Chromosome N-G banding method

Abstract

The application provides a chromosome N-G banding method, which comprises the following steps: S1: preparing a split-phase glass slide with good quality; S2: adding solution, sucking 2 drops of solution A and 4 drops of solution B into a test tube, and then dropping the test tube on a glass slide to be dyed after rapid mixing; S3: dyeing, covering a cover glass, and placing the glass slide on a preheated 70 DEG C baking plate for heating; the dyeing solution will become yellow within 30 s of heating, and will become dark golden brown within 2 min; S4: washing the glass slide, removing the cover glass, and then washing the glass slide with flowing distilled water, and then shaking off water drops, and baking the glass slide at 65 DEG C for 30 min; S5: banding, Giemsa banding, preparing six dyeing tanks, and repeatedly immersing and dyeing according to a predetermined order, and developing color after drying, so that the nucleolus region is dyed black, and the chromosome is purple. The method can simultaneously observe the NOR region and the variation of the chromosome band, and thus the accuracy of judging the variation of the NOR region is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to chromosome banding technology, in particular to a chromosome N-G banding method. BACKGROUND

[0002] After a certain special treatment or specific staining of chromosomes, a series of continuous light and dark stripes can be shown on the chromosomes, which are called banded chromosomes. Chromosome banding technology is a technology developed on the basis of displaying chromosomes, which has the advantage of showing the finer structure of chromosomes itself. Chromosome banding technology greatly promotes the development of cytogenetics, helps to more accurately identify each chromosome and chromosomal structural abnormalities, is suitable for various cell chromosome specimens, and also provides a basis for the study of gene localization.

[0003] The commonly used chromosome banding method in the prior art is G banding, which is to treat the chromosome specimen with trypsin, alkali, citrate or urea reagent, and then stain with Giemsa dye, as shown in Figure 3 The light and dark alternating horizontal stripes displayed are G bands, and the G bands of the chromosomes can be observed under an ordinary optical microscope. The G banding method is simple, the band is clear, and the chromosome specimen can be stored for a long time, so it is widely used in the diagnosis and research of chromosome diseases. In addition, there is an N banding method for the satellites and nucleolar organizer regions of chromosomes, which uses silver nitrate staining to make the satellites and nucleolar organizer regions (NOR) of chromosomes present specific black silver staining (special display of chromosome nucleolar organizer banding), as shown in Figure 4 .

[0004] However, in the case of needing to observe G banding and N banding at the same time, there is no staining method in the prior art that can simultaneously realize both kinds of banding. Only G banding and N banding can be performed on the sample separately, which not only increases the operation, but also may cause deviation between samples. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the present application provides a chromosome N-G banding method, which can simultaneously observe the variation of the NOR region and the chromosome banding, thereby improving the accuracy of judging the variation of the NOR region.

[0006] To achieve the above purpose, a chromosome N-G banding method according to the present application comprises the following steps:

[0007] S1: Preparing a split-phase glass slide with good quality;

[0008] S2: Adding liquid, taking 2 drops of solution A and 4 drops of solution B into a test tube, mixing quickly and then dropping onto the glass slide to be stained;

[0009] S3: Staining, cover the cover glass, put the glass on the preheated to 70 ℃ baking sheet heating; within 30s of heating, the dye will turn yellow, and will become dark brown within 2min;

[0010] S4: Washing the film, removing the cover glass, rinsing the glass slide with flowing distilled water, then shaking off the water droplets, baking the film at 65℃ for 30min;

[0011] S5: Banding, Giemsa banding staining, prepare 6 dyeing tanks, immerse and dye multiple times according to the established order, color development after drying, the nucleolus region should be dyed black, and the chromosomes should be purple.

[0012] Further, solution A is a colloidal developer, solution A is obtained by dissolving 2g of analytical pure gelatin powder in 100ml of double distilled water, adding 1ml of formic acid, and continuously stirring for 10min until the gelatin is completely dissolved, solution A can be used stably for 2 weeks, solution A needs to be stored in a brown glass bottle with a bottle cap, and stored in the dark.

[0013] Further, solution B is a silver nitrate solution, solution B is obtained by dissolving 4g of analytical pure AgNO3 in 8ml of double distilled water, solution B can be used stably, solution B needs to be stored in a brown glass bottle with a bottle cap, and stored in the dark.

[0014] Further, the 6 dyeing tanks include dyeing tank a, dyeing tank b, dyeing tank c, dyeing tank d, dyeing tank e and dyeing tank f, 200ml of trypsin working solution is added to dyeing tank a, 200ml of trypsin buffer is added to dyeing tank b and dyeing tank c respectively, 200ml of Giemsa working solution is added to dyeing tank d, 200ml of acetone working solution is added to dyeing tank e, and 200ml of distilled water is added to dyeing tank f.

[0015] Further, the trypsin buffer is obtained by adding 36g of NaCl, 15.52g of Na2HPO4-12H2O, and 0.96g of KH2PO4 to 4000ml of pure water.

[0016] Further, the trypsin working solution is obtained by mixing 20ml of trypsin stock solution with 180ml of trypsin buffer, and the trypsin stock solution is prepared by adding 0.25g of trypsin to 100ml of pure water.

[0017] Further, the Giemsa working solution is obtained by mixing 10ml of Giemsa stock solution with 190ml of Giemsa buffer, and the Giemsa buffer is prepared by adding 17g of KH2PO4 and 2.91g of NaOH to 2.5L of pure water.

[0018] Further, the acetone working solution is obtained by adding 1ml of acetone to 199ml of pure water.

[0019] Further, step S5 further includes the following substeps:

[0020] S501: Put the slide into the trypsin working solution in dye vat a for 2 minutes and 30 seconds, take it out, and drain water;

[0021] S502: Put the slide into the trypsin buffer solution in dye vat b for 3 seconds to terminate trypsin action, take it out, and drain water;

[0022] S503: Put the slide into the trypsin buffer solution in dye vat c again for 3 seconds to terminate trypsin action, take it out, and drain water;

[0023] S504: Put the slide into the Giemsa working solution in dye vat d for 3 minutes for staining, take it out, and drain water;

[0024] S505: Put the slide into the acetone working solution in dye vat e for cleaning, take it out, and drain water;

[0025] S506: Put the slide into the distilled water in dye vat f for cleaning, take it out, and drain water, and place it in a slide drying machine for drying.

[0026] The chromosome N-G banding method of the preferred embodiment of the present application combines the characteristics of NOR banding and G banding, can simultaneously observe NOR regions and variations of chromosome banding, and further improves the accuracy of judging NOR region variations; and each experimental link is optimized, and the technical prejudice that NOR banding and G banding cannot be simultaneously performed in the prior art is overcome. BRIEF DESCRIPTION OF DRAWINGS

[0027] The present application will be further described and explained with reference to the accompanying drawings.

[0028] Figure 1 is the chromosome N-G banding of the preferred embodiment of the present application.

[0029] Figure 2 is the NOR banding of the comparative example.

[0030] Figure 3 is the G banding in the prior art.

[0031] Figure 4 is the NOR banding in the prior art. DETAILED DESCRIPTION

[0032] The technical solutions of the present application will be more clearly and completely described by combining the accompanying drawings and the description of the preferred embodiment of the present application.

[0033] The reagents used in the chromosome N-G banding method of the preferred embodiment of the present application include:

[0034] Solution A: Colloidal developer. Solution A is prepared by dissolving 2g of analytical grade gelatin powder in 100ml of double-distilled water, adding 1ml of formic acid, and stirring continuously for 10min until the gelatin is completely dissolved. Solution A is stable for 2 weeks. Solution A needs to be stored in a capped brown glass bottle and protected from light.

[0035] Solution B: Silver nitrate solution. Solution B is obtained by dissolving 4g of analytical grade AgNO3 in 8ml of double-distilled water. Solution B is stable and should be stored in a capped brown glass bottle, protected from light.

[0036] Pancreatic enzyme stock solution: prepared by adding 0.25g of pancreatic enzyme to 100ml of pure water.

[0037] Trypsin buffer: 36g NaCl, 15.52g Na2HPO4-12H2O, and 0.96g KH2PO4 are added to 4000ml of pure water.

[0038] Pancreatic enzyme working solution: prepared by mixing 20 ml of pancreatic enzyme stock solution with 180 ml of pancreatic enzyme buffer solution.

[0039] Giemsa buffer solution: prepared by adding 17g KH2PO4 and 2.91g NaOH to 2.5L of pure water.

[0040] Giemsa working solution: prepared by mixing 10 ml of Giemsa stock solution with 190 ml of Giemsa buffer solution.

[0041] Acetone working solution: 1 ml of acetone is added to 199 ml of pure water.

[0042] The chromosome NG banding method of this embodiment includes the following steps:

[0043] S1: Slide preparation, preparing glass slides with good quality split phases;

[0044] S2: Add liquid, take 2 drops of solution A and 4 drops of solution B into a test tube, mix them quickly and then drop them onto the glass slide to be stained;

[0045] S3: Staining. Cover with a coverslip and place the slide on a preheated 70°C baking plate. The stain will turn yellow within 30 seconds and dark golden brown within 2 minutes.

[0046] S4: Wash the slide, remove the coverslip, rinse the slide with running distilled water, then shake off the water droplets, and bake the slide at 65℃ for 30 minutes.

[0047] S5: banding, Giemsa banding, prepare 6 dyeing tanks, 6 dyeing tanks include dyeing tank a, dyeing tank b, dyeing tank c, dyeing tank d, dyeing tank e and dyeing tank f, 200ml trypsin working solution is added in dyeing tank a, 200ml trypsin buffer solution is added in dyeing tank b and dyeing tank c respectively, 200ml Giemsa working solution is added in dyeing tank d, 200ml acetone working solution is added in dyeing tank e, and 200ml distilled water is added in dyeing tank f. Multiple times of immersion and dyeing are carried out in the order of a-f.

[0048] S501: put the slide into the trypsin working solution in dyeing tank a for 2 minutes and 30 seconds, take out and drain;

[0049] S502: put the slide into the trypsin buffer solution in dyeing tank b for 3 seconds to terminate the trypsin effect, take out and drain;

[0050] S503: put the slide into the trypsin buffer solution in dyeing tank c again for 3 seconds to terminate the trypsin effect, take out and drain;

[0051] S504: put the slide into the Giemsa working solution in dyeing tank d for 3 minutes for dyeing, take out and drain;

[0052] S505: put the slide into the acetone working solution in dyeing tank e for cleaning, take out and drain;

[0053] S506: put the slide into the distilled water in dyeing tank f for cleaning, take out and drain, and place it in a slide drying machine for drying.

[0054] According to the banding condition, the trypsin treatment time can be adjusted appropriately. The nucleolar region should be dyed black, and the chromosome should be purple.

[0055] The banding diagram obtained after the implementation of the embodiment is shown in Figure 1 It can identify the variation of NORs region, and can also prepare to identify the chromosome banding, so it can judge the variation of D group and G group chromosomes.

[0056] Comparative example: NOR banding is carried out for the same sample, and the NOR banding is shown in Figure 2 It can be seen that the conventional NOR banding can identify the variation of NORs region, but cannot prepare to identify the chromosome banding, so it cannot identify the chromosomes of D group and G group.

[0057] The above specific embodiments only describe the preferred embodiments of the present application, and do not limit the protection scope of the present application. Various modifications, substitutions and improvements of the technical solutions of the present application made by those skilled in the art according to the description and drawings of the present application should belong to the protection scope of the present application. The protection scope of the present application is determined by the claims.

Claims

1. A method for chromosome NG banding, characterized in that, Includes the following steps: S1: Slide preparation, preparing glass slides with good quality split phases; S2: Add liquid. Take 2 drops of solution A and 4 drops of solution B and put them into a test tube. Mix them quickly and then drop them onto the slide to be stained. Solution A is a colloidal developer. Solution A is obtained by dissolving 2g of analytical grade gelatin powder in 100ml of double-distilled water, adding 1ml of formic acid, and stirring continuously for 10min until the gelatin is completely dissolved. Solution B is a silver nitrate solution. Solution B is obtained by dissolving 4g of analytical grade AgNO3 in 8ml of double-distilled water. S3: Staining. Cover with a coverslip and place the slide on a preheated 70°C baking plate. The stain will turn yellow within 30 seconds and dark golden brown within 2 minutes. S4: Wash the slide, remove the coverslip, rinse the slide with running distilled water, then shake off the water droplets, and bake the slide at 65℃ for 30 minutes. S5: Development, Giemsa development and dyeing, prepare 6 dyeing vats, the 6 dyeing vats include dyeing vat a, dyeing vat b, dyeing vat c, dyeing vat d, dyeing vat e and dyeing vat f; The dyeing vat a contains 200 ml of pancreatic enzyme working solution, which is prepared by mixing 20 ml of pancreatic enzyme stock solution with 180 ml of pancreatic enzyme buffer solution. The pancreatic enzyme stock solution is prepared by adding 0.25 g of pancreatic enzyme to 100 ml of pure water. Each of the dyeing vat b and dyeing vat c contains 200 ml of trypsin buffer solution, which is obtained by adding 36 g NaCl, 15.52 g Na2HPO4-12H2O, and 0.96 g KH2PO4 to 4000 ml of pure water. The dyeing vat d contains 200 ml of Giemsa working solution, which is prepared by mixing 10 ml of Giemsa stock solution with 190 ml of Giemsa buffer solution. The Giemsa buffer solution is prepared by adding 17 g of KH2PO4 and 2.91 g of NaOH to 2.5 L of pure water. The dyeing vat e contains 200 ml of acetone working solution, which is obtained by adding 1 ml of acetone to 199 ml of pure water. The dye vat f contains 200ml of distilled water; Multiple immersions in a predetermined order: S501: Place the glass slide into the trypsin working solution in staining jar a for 2 minutes and 30 seconds to digest, then remove and drain. S502: Place the glass slide into the trypsin buffer solution in staining tank b for 3 seconds to stop the trypsin action, then remove it and drain. S503: Place the slide back into the trypsin buffer solution in staining tank c for 3 seconds to stop the trypsin action, remove it and drain. S504: Place the glass slide into the Giemsa working solution in staining tank d and stain for 3 minutes, then remove and drain. S505: Wash the glass slide in the acetone working solution of staining tank e, remove it and drain it; S506: Wash the glass slides with distilled water in dyeing vat f, remove them, drain the water, and place them in a slide drying machine to dry; After drying, the nucleolus region should be stained black and the chromosomes should be stained purple.

2. The chromosome NG banding method according to claim 1, characterized in that, Solution A can be used stably for 2 weeks. Solution A needs to be stored in a capped brown glass bottle and protected from light.

3. The chromosome NG banding method according to claim 2, characterized in that, Solution B is stable and should be stored in a capped brown glass bottle, protected from light.

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