A method for rapid identification of exogenous cop-gfp

Abstract

The application belongs to the technical field of gene detection, and provides a method for rapidly identifying exogenous cop-GFP. The method comprises the following steps: a digoxin probe is designed and synthesized according to the DNA sequence of cop-GFP; DNA of a sample to be detected is extracted by using a centrifugal column purification method; the DNA of the sample to be detected is boiled and then added to a film; a DNA surface of the film is mixed with pre-hybridization solution for pre-hybridization; the film is taken out and mixed with hybridization solution containing the digoxin probe for hybridization overnight; the film is rinsed to obtain a probe cross-linked film; finally, a nucleic acid detection kit is used for developing the probe cross-linked film to determine the result. The application can quickly detect the expression of cop-GFP green fluorescence in cells. Moreover, the application can detect multiple samples at a time, and subsequent dot blotting can be used to analyze the expression amount of cop-GFP in cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene detection, in particular to a method for rapidly identifying exogenous cop-GFP. BACKGROUND

[0002] cop-GFP is a kind of enhanced fluorescent protein, which emits green fluorescence after expression in animals. It is usually used as a reporter gene to detect gene expression, regulation, cell differentiation, and protein localization and transport in organisms. The expression identification method of cop-GFP mainly includes: after the exogenous gene is transferred into the animal body, green fluorescent protein expression is observed using a fluorescent inverted microscope at 48-72 hours, or a flow cytometer or a UV spectrophotometer is used to determine the expression of fluorescent protein. However, although the use of a fluorescent microscope can determine whether the cell has green fluorescent protein expression, it cannot quantitatively determine the expression level of the fluorescent gene. Although the use of a flow cytometer to detect the expression of cop-GFP green fluorescent protein has high accuracy and can quantitatively analyze and accurately determine the expression level of the fluorescent protein, it requires antibodies and flow cytometers, which are relatively harsh in terms of equipment conditions, and cannot detect the expression of exogenous cop-GFP in vitro. Therefore, a relatively fast, stable, qualitative and quantitative method for detecting the expression of cop-GFP gene is needed. SUMMARY

[0003] To overcome the above-mentioned defects in the prior art, the present application provides a method for rapidly identifying exogenous cop-GFP.

[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0005] The present application provides a method for rapidly identifying exogenous cop-GFP, comprising the following steps:

[0006] (1) designing and synthesizing a digoxin probe according to the DNA sequence of cop-GFP;

[0007] (2) using a centrifugal column purification method to extract DNA from the sample to be tested;

[0008] (3) boiling the DNA of the sample to be tested and adding it to the film;

[0009] (4) after the film is air-dried, it is immersed in 2xSSC buffer;

[0010] (5) after soaking, use UV crosslinking to fix the DNA on the film;

[0011] (6) after fixation, mix the DNA side of the film with pre-hybridization solution for pre-hybridization to obtain a pre-hybridized film;

[0012] (7) boiling the digoxin probe obtained in step (1) to denature, then cooling and mixing with hybridization solution to obtain a mixed solution 2;

[0013] (8) mixing the pre-hybridization membrane obtained in step (6) with the mixed solution obtained in step (7) to hybridize overnight, then rinsing with 0.5×SSC solution to obtain a probe cross-linking membrane;

[0014] (9) developing the probe cross-linking membrane using a nucleic acid detection kit.

[0015] Preferably, the DNA sequence of the cop-GFP in step (1) is shown as SEQ ID NO. 1.

[0016] Preferably, the DNA sequence of the digoxin probe in step (1) is shown as SEQ ID NO. 2.

[0017] Preferably, the extraction concentration of the DNA of the sample to be tested in step (2) is ≥100 ng / μl.

[0018] Preferably, the boiling treatment time in step (3) is 4-6 min, the film in step (3) includes a nitrocellulose membrane or a nylon membrane, and the soaking time in step (4) is 15-25 min.

[0019] Preferably, the ultraviolet cross-linking time in step (5) is 55-65 s.

[0020] Preferably, the pre-hybridization solution in step (6) includes the following components: 25-35 ml 20×SSC buffer, 8-12 ml 50×Denhardt’s, 4-6 ml 10×SDS, 0.5-1.5 ml Salmon DNA, and 50-55 ml dH2O, the concentration of the Salmon DNA is 8-12 mg / ml, the amount of the pre-hybridization solution is 1-2 ml / cm2 of the membrane area, the pre-hybridization temperature is 55-65℃, and the pre-hybridization time is 3.5-4.5 h. 2

[0021] Preferably, the boiling denaturation time in step (7) is 4-6 min, and the hybridization solution includes the following components: 25-35 ml 20×SSC buffer, 8-12 ml 50×Denhardt’s, 4-6 ml 10×SDS, and 50-55 ml dH2O, the amount of the hybridization solution is 4-6 ml / cm2 of the membrane area. 2

[0022] ​​Preferably, the hybridization temperature in step (8) is 55-65 DEG C, the 0.5xSSC solution contains 8-12% SDS, and the number of rinsing times is 1-3 times, and the time for each rinsing is 15-25 min.

[0023] Preferably, the nucleic acid detection kit in step (9) comprises a chemiluminescence biotin-labeled nucleic acid detection kit.

[0024] Compared with the prior art, the present application has the following advantages:

[0025] The present application can quickly detect the expression of cop-GFP green fluorescence in cells. The present application can detect multiple samples at one time, and the expression amount of cop-GFP in cells can be analyzed by dot blotting subsequently. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of the provided drawings.

[0027] Figure 1 The sequence of the digoxin probe is designed;

[0028] Figure 2 The developing result of the embodiment 1 of the present application. DETAILED DESCRIPTION

[0029] The present application provides a method for quickly identifying exogenous cop-GFP, comprising the following steps:

[0030] (1) designing and synthesizing a digoxin probe according to the DNA sequence of cop-GFP;

[0031] (2) extracting the DNA of the sample to be tested by using a centrifugal column purification method;

[0032] (3) boiling the DNA of the sample to be tested and then adding it to a film;

[0033] (4) after the film is air-dried, it is soaked in 2xSSC buffer;

[0034] (5) after the soaking is completed, the DNA is fixed on the film by using ultraviolet crosslinking;

[0035] (6) after the fixing is completed, the film DNA side is mixed with a pre-hybridization solution to perform pre-hybridization, and a pre-hybridization film is obtained;

[0036] (7) boiling and denaturing the digoxin probe obtained in step (1) and then cooling, and mixing with hybridization solution to obtain a mixed solution 2;

[0037] (8) mixing the pre-hybridization membrane obtained in step (6) with the mixed solution obtained in step (7) to perform hybridization overnight, and then using 0.5×SSC buffer and SDS solution to perform rinsing to obtain a probe cross-linking membrane;

[0038] (9) using a nucleic acid detection kit to perform developing experiment on the probe cross-linking membrane.

[0039] In the present application, the DNA sequence of the cop-GFP in step (1) is preferably as shown in SEQ ID NO. 1.

[0040] In the present application, the DNA sequence of the digoxin probe in step (1) is preferably as shown in SEQ ID NO. 2.

[0041] In the present application, the extraction concentration of the DNA of the sample to be tested in step (2) is preferably ≥100 ng / μl.

[0042] In the present application, the boiling treatment time in step (3) is preferably 4-6 min, further preferably 5 min; the film in step (3) preferably comprises a nitrocellulose membrane or a nylon membrane, further preferably a nylon membrane; the soaking time in step (4) is preferably 15-25 min, further preferably 18-22 min, and more further preferably 20 min.

[0043] In the present application, the ultraviolet cross-linking time in step (5) is preferably 55-65 s, further preferably 58-62 s, and more further preferably 60 s.

[0044] In the present application, the pre-hybridization solution in step (6) preferably comprises the following components: 25-35 ml 20×SSC buffer, 8-12 ml 50×Denhardt’s, 4-6 ml 10×SDS, 0.5-1.5 ml Salmon DNA and 50-55 ml dH2O, further preferably the following components: 30 ml 20×SSC buffer, 10 ml 50×Denhardt’s, 5 ml 10×SDS, 1 ml Salmon DNA and 54 ml dH2O; the concentration of the Salmon DNA is preferably 8-12 mg / ml, further preferably 10 mg / ml; the amount of the pre-hybridization solution is preferably 1-2 ml / cm2of the membrane area, further preferably 1.5 ml / cm2of the membrane area. 2 2 ​The pre-hybridization temperature is preferably 55-65℃, further preferably 58-62℃, and more preferably 60℃; the pre-hybridization time is preferably 3.5-4.5h, further preferably 3.8-4.2h, and more preferably 4h.

[0045] In the present application, the boiling denaturation time in step (7) is preferably 4-6min, and further preferably 5min; the hybridization solution preferably comprises the following components: 25-35ml 20×SSC buffer, 8-12ml 50×Denhardt's, 4-6ml 10×SDS and 50-55ml dH2O, and further preferably the following components: 30ml 20×SSC buffer, 10ml 50×Denhardt's, 5ml 10×SDS and 54ml dH2O; the amount of the hybridization solution is preferably 4-6ml / cm2 of membrane area, and further preferably 5ml / cm2 of membrane area. 2 2 .

[0046] In the present application, the hybridization temperature in step (8) is preferably 55-65℃, further preferably 58-62℃, and more preferably 60℃; the 0.5×SSC solution preferably contains 8-12% SDS, and further preferably 10% SDS; the number of rinsing is preferably 1-3 times, and further preferably 2 times; the rinsing time is preferably 15-25min, further preferably 18-22min, and more preferably 20min.

[0047] In the present application, the nucleic acid detection kit in step (9) preferably comprises a chemiluminescence biotin-labeled nucleic acid detection kit.

[0048] The technical solutions provided by the present application will be described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0049] ​The consumables and materials required for the following examples are: nitrocellulose membrane, 20×SSC (3M NaCl, 0.3M sodium citrate, pH adjusted to 7.0 with NaOH, autoclaved, and stored at room temperature) (Solebo product number: S1030); prehybridization solution (Solebo product number: S8010); 5×Denhardt's solution (Solebo product number: D1080); 100 μg / ml denatured, sheared salmon sperm DNA (Solebo product number: H1060); hybridization solution (Solebo product number: S8010); 5×Denhardt's solution (Solebo product number: D1080); digoxigenin probe synthesized with labeled probe (Qingke Company); chemiluminescent biotin-labeled nucleic acid detection kit (Beyotime D3308).

[0050] Example 1

[0051] A rapid method for identifying exogenous cop-GFP, comprising the following steps:

[0052] 1. DNA was extracted from four groups of chicken blood cells using a DNA extraction kit (to be used as the basis for subsequent analysis). Figure 2 Groups 3, 4, 5, and 6 in the list):

[0053] (1) Use a pipette to draw 20 μl of blood from a positive chicken, add GA solution to make up to 200 μl, and shake thoroughly until homogeneous.

[0054] (2) Add 20 μl Proteinase K solution and mix well.

[0055] (3) Add 200 μl of buffer GB, mix thoroughly by inverting, place at 70°C for 10 min, the solution should become clear, and then briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0056] (4) Add 200 μl of anhydrous ethanol and shake well for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0057] (5) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0058] (6) Add 500 μl of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0059] (7) Add 600 μl of rinse solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 sec, discard the waste, and place the adsorption column CB3 in a collection tube.

[0060] (8) Repeat the previous step.

[0061] (9) Place the adsorption column CB3 back in the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste. Place the adsorption column CB3 at room temperature for several minutes to dry the adsorption material completely.

[0062] (10) Transfer the adsorption column CB3 to a clean centrifuge tube, and add 200 μl of elution buffer to the middle of the adsorption membrane, and centrifuge at 12,000 rpm for 1 min at room temperature.

[0063] 2. Dot blot detection:

[0064] (1) Boil the DNA sample (50 ng, 5 μl) for 5 min, and cool it in ice water.

[0065] (2) Dot the DNA to be detected, positive and negative sample DNAs on a nylon membrane, and mark them with a pencil. Cut the corner of the membrane to distinguish the front and back sides.

[0066] (3) After the membrane is dried, immerse it in 2 x SSC for 20 min.

[0067] (4) Place the membrane with the DNA side up on filter paper, cover it with filter paper, and cross-link it under UV light for 60 s. Immerse the cross-linked membrane in 2 x SSC.

[0068] (5) Place the membrane with the DNA side up in a hybridization tube, and add pre-hybridization solution (1.5 ml / cm 2 ) to the membrane. Pre-hybridize it at 60°C for 4 hours to obtain a pre-hybridized membrane. The composition of the pre-hybridization solution is 30 ml of 20 x SSC buffer, 10 ml of 50 x Denhardt's, 5 ml of 10 x SDS, 1 ml of Salmon DNA, and 54 ml of dH2O.

[0069] (6) Denature the labeled probe DNA (20 ng / ml) by boiling for 5 min, and cool it in ice water. Add it to the pre-warmed hybridization solution to obtain a mixture. The composition of the hybridization solution is 30 ml of 20 x SSC buffer, 10 ml of 50 x Denhardt's, 5 ml of 10 x SDS, and 54 ml of dH2O. The amount of the hybridization solution is 5 ml / cm 2 .

[0070] (7) Add the pre-hybridized membrane and the mixture to a hybridization instrument, and hybridize it overnight at 60°C.

[0071] (8) Rinse twice with 0.5x SSC solution (containing 10% SDS) for 20 min each.

[0072] 3. Steps of Biyun Tian chemiluminescence kit:

[0073] Use the membrane which has finished probe cross-linking to carry out developing experiment:

[0074] (1) Dissolve blocking solution and washing solution in 45℃ water bath.

[0075] Note: The blocking solution and washing solution must be completely dissolved before use. The blocking solution and washing solution can be used at room temperature to 50℃, but it must be ensured that there is no precipitation in the two solutions, and special attention should be paid in winter.

[0076] (2) Take a suitable container and add 15 ml of blocking solution, and then put the cross-linked nylon membrane containing sample. Slowly shake on the horizontal shaker for 15 minutes.

[0077] (3) Take 7.5 μl Streptavidin-HRP Conjugate and add to 15 ml of blocking solution (1:2000 dilution), mix well for standby.

[0078] (4) Remove the blocking solution, add 15 ml of blocking solution containing Streptavidin-HRP Conjugate prepared in the previous step. Slowly shake on the horizontal shaker for 15 minutes.

[0079] (5) Take 25 ml of washing solution (5x), add 100 ml of distilled water or Milli-Q grade pure water, mix well to prepare 125 ml of washing solution.

[0080] (6) Transfer the nylon membrane to another container containing 18 ml of washing solution, and rinse for 1 minute.

[0081] (7) Remove the washing solution, add 18 ml of washing solution, and slowly wash on the horizontal shaker for 5 minutes.

[0082] (8) Repeat step (7) three times (a total of four times), and each washing time is 5 minutes.

[0083] (9) Transfer the nylon membrane to another container containing 25 ml of detection equilibration solution, and slowly shake on the horizontal shaker for 5 minutes.

[0084] (10) Mix 5 ml of BeyoECL Moon A solution and 5 ml of BeyoECL Moon B solution to prepare BeyoECL Moon working solution. Note: The BeyoECL Moon working solution must be prepared immediately before use. Note: From this step, the operation method and matters needing attention are the same as the fluorescence detection of Western experiment.

[0085] (11) Remove the nylon membrane and absorb excess liquid with absorbent paper. Immediately place the membrane, sample side up, into a clean container or on plastic wrap on a level table.

[0086] (12) Carefully add 10 ml of the BeyoECL Moon working solution prepared in step (10) to the surface of the nylon membrane, ensuring that the working solution completely covers the nylon membrane. Let it stand at room temperature for 3 minutes.

[0087] (13) Remove the nylon film and absorb excess liquid with absorbent paper. Place the nylon film between two pieces of plastic wrap and secure it in the film cassette (also called film holder).

[0088] (14) Press the X-ray film for 1 minute and immediately develop and fix it. Then adjust the pressing time according to the results (or press the film separately for 30 seconds, 1, 3, 5 minutes or longer, and then develop and observe the results together). Qualitative or quantitative analysis can be performed on the bands displayed after development. For qualitative analysis, the presence or absence of a band on the nylon film is used as the standard. When the test result is positive, a black imprint band can be seen after development; when the test result is negative, no band is visible. The development results of this embodiment are as follows: Figure 2 As shown, by Figure 2 It is known that when the cop-GFP gene is stably inserted into the animal genome, the insertion of the exogenous cop-GFP gene can be detected by the method of the present invention; after development, a circular black mark can be observed on the membrane, and when there is no exogenous gene insertion, no mark can be observed after development.

[0089] If it is necessary to detect the expression level, imageJ software should be used to perform grayscale analysis of the bands. This method can perform both qualitative and quantitative analysis of cop-GFP.

[0090] The above description 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 principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapid identification of exogenous cop-GFP, characterized in that, Includes the following steps: (1) Design and synthesize a digoxigenin probe based on the DNA sequence of cop-GFP; the DNA sequence of cop-GFP is shown in SEQ ID NO.1; the DNA sequence of the digoxigenin probe is shown in SEQ ID NO.2; (2) Extract DNA from the sample to be tested using a centrifugal column purification method; (3) The DNA of the sample to be tested is boiled and then added to the membrane; (4) After the membrane has air-dried, immerse it in 2×SSC buffer solution; (5) After soaking, the DNA was fixed onto the membrane using ultraviolet crosslinking; (6) After fixation, the membrane DNA side is mixed with the prehybridization solution for prehybridization to obtain a prehybridization membrane; the prehybridization solution includes the following components: 25~35mL 20×SSC buffer, 8~12mL 50×Denhardt's, 4~6mL 10×SDS, 0.5~1.5mL Salmon DNA and 50~55mL dH2O, the concentration of the Salmon DNA is 8~12mg / mL, and the prehybridization temperature is 55~65℃; (7) The digoxigenin probe obtained in step (1) is boiled and denatured, then cooled and mixed with the hybridization solution to obtain a mixture; the hybridization solution includes the following components: 25~35mL 20×SSC buffer, 8~12mL 50×Denhardt's, 4~6mL 10×SDS and 50~55mL dH2O; (8) Mix the prehybridized membrane obtained in step (6) with the mixture obtained in step (7) and hybridize overnight. Then rinse with 0.5×SSC solution to obtain the probe crosslinked membrane. The hybridization temperature is 55~65℃; (9) Use a nucleic acid detection kit to perform a development experiment on the probe cross-linked membrane.

2. The method for rapid identification of exogenous cop-GFP according to claim 1, characterized in that, The DNA extraction concentration of the sample to be tested in step (2) is ≥100ng / μL.

3. The method for rapid identification of exogenous cop-GFP according to claim 1, characterized in that, The boiling treatment time in step (3) is 4 to 6 minutes, the film in step (3) includes nitrocellulose film or nylon film, and the soaking time in step (4) is 15 to 25 minutes.

4. The method for rapid identification of exogenous cop-GFP according to claim 1, characterized in that, The UV crosslinking time in step (5) is 55~65s.

5. The method for rapid identification of exogenous cop-GFP according to claim 1, characterized in that, The amount of prehybridization solution used in step (6) is 1-2 mL / cm² of the membrane area. 2 The pre-hybridization time is 3.5~4.5h.

6. The method for rapid identification of exogenous cop-GFP according to claim 1, characterized in that, The boiling denaturation time in step (7) is 4-6 minutes, and the amount of hybridization solution used is 4-6 mL / cm², which covers the membrane area. 2 .

7. The method for rapid identification of exogenous cop-GFP according to claim 1, characterized in that, The 0.5×SSC solution in step (8) contains 8~12% SDS, and the rinsing is performed 1~3 times, with each rinsing lasting 15~25 minutes.

8. The method for rapid identification of exogenous cop-GFP according to claim 1, characterized in that, The nucleic acid detection kit mentioned in step (9) includes a chemiluminescent biotin-labeled nucleic acid detection kit.

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