 cm is cut out.

[0014] Preferably, each side of the absorbent paper is 0.5 to 1 cm longer than each side of the rubber block; and the number of the absorbent paper is 70 to 80 sheets.

[0015] Preferably, the thickness of the lower glass plate and the upper glass plate is 8 mm.

[0016] Preferably, the thickness of the first filter paper is 0.53 mm; the thickness of the second filter paper and the third filter paper are both 0.2 mm.

[0017] Preferably, the vertical centers of the first filter paper, the second filter paper, the rubber block, the nylon membrane, the third filter paper, the absorbent paper, the upper glass plate and the weight are on the same vertical line.

[0018] Preferably, there are no bubbles between the first filter paper, the second filter paper, the rubber block, the nylon membrane, the third filter paper, the absorbent paper, the upper glass plate and the weight, and they are all flat.

[0019] Preferably, the transfer buffer is a sodium hydroxide buffer; and the amount of the transfer buffer added is 2 / 3 of the volume of the transfer device.

[0020] The present invention provides a high-resolution DNA topoisomerism analysis method. The method of the present invention can realize high-resolution DNA topoisomerism analysis. It is of great significance to the development of genetic disease analysis, gene therapy, DNA vaccine research, DNA map analysis, DNA and its content in detection samples, PCR product analysis and other fields. The test results show that the bands obtained by the method of the present invention are very clear, the band separation effect is excellent, there is no overlap between the bands, the bands are straight, there is no tilt, there are no curved bands, and the background is light, there is no need to increase the exposure to display the bands, and the detailed information of each band can be very well judged. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0022] Figure 1 A schematic diagram of a transfer device provided in Example 1 of the present invention;

[0023] Figure 2 The tooth comb model for making glue holes provided in Example 1 of the present invention;

[0024] Figure 3 The agarose gel electrophoresis diagram provided in Example 1 of the present invention;

[0025] Figure 4 A diagram of the gel cutting process after electrophoresis provided in Example 1 of the present invention;

[0026] Figure 5 The membrane transfer flow chart provided in Example 1 of the present invention;

[0027] Figure 6 The hybridization result diagram provided in Example 1 of the present invention;

[0028] Figure 7 The hybridization result diagram provided in Example 2 of the present invention;

[0029] Figure 8 The hybridization result diagram provided in Example 3 of the present invention;

[0030] Fig. 9 The hybridization result diagram provided in Example 4 of the present invention;

[0031] Fig.10 The hybridization result diagram provided in Example 5 of the present invention;

[0032] Fig.11The hybridization result diagram provided for Comparative Example 1 of the present invention;

[0033] Fig.12 The hybridization result diagram provided for Comparative Example 2 of the present invention;

[0034] Fig.13 The hybridization result diagram provided for Comparative Example 3 of the present invention;

[0035] Fig.14 The hybridization result diagram provided for Comparative Example 4 of the present invention;

[0036] Fig.15 The hybridization result diagram provided for Comparative Example 5 of the present invention;

[0037] Fig.16 The hybridization result diagram provided for Comparative Example 6 of the present invention;

[0038] Fig.17 This is a hybridization result diagram provided for Comparative Example 7 of the present invention. DETAILED DESCRIPTION

[0039] The present invention provides a high-resolution DNA topoisomerism analysis method, comprising the following steps:

[0040] Extract DNA and detect DNA concentration;

[0041] Prepare agarose gel;

[0042] Load the DNA into the wells of the agarose gel in the same total amount;

[0043] After setting the voltage intensity, electrophoresis time and ambient temperature, perform agarose gel electrophoresis;

[0044] After electrophoresis, the gel is cut and transferred to a membrane; the transfer device is filled with a transfer buffer, a lower glass plate for bridging is placed parallel to the short side of the transfer tank and through the center of the transfer tank, and a first filter paper is placed on the lower glass plate to form a bridge to obtain a thick filter paper bridge; a second filter paper of the same size as the gel block is laid flat on the thick filter paper bridge; the cut gel block is placed on the second filter paper and laid flat; a nylon membrane of the same size as the gel block is placed on top of the gel block and laid flat; a third filter paper of the same size as the gel block is placed on top of the nylon membrane and laid flat; an absorbent paper of a size greater than or equal to the size of the gel block is placed on top of the third filter paper and laid flat; an upper glass plate is placed on top of the absorbent paper, and then a weight is placed;

[0045] After transfer, the nylon membrane is removed for radiation hybridization, scanning imaging, and analysis and interpretation of DNA topological isomerism information.

[0046] Extract DNA and detect DNA concentration. In the present invention, DNA can be extracted from plants, animals, yeast, etc. In a specific embodiment, the sample DNA is extracted using the classic phenol-chloroform extraction method. In a specific embodiment, the DNA concentration is double-detected using a Nanodrop instrument and a Qubit4 instrument. The Nanodrop instrument is a physical method, and the Qubit4 instrument is a chemical method. Two methods are used to determine the concentration and purity of the extracted DNA. The DNA quality is detected from the perspective of different detection principles of ultraviolet absorption and fluorescence intensity, and the amount of DNA loaded in each loading well can be accurately determined.

[0047] The present invention prepares agarose gel. The present invention uses a gel-making mold for preparation. In a specific embodiment, the gel-making mold has a length of 21.6 cm, 20 gel holes, each hole has a width of 0.7 cm, and each hole is 0.3 cm apart. The size of the actual produced gel block is 19.7 cm×30 cm. In a specific embodiment, 1% to 2% agarose gel can be prepared. In a specific embodiment, a microwave heating method can be used to prepare agarose gel. The agarose must be fully melted, low heat is used, and it is taken out and rotated and shaken several times until the boiled gel is clear, transparent, and uniform; or a magnetic stirring bar can be placed in a conical bottle for gel making, taken out halfway and placed on a magnetic stirrer and stirred slowly at a low speed to avoid bubbles, so as to prepare agarose gel with high uniformity and no bubbles, and the gel is poured after the gel is cooled to about 55°C, and the gel is solidified, and then the sample is loaded and electrophoresed. In a specific embodiment, the ambient temperature for loading and electrophoresis is 4°C.

[0048] After detecting the DNA concentration and obtaining the agarose gel, the present invention loads the DNA into the agarose gel wells with the same total amount. The present invention accurately determines the DNA loading amount. The present invention detects the DNA concentration of different samples, adjusts the DNA of all samples to the same concentration for loading, and the accurate total loading amount can make the brightness of the DNA bands of all samples close, the background noise signal is low, the bands are clear, and high-resolution DNA topoisomerism results can be obtained. The loading amount is arbitrary, and the loading amount of each sample is determined simply by observing and estimating the DNA concentration of electrophoresis under ultraviolet light. It is difficult to eliminate the low band resolution caused by inconsistent concentrations of different samples.

[0049] After setting the voltage intensity, electrophoresis time and ambient temperature, agarose gel electrophoresis is performed. The present invention performs electrophoresis, and it is necessary to ensure that the laboratory desktop and the electrophoresis tank are level. The present invention uses a spirit level to measure whether the electrophoresis tank is level, and the knob at the bottom of the electrophoresis tank can be adjusted to ensure that the overall structure of the electrophoresis tank on the desktop is horizontal, which is convenient for improving the agarose gel electrophoresis effect. If the desktop or the electrophoresis tank is not level, the DNA bands on the agarose gel may tend to one direction, resulting in uneven DNA bands, affecting band identification, and ultimately affecting the interpretation of the size of DNA topoisomerism bands. In a specific embodiment, the voltage intensity is 50V; the electrophoresis time is 48h; and the ambient temperature is 4°C. The above-mentioned voltage and electrophoresis time of the present invention can ensure that the DNA bands are fully separated. High voltage can easily cause DNA bands to shift crooked and can also easily cause DNA degradation due to heat generation, seriously affecting the topological isomerism structure of DNA; short electrophoresis time will result in insufficient band separation, insufficient separation of the DNA superhelical structure, and difficulty in interpretation, while long electrophoresis time will cause the DNA bands to run out of the agarose gel; an ambient temperature of 4°C indicates that electrophoresis must be performed in a 4°C cold room, and staff are required to wear cotton clothes and enter the 4°C cold room to load samples, and then leave the cold room after starting the electrophoresis, thereby avoiding DNA degradation due to excessively high ambient temperature, which affects the interpretation of the DNA superhelical structure.

[0050] After electrophoresis of the present invention, gel cutting is performed, and transfer is performed. The transfer film is filled with transfer buffer in the transfer device, and a lower glass plate for bridging is placed parallel to the short side of the transfer tank and through the center of the transfer tank, and a first filter paper is placed on the lower glass plate to bridge to obtain a thick filter paper bridge; a second filter paper of the same size as the gel block is laid flat on the thick filter paper bridge; the cut gel block is placed on the second filter paper and laid flat; a nylon membrane of the same size as the gel block is placed above the gel block and laid flat; a third filter paper of the same size as the gel block is placed above the nylon membrane and laid flat; an absorbent paper of a size greater than or equal to the size of the gel block is placed above the third filter paper and laid flat; an upper glass plate is placed above the absorbent paper, and then a weight is placed. In a specific embodiment, the position of the gel cutting includes: gel cutting from 1.5 cm below the linear genomic DNA band observed from a distance until a length of 10 cm is cut out. The present invention only retains this 10 cm length, and the rest is removed, and the blank gel part without DNA bands on the left and right sides of the gel electrophoresis channel is also cut off. Specifically, the present invention can be observed under the ultraviolet light of the gel imager, and at the same time, a special ruler that can transmit ultraviolet light is placed on the gel to measure, so that the position of the genomic DNA can be locked. Then the gel is taken out of the imager. Although the DNA bands cannot be seen without ultraviolet light at this time, the position of the DNA can be determined using the same ruler. If it is too close to the DNA bands, the supercoiled DNA will be affected by the genomic DNA. Extending 1.5cm downward to cut the gel can avoid the influence of the genomic DNA. Cutting down a length of about 10cm, if the length is less than 10cm, will cause part of the supercoiled DNA to not be included in the gel. If the length exceeds the range of 10cm, in addition to the supercoil, other bands may be included, which will affect the judgment of the experimental results. In a specific embodiment, the thickness of the lower glass plate and the upper glass plate is 8mm. The present invention places a glass plate (lower glass plate) above the electrophoresis tank. The glass plate needs to be very flat and able to withstand a certain weight pressure. The area needs to be larger than the area of ​​the gel block. The uneven glass plate will cause the filter paper, gel block, transfer film, absorbent paper, etc. to be uneven, and the bands will also be deformed. Insufficient thickness of the glass plate will cause the glass to break, because the glass plate needs to carry many items, such as filter paper, glue block, transfer film, absorbent paper, weights, etc., and in the whole transfer process, it will continue to absorb water and the weight will increase, so the glass plate needs to bear a certain weight. After placing the glass plate, the present invention places the first filter paper on the glass plate, and the first filter paper can also be called bridging filter paper. In a specific embodiment, the thickness of the first filter paper is 0.53mm. In a specific embodiment, the thickness of the second filter paper and the third filter paper is 0.2mm respectively. The present invention uses thick bridging filter paper for bridging. In a specific embodiment, a bridging filter paper with a width less than the width of the electrophoresis tank is used for bridging, and a large filter paper can also be used to meet the width requirement by cutting, and the filter paper is placed above the glass plate in a cross-shaped manner with the glass plate, and the two ends of the filter paper are immersed in the buffer solution of the electrophoresis tank.If the filter paper is too thin, it is easy to break during the transfer process. Both ends of the filter paper need to be soaked in the buffer of the electrophoresis tank at the same time to fully absorb the infiltration of the buffer. The width of the filter paper should be slightly smaller than the width of the inner diameter of the transfer tank, otherwise it will affect the uniform absorption of the buffer by the filter paper and the transfer cannot be completed. After placing the bridge filter paper, lay a second filter paper of the same size as the glue on the thick filter paper bridge. The second filter paper is ordinary filter paper; then place the cut glue block, specifically a 10cm long glue block (the width is not limited, it is determined according to the amount of sample loaded, but it cannot exceed the width of the bridge) flatly on the second filter paper. Make sure it is flat, wrinkles will cause wrinkles to appear on the membrane at this position, ensure that there are no bubbles between the glue block and the filter paper, and ensure that the transferred glue is complete. If the glue breaks during the transfer process, the DNA supercoil topoisomer presented at the end is also broken and discontinuous, which will affect the accuracy of DNA topoisomer interpretation. Afterwards, the present invention places a nylon membrane with the same area and size as the glue block on top of the glue block, and ensures that the size of the membrane and the glue block is consistent by determining that the length and width are completely consistent, accurate to the millimeter level. Transferring the membrane is to transfer all the genetic information on the glue block to the membrane, so it is necessary to ensure that the size of the membrane and the glue block is completely consistent. If the membrane is smaller than the glue block, some information on the glue block will be missed. If the membrane is larger than the glue block, it will cause waste, and after the hybridization is completed, the membrane needs to be cut again, otherwise the hybridization effect will be affected. The present invention needs to place a third filter paper on top of the nylon membrane, and the third filter paper is also an ordinary filter paper, or a small filter paper. In the present invention, the number of sheets of the third filter paper is one. Similarly, as mentioned above, it is necessary to ensure that the area of ​​the third filter paper and the nylon membrane are consistent. The present invention places absorbent paper on the third filter paper. In a specific embodiment, each side length of the absorbent paper is 0.5 to 1 cm longer than each side length of the glue block; the number of absorbent papers is 70 to 80 sheets. Each side length of the absorbent paper can ensure complete coverage below, which can prevent the absorbent paper from shrinking due to water absorption and affecting the transfer effect of the leftmost and rightmost bands of the gel. Placing about 70 to 80 sheets of absorbent paper can ensure that the electrophoresis buffer is just sucked dry and the DNA genetic information is just transferred to the nylon membrane. The number of absorbent papers is also a very important link. Too much absorbent paper can easily cause insufficient buffer and failure of membrane transfer; too little absorbent paper can easily cause insufficient membrane transfer and affect the experimental results. The present invention places an upper glass plate (flat glass plate) on top of the absorbent paper with a thickness of 8mm and an area larger than the absorbent paper to ensure that all items below can be fully covered. In a specific embodiment, the center of gravity of the first filter paper, the second filter paper, the glue block, the nylon membrane, the third filter paper, the absorbent paper, the upper glass plate and the weight are on the same vertical line. The first filter paper, second filter paper, glue block, nylon membrane, third filter paper, and absorbent paper between the lower glass plate above the electrophoresis tank and the upper glass plate above the absorbent paper are vertical and neatly stacked, that is, to ensure that the center of gravity of all items is consistent. If the center of gravity is tilted, it is easy to collapse, resulting in transfer failure. The glass plate above the absorbent paper needs to be flat and fully covered with absorbent paper to ensure uniform force.The two glass plates are of the same size, and everything between the glass plates needs to be vertical, otherwise it will cause uneven force and failure of transfer. The present invention selects weights of appropriate weight to be placed on the glass plate to ensure that the pressure is appropriate and the gap between the absorbent papers can be compressed. Compressing the gap between the absorbent papers can increase the transfer effect, and too much or too little pressure will affect the transfer effect. In a specific embodiment, there are no bubbles between the first filter paper, the second filter paper, the glue block, the nylon membrane, the third filter paper, the absorbent paper, the upper glass plate and the weights, and they are all flat. In a specific embodiment, the transfer buffer is a sodium hydroxide buffer; the amount of the transfer buffer added is 2 / 3 of the volume of the transfer device. In a specific embodiment, transfer is a very critical step, and 2 / 3 of the volume of the electrophoresis tank is added during transfer. If too much transfer buffer is added to the electrophoresis tank, the absorbent paper will continue to absorb water, or cause transfer transition, affecting the transfer effect; if too little transfer buffer is added to the electrophoresis tank, the absorbent paper will not absorb enough water, and the DNA bands on the gel block will not be transferred to the membrane before they are transferred to the membrane, and the transfer cannot be completed. After all the lower glass plate, first filter paper, second filter paper, agarose gel, nylon membrane, third filter paper, absorbent paper, upper glass plate, and weights are placed, the transfer begins and is observed for 24 hours to ensure that the buffer is not absorbed dry, and the transfer is stopped until all the absorbent paper is completely wet.

[0051] After the transfer, the nylon membrane is taken out for radiation hybridization, scanning and imaging, and DNA topological isomerism is analyzed. The present invention first removes all the objects above the nylon membrane, and then peels off the nylon membrane with tweezers for radiation hybridization. In a specific embodiment, the radioactive hybridization uses the RadPrime DNA Labeling System (Cat. No. 18428-011) kit of Invitrogen, and radioactive hybridization is performed according to the steps of the instructions. Specifically, the present invention carefully places the nylon membrane into a hybridization tube to which the hybridization solution has been added in advance, and then places the hybridization tube into a hybridization box, starts the rotation button of the hybridization box, and makes the nylon membrane rotate repeatedly 360° in a stable and regular manner in the hybridization box, so as to achieve the effect of full hybridization. After hybridization, the present invention elutes the hybridized membrane, then transfers it to a dark box for 24 hours, and finally scans and images it in the Personal Molecular Imager (PMI) System imaging system of BIO-RAD.

[0052] Accurate determination of DNA concentration, fine gel preparation, accurate loading amount, electrophoresis, voltage, electrophoresis time, gel cutting, membrane transfer and other links will affect the experimental results. The present invention determines the DNA concentration to ensure uniform DNA loading amount; makes highly uniform gel; selects appropriate voltage intensity, electrophoresis time, and ambient temperature (constant voltage is 50V, electrophoresis time is 48h, and ambient temperature is 4°C); after the electrophoresis, grasps the accurate position of gel cutting (cuts the gel from 1.5cm below the observed genomic DNA band until a length of 10cm is cut); determines the volume of the buffer solution, and adds 2 / 3 of the volume of the electrophoresis tank NaOH solution; the size of the absorbent paper, each side of the absorbent paper is longer than each side of ordinary filter paper The side length is about 1 cm, and 70 to 80 sheets are placed; the glass plate above the absorbent paper is the key to whether the transfer can be uniform (a flat glass plate is placed above the absorbent paper, with a thickness of 8 mm and an area larger than the absorbent paper, ensuring that the first filter paper, the second filter paper, the gel block, the nylon membrane, the third filter paper, and the absorbent paper between the glass plate above the electrophoresis tank (lower glass plate) and the glass plate above the absorbent paper (upper glass plate) are neatly stacked, vertically vertically, and the center of gravity is on a line. The setting of the conditions of the above method of the present invention is conducive to the analysis of high-resolution DNA topoisomerism.

[0053] In order to further illustrate the present invention, a high-resolution DNA topoisomerism analysis method provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] Product composition:

[0056] Gel mold, electrophoresis tank, glass bridge, thick filter paper (first filter paper), ordinary filter paper (second filter paper), agarose gel, nylon membrane, ordinary filter paper (third filter paper), absorbent paper, glass plate and weights, Figure 1 Schematic diagram of the transfer device.

[0057] Specific parameters:

[0058] Electrophoresis tank: 47cm×24cm;

[0059] Glass plate (lower glass plate): 25cm×20cm, 325g, 8mm thick;

[0060] Thick filter paper bridge: 30cm×15cm, 0.53mm thick;

[0061] Gel block size: 19.7cm×10cm;

[0062] Nylon membrane size: 19.7cm×10cm;

[0063] Ordinary filter paper (second filter paper, third filter paper) size: 19.7cm×10cm, 0.2mm thick;

[0064] Absorbent paper: 20.2cm×10.5cm;

[0065] Glass plate (upper glass plate): 25cm×20cm, 325g, 8mm thick;

[0066] Weight: The weight that compresses the gap between absorbent papers.

[0067] Step 1: Make agarose gel

[0068] The glue mold was selected, with a length of 21.6 cm, 20 glue holes, each hole was 0.7 cm wide, and the interval between each hole was 0.3 cm. The actual size of the glue block was 19.7 cm × 30 cm. Figure 2 To make a tooth comb model of gel holes. Use microwave heating method to make 1% agarose gel. The agarose should be fully melted. Use low heat and take it out for many times to shake it evenly or stir it slowly with a magnetic stirrer to avoid bubbles. This will make a highly uniform and bubble-free agarose gel. When the gel cools to about 55°C, pour the gel. After the gel solidifies, take the gel into a 4°C cold room for sample loading and electrophoresis.

[0069] Step 2: Agarose gel electrophoresis

[0070] Using yeast as a sample, the classic phenol-chloroform extraction method was used to extract sample DNA. The Nanodrop instrument (physical method) and the Qubit4 instrument (chemical method) were used to determine the concentration and purity of the extracted DNA. The DNA quality was tested from the perspective of different detection principles of ultraviolet absorption and fluorescence intensity to determine the amount of DNA loaded in each loading well. Ensure that the laboratory desktop and the electrophoresis tank are level. Use a level to measure whether the electrophoresis tank is level. Adjust the 3 knobs at the bottom of the electrophoresis tank to ensure that the overall structure of the electrophoresis tank on the desktop is level. Add the target DNA sample to the corresponding gel well, adjust the voltage: 50V, the electrophoresis time: 48h, and the ambient temperature to 4°C. Figure 3 Agarose gel electrophoresis.

[0071] Step 3: Cutting the glue

[0072] After the electrophoresis is finished, cut the gel 1.5cm below the observed genomic DNA band, cut out a length of 10cm, and only keep this 10cm length, and remove the rest, and also cut out the blank gel parts without DNA bands on the left and right sides of the gel electrophoresis channel. The DNA on the gel can be observed under the ultraviolet light of the UVP gel imager, and a special ruler that can transmit ultraviolet light is placed on the gel for measurement, so that the distance between the position of the genomic DNA and the gel hole can be measured. Then take the gel out of the imager. Although there is no ultraviolet light and the DNA bands cannot be seen, the position of the DNA can be determined based on the length measured by the ruler just now. Figure 4 This is a diagram of the gel cutting process after electrophoresis. Figure 4 Cut as shown and proceed to the next step of transfer printing, with a size of 19.7cm×10cm.

[0073] Step 4: Make a transfer device and transfer the film

[0074] Use a rectangular transfer tank with a size of 47cm×24cm, fill it with 2 / 3 transfer buffer (0.4MNaOH solution), and place a bridge glass plate (thickness 8mm) parallel to the short side of the transfer tank and across the center of the transfer tank. The size of the bridge glass plate is 25cm×20cm (lower glass plate). Place thick filter paper (first filter paper) on the glass plate to bridge, the size is 30cm×15cm. Lay a piece of ordinary filter paper (second filter paper) of the same size as the glue on the filter paper bridge, and place the cut glue block on the second filter paper and flatten it. Place the nylon membrane (size = glue block size) on the glue block and flatten it. Place the third filter paper (size = glue block size) on the nylon membrane and flatten it. Place 80 pieces of absorbent paper (size ≥ glue block size) on the third filter paper and flatten it. Stack the absorbent paper one by one. Because the absorbent paper is nested with each other, it is not easy to adjust if it is stacked all at once, and it needs to be adjusted very vertically. Place a 25cm×20cm glass plate (upper glass plate) weighing 325g on top of the absorbent paper, and then add weights of appropriate weight to ensure that there are no bubbles, the glue is intact, and the center of gravity of the glass plate and all items between them are on the same vertical line. After all the lower glass plate, the first thick filter paper for bridging, the ordinary second filter paper, agarose gel, nylon membrane, the ordinary third filter paper, absorbent paper, upper glass plate and weights are placed, start the transfer and observe for 24 hours to ensure that the buffer solution is not absorbed dry, until all the absorbent paper is completely wet, and stop the transfer. Figure 5 The figure is the flow chart of membrane transfer.

[0075] Step 5: Radiation hybridization

[0076] Radioactive hybridization was performed using the RadPrime DNA Labeling System (Cat. No. 18428-011) kit from Invitrogen according to the instructions. All objects above the nylon membrane were removed, the nylon membrane was peeled off with tweezers, and carefully placed in a hybridization tube that had been pre-added with hybridization solution, and then the hybridization tube was placed in a hybridization box, and the rotation button of the hybridization box was turned on to allow the nylon membrane to rotate 360° repeatedly in the hybridization box in a stable and regular manner, thereby achieving a sufficient hybridization effect. The hybridized membrane was eluted, then transferred to a dark box and placed for 24 hours, and finally scanned and imaged on the Personal Molecular Imager (PMI) System imaging system of BIO-RAD.

[0077] The hybridization results are as follows Figure 6 As shown, according to Figure 6 It can be seen that the topological isomerism bands of supercoiled DNA have high resolution, clear band separation, no overlapping between bands, straight bands without tilt or bending, and a lighter background. There is no need to increase exposure to display the bands, and the detailed information of each band can be judged very well.

[0078] Example 2

[0079] The operation method is the same as in Example 1, and the DNA sample is also from yeast. Figure 7 shown.

[0080] Example 3

[0081] The operation method is the same as in Example 1, and the DNA sample is also from yeast. Figure 8 shown.

[0082] Example 4

[0083] The operation method is the same as in Example 1, and the DNA sample is also from yeast. Fig. 9 shown.

[0084] Example 5

[0085] The operation method is the same as in Example 1, and the DNA sample is also from yeast. Fig.10 shown.

[0086] Comparative Example 1

[0087] Taking the prior art as a comparative example, the results are as follows Fig.11 As shown. Fig.11It can be seen that the supercoiled DNA image presented is obviously severely deformed, the strips are not horizontal, the left half of the strips are tilted to the lower left, and the right half of the strips are tilted to the lower right. In addition, the background of the image is too dark and unclear, the electrophoresis strips are black, and some are too thick and some are too thin.

[0088] Literature from: Zou Y, Yu Q, Bi

[0089] Comparative Example 2

[0090] Taking the prior art as a comparative example, the results are as follows Fig.12 As shown. Fig.12 It can be seen that the supercoiled DNA image presented is also seriously deformed, especially the middle image and the image on the right. The middle image is curved, and the image on the right is obviously tilted to the upper right corner, which affects the reading of the bands and causes large errors in band identification. In addition, the background of the image is too dark and unclear, the electrophoresis bands are black, and some are too thick, while others are too thin.

[0091] Literature from: Zou Y, Yu Q, Bi

[0092] Comparative Example 3

[0093] Taking the prior art as a comparative example, the results are as follows Fig.13 As shown. Fig.13 It can be seen that the resolution of the image on the left is too low and the stripes are almost invisible; in the image on the right, only thick stripes can be seen, and some fine strips are not displayed due to poor resolution.

[0094] Literature from: (1) Bi

[0095] (2) Zhang

[0096] Comparative Example 4

[0097] Taking the prior art as a comparative example, the results are as follows Fig.14 As shown. Fig.14 It can be seen that due to the lack of accurate measurement of the sample amount, the DNA sample amount was excessive, the scanned bands were too thick and too dark, and the bands overlapped and piled together, affecting the identification of the band type.

[0098] Literature from: Yu Q, Zhang X, Bi

[0099] Comparative Example 5

[0100] Taking the prior art as a comparative example, the results are as follows Fig.15 As shown. Fig.15 It can be seen that the amount of DNA loaded is obviously insufficient, resulting in unclear bands. Attempts to increase the brightness by forcibly increasing the exposure resulted in insufficient resolution of the bands, making them look particularly unclear.

[0101] Literature from: Yu Q, Sandmeier J, Xu H, et al. Mechanism of the long range anti-silencing function of targeted histone acetyltransferases in yeast [J]. Journal of Biological Chemistry, 2006, 281(7): 3980-3988.

[0102] Comparative Example 6

[0103] Taking the prior art as a comparative example, the results are as follows Fig.16 As shown. Fig.16 It can be seen that there are serious impurities in the background.

[0104] Literature from: Yu Q, Kuzmiak H, Zou Y, et al. Saccharomyces cerevisiae linkerhistone Hho1p functionally interacts with core histone H4 and negatively regulates the establishment of transcriptionally silent chromatin[J]. Journal of Biological Chemistry, 2009, 284(2):740-750.

[0105] Comparative Example 7

[0106] Taking the prior art as a comparative example, the results are as follows Fig.17 As shown. Fig.17 It can be seen that insufficient membrane washing resulted in severe background on the membrane, affecting the interpretation of the bands.

[0107] Literature source: Zou Y, Bi

[0108] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A high-resolution DNA topoisomerism analysis method, characterized in that: The following steps are involved: Extract DNA and detect DNA concentration; Prepare agarose gel; Load the DNA into the wells of agarose gel in the same total amount; After setting the voltage intensity, electrophoresis time and ambient temperature, perform agarose gel electrophoresis; After electrophoresis, cut the gel and transfer to the membrane; The transfer membrane is prepared by adding transfer buffer into the transfer device, placing a lower glass plate for bridging parallel to the short side of the transfer tank and through the center of the transfer tank, placing a first filter paper on the lower glass plate to form a bridge, and obtaining a thick filter paper bridge; laying a second filter paper of the same size as the rubber block on the thick filter paper bridge; placing the cut rubber block on the second filter paper and laying it flat; placing a nylon membrane of the same size as the rubber block on top of the rubber block and laying it flat; placing a third filter paper of the same size as the rubber block on top of the nylon membrane and laying it flat; Place absorbent paper of a size equal to or larger than the size of the glue block on top of the third filter paper and lay it flat; place the upper glass plate on top of the absorbent paper and then place the weights; After transfer, the nylon membrane is removed for radiation hybridization, scanning and imaging, and analysis and interpretation of DNA topological isomerism information.

2. The high-resolution DNA topoisomerism analysis method according to claim 1, characterized in that: DNA concentration was measured using a Nanodrop instrument and a Qubit4 instrument.

3. The high-resolution DNA topoisomerism analysis method according to claim 1, characterized in that: The voltage intensity is 50V; the electrophoresis time is 48h; and the ambient temperature is 4°C.

4. The high-resolution DNA topoisomerism analysis method according to claim 1, characterized in that: The gel cutting position includes: cutting from 1.5 cm below the observed genomic DNA band until a length of 10 cm is cut out.

5. The high-resolution DNA topoisomerism analysis method according to claim 1, characterized in that: Each side of the absorbent paper is 0.5 to 1 cm longer than each side of the rubber block; the number of absorbent papers is 70 to 80 sheets.

6. The high-resolution DNA topoisomerism analysis method according to claim 1, characterized in that: The thickness of the lower glass plate and the upper glass plate is 8 mm.

7. The high-resolution DNA topoisomerism analysis method according to claim 1, characterized in that: The thickness of the first filter paper is 0.53 mm; the thickness of the second filter paper and the third filter paper are both 0.2 mm.

8. The high-resolution DNA topoisomerism analysis method according to claim 1, characterized in that: The vertical centers of the first filter paper, the second filter paper, the rubber block, the nylon membrane, the third filter paper, the absorbent paper, the upper glass plate and the weight are on the same vertical line.

9. The high-resolution DNA topoisomerism analysis method according to claim 1, characterized in that: There are no bubbles between the first filter paper, the second filter paper, the rubber block, the nylon membrane, the third filter paper, the absorbent paper, the upper glass plate and the weight, and they are all flat.

10. The high-resolution DNA topoisomerism analysis method according to claim 1, characterized in that: The transfer buffer is a sodium hydroxide buffer; the amount of the transfer buffer added is 2 / 3 of the volume of the transfer device.

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