CN112626180A - DNA hybridization enhancing solution and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a DNA hybridization enhancing solution and a preparation method and application thereof. The DNA hybridization enhancing solution comprises the following components in concentration: a solution of a buffer as a solute; hybridization accelerator: 2-60 g/100ml of polymer, 10-80% of organic solvent by volume and 0.5-10 g/100ml of protein by mass; salts: 1mM to 750 mM. The preparation method comprises the following steps: and adding the hybridization accelerator into the buffer solution, mixing, and finally adding the salt, mixing to obtain the DNA hybridization enhancing solution. The DNA hybridization enhancing solution is applied to DNAFSH products. The invention shortens the hybridization time, has good hybridization effect and very high specificity and sensitivity, reduces the probe dosage to one fifth of the original dosage and saves the probe cost.
Description
Technical Field
The invention relates to the field of molecular analysis of in-situ hybridization, relates to a DNA hybridization enhancing solution, a preparation method and application thereof, and particularly relates to a DNA hybridization enhancing solution and a preparation method of the DNA hybridization enhancing solution which can be combined with a DNA molecular probe for use.
Background
In Situ Hybridization (ISH) is a combination of molecular biology, histochemistry, and cytology.
The in situ hybridization techniques can be divided into genome in situ hybridization techniques, fluorescence in situ hybridization techniques, multicolor fluorescence in situ hybridization techniques, and in situ PCR.
Fluorescence In Situ Hybridization (FISH) is a new in situ hybridization method formed by replacing isotope labeling with fluorescent labeling based on a nonradioactive molecular cytogenetic technology developed on the basis of a radioactive in situ hybridization technology in the end of the 80 th 20 th century.
The basic principle of FISH is to label a DNA (or RNA) probe with a special nucleotide molecule, then directly hybridize the probe to a chromosome or a DNA fiber slice, and then detect qualitative, positioning and relative quantitative analysis of a DNA sequence on the chromosome or the DNA fiber slice by the specific binding of a monoclonal antibody coupled with a fluorescein molecule and the probe molecule.
FISH has the advantages of safety, rapidness, high sensitivity, long-term preservation of probes, simultaneous display of various colors and the like, can display metaphase mitosis and interphase nuclei, and develops a multicolor fluorescence in situ hybridization technology and a chromatin fiber fluorescence in situ hybridization technology on the basis of fluorescence in situ hybridization.
In the molecular analysis techniques currently used in oncology, Fluorescence In Situ Hybridization (FISH) is widely used to detect chromosomal abnormalities, such as amplification, aneuploidy, fragmentation, deletion, rearrangement. The sensitivity and specificity of FISH depends primarily on at least one of three factors: (A) denaturing conditions; (B) hybridization conditions; and (C) post-hybridization wash conditions. Hybridization after denaturation is the most time consuming, and in the traditional Fluorescence In Situ Hybridization (FISH) protocol, hybridization takes 14 to 24 hours, and possibly even 72 hours.
The use of formamide, which interferes with the Watson-Crick binding sites of nucleic acid bases, and thus hydrogen bonding between complementary nucleic acid bases, is the only way to lower the annealing Temperature (TM) of the complementary strand, which is necessary for the denaturation step. However, although the use of the crossover agent reduced the TM, formamide significantly extended the hybridization time.
Disclosure of Invention
The invention aims to provide a DNA hybridization enhancing solution, a preparation method and application thereof, which can greatly promote the efficiency of DNA molecular hybridization and overcome the defect of overlong hybridization time in fluorescence in situ hybridization.
The invention provides a DNA hybridization enhancing solution, which comprises the following components in concentration:
a solution of a buffer as a solute;
hybridization accelerator: 2-60 g/100ml of polymer, 10-80% of organic solvent by volume and 0.5-10 g/100ml of protein by mass;
salts: 1mM to 750 mM.
In the above-mentioned DNA hybridization enhancing solution, the buffer may comprise at least one of SSC, HEPES, TRIS, potassium phosphate and citric acid to provide a desired pH in the hybridization reaction system;
the final concentration of the buffer in the DNA hybridization enhancing solution may be 0.4% to 8.8% by mass volume.
Further, the final pH value of the DNA hybridization enhancing solution can be 6-8.
In the DNA hybridization enhancing solution, the polymer is at least one selected from Ficoll, PVP, heparin and dextran sulfate;
the protein comprises bovine serum albumin BSA;
the organic solvent is at least one selected from ethylene glycol, glycerol, 1, 3-propylene glycol, formamide, diethylene glycol formamide, dimethylformamide and DMSO;
the salts comprise at least one of sodium chloride, magnesium chloride, sodium phosphate and magnesium phosphate;
the concentration of the salt is 10 mM-500 mM.
The DNA hybridization enhancing solution specifically comprises the following components in concentration:
2 XSSC buffer or Tris buffer is used as a solvent;
the mass volume percentage of the dextran sulfate is 40g/100ml, the mass volume percentage of the formamide is 10 percent, and the mass volume percentage of the BSA is 1g/100 ml;
300mM sodium chloride and 10mM magnesium chloride.
In the DNA hybridization enhancing solution, the solvent is the 2 XSSC buffer solution or the Tris buffer solution;
the 2 XSSC buffer solution consists of sodium chloride, sodium citrate (or sodium citrate dihydrate) and water, specifically 17.6g of sodium chloride and 8.8g of sodium citrate (or sodium citrate dihydrate), and double distilled water is added to the buffer solution to a constant volume of 1L;
the Tris buffer solution is prepared from 3-60 g of Tris and double distilled water with constant volume of 1L, and specifically can be Tris6.058g of double distilled water with constant volume of 1L.
In the enhancement solution, the organic solvent such as formamide plays a role of a denaturant in the hybridization stage to interfere with hydrogen bonds between complementary nucleic acid bases, so that the hybridization time is obviously prolonged in the hybridization process, and the hybridization time can be shortened by reducing the content of formamide; the hybridization accelerator can be combined with water, so that the effective volume of a hybridization solution is reduced, the concentration of the probe is relatively improved, and the hybridization rate is greatly improved; dextran sulfate and BSA can improve signal intensity and background staining; in general, as dextran sulfate and BSA concentrations increased, signal intensity increased and background decreased.
In the invention, the salt is the combination of 300mM sodium chloride and 10mM magnesium chloride, and the hybridization of the nucleic acid molecule depends on the concentration of salt ions, so that the charge of the oligonucleotides is reduced, and the contact chance between the oligonucleotides is increased.
The invention also provides a preparation method of the DNA hybridization enhancing solution, which comprises the following steps: and adding the hybridization accelerator into the buffer solution, mixing, and finally adding the salt, mixing to obtain the DNA hybridization enhancing solution.
The specific method of the invention is that the hybridization enhancer of 40 percent dextran sulfate, 10 percent formamide and 1 percent BSA are added into 2 XSSC buffer solution, and then salts are added to lead the final concentration of sodium chloride to be 300mM and the final concentration of magnesium chloride to be 10mM, thus obtaining the hybridization enhancement solution of the invention.
The DNA hybridization enhancing solution is applied to DNAFSH products.
In the above application, the volume ratio of the hybridization enhancing solution to the commercial probe containing the hybridization solution may be 2-8: 1, specifically 4: 1.
the invention has the following advantages:
1. the DNA molecular hybridization enhancing solution of the invention is added with high molecular polymer dextran sulfate on the basis of the traditional FISH hybridization solution, and can form a three-dimensional pore structure, thereby promoting the efficiency of fluorescence in situ hybridization, greatly shortening the hybridization time from 14-24 hours to 1-2 hours.
2. The hybrid liquid of the invention is added with dextran sulfate, and the concentration of the traditional components of potassium chloride, sodium citrate and formamide is adjusted; the obtained hybridization mixed solution has better hybridization effect, and the probe result after hybridization still has extremely high signal to noise ratio and has very high specificity and sensitivity.
3. The probe dosage is reduced to one fifth of the original dosage, and the probe cost is saved.
Drawings
FIG. 1 shows the result of hybridization of the DNA hybridization enhancing solution with the FISH detection reagent ERBB2/CCP17 probe 3:1 for 2 hours on paraffin wax samples in example 1 of the present invention.
FIG. 2 is a graph showing the results of hybridization of the DNA hybridization enhancing fluid with the FISH detection IGH Break Apart probe 3:1 for 1.5 hours on a cytological specimen in example 2 of the present invention.
FIG. 3 shows the results of the FISH detection of ERBB2/CCP17 probe in comparative example 1 hybridized overnight for 18 hours on paraffin wax samples.
FIG. 4 is a graph showing the results of detection of the IGH Break Apart probe in comparative example 2 by FISH overnight hybridization on a cytological specimen for 18 hours.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, the cells were normal human EBV transformed lymphocytes and the source was Kunming cell bank, Chinese academy of sciences.
In the following examples, the DNA hybridization enhancing solution of the present invention was prepared from the following components in parts by weight:
buffer solution: 2 XSSC (sodium chloride 17.6g, sodium citrate (sodium citrate dihydrate) 8.8g, double distilled water constant volume to 1L);
hybridization accelerator: dextran sulfate 40g/100ml by mass, formamide 10% by mass, and BSA (bovine serum albumin) 1g/100ml by mass;
salts: final concentrations 300mM sodium chloride and 10mM magnesium chloride;
the preparation method of the DNA hybridization enhancing solution comprises the following steps: the DNA hybridization enhancing solution is obtained by adding hybridization accelerator 40% dextran sulfate, 10% formamide and 1% BSA to a buffer solution of 2 XSSC and 1 XSTris, and adding salts to make the final concentration of sodium chloride 300mM and the final concentration of magnesium chloride 10 mM.
Examples 1,
Paraffin section samples were prepared as follows:
1. slicing paraffin wax to 4-5 μm thickness, taking out the slices with in situ hybridization special glass slide, slicing, and baking at 65 deg.C; immersing the slices in xylene for 10min × 2 and absolute ethyl alcohol for 5min × 2 in sequence, and air-drying the slices for 2-5 min;
2. microwave oven heating, the copulin bottles were placed in a microwave oven and heated at 100% power until the solution began to boil. When the solution starts to boil, it is immediately heated in a microwave oven for 15min at a power of 10-20% to keep the solution at a temperature slightly below the boiling point. Then transferring into purified water at room temperature for soaking for 3 min;
3. immersing the slide in a protease working solution (purchased from Sigma company, catalog No. 77160-100G) preheated at 37 ℃ for incubation for 20min, observing under a microscope until the cell outline is clearly and uniformly dispersed, and then transferring the slide into purified water at room temperature for immersion for 3 min; immersing the slices in 70 percent, 85 percent and 100 percent ethanol in volume percentage for 2min respectively, and then air-drying the slices for 2-5 min;
4. the Probe used in the invention is ERBB2/CCP17 FISH Probe Kit, which is commercially available from CytoTest company, Cat: CT-PAC001-10-OG), fully and uniformly mixing the probe and the hybridization enhancement solution in a ratio of 1:4, dripping 10 mu L of probe mixture into the center of a tissue, covering a cover glass to ensure that the liquid is immersed in the surface of the tissue and bubbles are exhausted, and sealing the edge of the cover glass by using mounting glue; placing the slide in a hybridization instrument, performing covariation at 83 ℃ for 5min, and incubating at 37 ℃ for 2 h; simultaneous hybridization with conventional experiments using only probes;
5. carefully remove the mounting gel with ophthalmic forceps after hybridization; the cover slips were separated from it by immersion in a buffer (2 XSSC) at room temperature and gentle shaking; immersed in 70 ℃ wash (0.3% NP-40) for 2min and gently shaken; immersed in room temperature wash (0.1% NP-40) for 1min and gently shaken; sequentially immersing in 70%, 85% and 100% ethanol for 1min respectively, gently shaking, and naturally drying at room temperature in dark place;
6. and dripping 10-20 mu L of DAPI sealing tablets into the center of the hybridization area, covering a cover glass, exhausting bubbles, keeping away from light, standing for at least 30min, and selecting a proper light filtering module and observing the result by a magnification under a fluorescence microscope. The red and green signals of the glass slide added with the hybridization enhancing liquid have better hybridization effect, strong signal brightness and good specificity, and the comparison result is shown in the following table 1.
TABLE 1 DNA hybridization results for paraffin section samples
The result of hybridization of the DNA hybridization enhancing solution of the present invention with the FISH detection reagent ERBB2/CCP17 probe 4:1 on paraffin wax samples for 2 hours is shown in FIG. 1.
Examples 2,
Cytological specimens were prepared as follows:
1. harvesting cells: collecting the cultured or uncultured cells into a graduated centrifuge tube, centrifuging and removing supernatant;
2. hypotonic: adding 0.075mol/L KCl solution pre-warmed at 37 ℃, lightly blowing and beating the cell clusters by using a suction pipe, uniformly mixing, and placing the cell clusters in a constant-temperature water bath cabinet at 37 ℃ for hypotonic treatment for 20-30 minutes;
3. pre-fixing: the freshly prepared fixative (methanol: glacial acetic acid 3:1) was added, carefully mixed with a pipette, centrifuged and the supernatant discarded. Fixing: adding the fixing liquid, blowing, beating and mixing uniformly, and fixing for 20 minutes. The supernatant was discarded by centrifugation. And (3) fixing again: repeating the step 4 for 2 times or more until the cell sediment is washed white and clean.
4. Preparation of cell suspension: adding a proper amount of newly prepared fixing liquid according to the number of the cells, and blowing and beating the cells to prepare suspension. And (3) sucking a small amount of cell suspension, dripping the cell suspension onto a clean glass slide, dripping 2-3 drops of the cell suspension into each glass slide, fully diffusing, and airing at room temperature.
5. The slides were immersed in 37 ℃ buffer (2 XSSC) and equilibrated for 5 min; immersing the slices in a protease working solution preheated at 37 ℃ to incubate for 10min, and observing the slices under a microscope until the cell outline is clear;
6. immersing in buffer solution (2 XSSC), washing at room temperature for 5 min; immersing in formaldehyde fixing solution, and fixing at room temperature for 5 min; immersing in buffer solution (2 XSSC), washing at room temperature for 5 min; sequentially immersing in 70%, 85% and 100% ethanol for 2min, and naturally drying.
7. The Probe used in the invention is an IGH Break Apart FISH Probe Kit, which is commercially available from CytoTest company, Cat: CT-PAC201-10-OG), the probe and the hybridization enhancement solution are fully and uniformly mixed according to the proportion of 1:4, 10 mu l of probe mixture is dripped into a sample, a cover glass is covered to ensure that the liquid is immersed in the tissue surface and bubbles are exhausted, and the edge of the cover glass is sealed by mounting glue; placing the slide in a hybridization instrument, performing covariation for 2min at 80 ℃, and incubating for 2h at 37 ℃; simultaneous hybridization with conventional experiments using only probes;
8. carefully remove the mounting gel with ophthalmic forceps after hybridization; the cover slips were separated from it by immersion in a buffer (2 XSSC) at room temperature and gentle shaking; immersed in 70 ℃ wash (0.3% NP-40) for 2min and gently shaken; immersed in room temperature wash (0.1% NP-40) for 1min and gently shaken; sequentially immersing in 70%, 85% and 100% ethanol for 1min respectively, gently shaking, and naturally drying at room temperature in dark place;
9. and dripping 10-20 mul of DAPI sealing tablets into the center of the hybridization area, covering a cover glass, exhausting bubbles, keeping away from light, standing for at least 30min, and selecting a proper light filtering module and observing the result by a magnification under a fluorescence microscope. The red and green signals of the glass slide added with the hybridization enhancing liquid have better hybridization effect, strong signal brightness and good specificity, and the comparison result is shown in the following table 2.
TABLE 2 cytological specimen DNA hybridization results
The detection result of hybridization of the IGH Break Apart probe 4:1 on the cytological specimen for 1.5 hours was detected by using the DNA hybridization enhancing fluid of the present invention and FISH, as shown in FIG. 2.
Comparative examples 1,
The same procedure as in example 1 of the present invention was followed, except that only the ERBB2/CCP17 probe was hybridized overnight for 18 hours on the paraffin wax sample by FISH detection, and the results are shown in FIG. 3.
Comparative examples 2,
The same method as in example 2 of the present invention, except that only the FISH detection IGH Break Apart probe was hybridized overnight for 18 hours on the cytological specimen, the detection results are shown in FIG. 4.
By comparing the results of example 1 and comparative example 1 of the present invention and example 2 and comparative example 2 (from fig. 1 and 3, fig. 2 and 4), it is apparent that the results of example 1 and example 2 are superior to those of comparative example 1 and comparative example 2 in both signal intensity and background interference.
The statistical results of the comparative data of the ERBB2/CCP17 with and without DNA hybridization enhancing solution added in the experiment from 6 months 2020 to 12 months 2020 of the Beijing Sicheng Biotechnology company Limited are shown in the following Table 3:
TABLE 3 ERBB2/CCP17 results of experiments with and without DNA hybridization enhancing solution
| Type of reagent | Positive for | Percentage of positivity | Negative of | Percent negative | Total number of |
| ERBB2/CCP17 | 39 | 34％ | 77 | 66％ | 116 |
| ERBB2/CCP17+ DNA hybridization enhancing solution | 39 | 34％ | 77 | 66％ | 116 |
From the data in Table 3 above, it was confirmed that the use of the DNA hybridization enhancing solution of the present invention together with the FISH detection kit has no effect on the interpretation of the results in the FISH detection of cytological specimens and histological specimens, and that the effects of reducing the amount of probe used, enhancing the signal intensity, and reducing the background interference are effective in ten parts. Has the following beneficial effects:
1. the addition of the probe of the invention greatly shortens the hybridization time from 14-24 hours to 1-2 hours.
2. The probe of the invention has extremely high signal-to-noise ratio, and has high specificity and sensitivity.
3. The probe dosage is reduced to one fifth of the original dosage, and the probe cost is saved.
While the invention has been described in detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that many more modifications and variations are possible in light of the above teaching. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A DNA hybridization enhancing solution, comprising: the DNA hybridization enhancing solution comprises the following components in concentration:
a solution of a buffer as a solute;
hybridization accelerator: 2-60 g/100ml of polymer, 10-80% of organic solvent by volume and 0.5-10 g/100ml of protein by mass;
salts: 10mM to 500 mM.
2. The DNA hybridization enhancing solution according to claim 1, wherein: the buffer is selected from at least one of SSC, HEPES, TRIS, potassium phosphate and citric acid;
the final mass volume concentration of the buffer in the DNA hybridization enhancing solution is 0.4-8.8%;
the final pH value of the DNA hybridization enhancing solution is 6-8
3. The DNA hybridization enhancing solution according to claim 1 or 2, wherein: the polymer is selected from at least one of Ficoll, PVP, heparin and dextran sulfate;
the protein comprises bovine serum albumin;
the organic solvent is at least one selected from the group consisting of ethylene glycol, glycerol, 1, 3-propanediol, propylene glycol, formamide, diethylene glycol formamide, dimethylformamide and DMSO.
4. The DNA hybridization enhancing solution according to any one of claims 1 to 3, wherein: the salts comprise at least one of sodium chloride, magnesium chloride, sodium phosphate and magnesium phosphate;
the concentration of the salt is 10 mM-500 mM.
5. The DNA hybridization enhancing solution according to any one of claims 1 to 4, wherein: the DNA hybridization enhancing solution consists of the following concentration components:
2 XSSC buffer or Tris buffer is used as a solvent;
the mass volume percentage of the dextran sulfate is 40g/100ml, the mass volume percentage of the formamide is 10 percent, and the mass volume percentage of the BSA is 1g/100 ml;
300mM sodium chloride and 10mM magnesium chloride.
6. A method for preparing the DNA hybridization enhancing solution according to any one of claims 1 to 5, comprising the steps of: and adding the hybridization accelerator into the buffer solution, mixing, adding the salt, mixing, and finally adjusting the pH value to obtain the DNA hybridization enhancement solution.
7. Use of the DNA hybridization enhancing solution according to any one of claims 1 to 6 in DNA FISH products.
8. Use according to claim 7, characterized in that: the volume ratio of the hybridization enhancing solution to the commercial probe containing the hybridization solution is 2-8: 1.
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| CN117887813A (en) * | 2023-11-28 | 2024-04-16 | 首都医科大学附属北京口腔医院 | Hybridization buffer solution and application |
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