Chromosome N-G banding method

Abstract

The invention provides a chromosome N-G banding method, which comprises the following steps: S1, preparing a slide: preparing a split phase glass slide with better quality; s2, adding the solution, sucking two drops of the solution A and four drops of the solution B, dripping into a test tube, quickly mixing, and dripping on a glass slide to be dyed; s3, dyeing, covering with cover glass, and heating the cover glass on a sheet baking plate preheated to 70 DEG C; the dye liquor becomes yellow within 30 seconds of heating and becomes deep golden brown within 2 minutes; s4, washing the slide, uncovering the cover glass, washing the slide with flowing distilled water, then throwing off water drops, and baking the slide at 65 DEG C for 30 minutes; S5, banding, Giemsa banding and dyeing, namely preparing six dye vats, dip-dyeing for multiple times according to a set sequence, drying and then developing, dyeing a nucleolus area into black, and dyeing chromosomes into purple. According to the method, the variation of the NOR region and the chromosome stripe can be observed at the same time, so that the accuracy of judging the variation of the NOR region is improved.

Description

Technical Field

[0001] This invention relates to chromosome banding technology, and more specifically to a method for chromosome NG banding. Background Technology

[0002] After special treatment or specific staining, chromosomes can display a series of continuous light and dark stripes, known as banded chromosomes. Chromosome banding technology is a technique developed based on chromosome visualization, and its advantage is that it can reveal more subtle structures of the chromosomes themselves. Chromosome banding technology has greatly promoted the development of cytogenetics, helping to more accurately identify each chromosome and chromosome structural abnormalities. It is applicable to various cell chromosome specimens and also provides a foundation for gene localization research.

[0003] Commonly used chromosome banding methods in the prior art include G banding, which involves treating chromosome specimens with reagents such as trypsin, alkali, citrate, or urea, followed by staining with Giemsa stain. Figure 3 As shown, the alternating light and dark horizontal bands are G-bands, which can be observed under a regular optical microscope. The G-banding method is simple, produces clear bands, and allows for long-term preservation of chromosome specimens, thus it is widely used in the diagnosis and research of chromosomal diseases. In addition, there is the N-banding method, which targets the satellites and nucleolar organizer regions of chromosomes. Using silver nitrate staining, the satellites and nucleolar organizer regions (NORs) of chromosomes exhibit a specific black silver stain (specifically displaying the bands in the nucleolar organizer regions), such as... Figure 4 As shown.

[0004] When it is necessary to observe both G and N banding simultaneously, there is no existing staining method that can simultaneously achieve both banding. Instead, G and N banding can be performed on the samples separately, which not only increases the workload but may also lead to deviations between samples. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention proposes a chromosome NOR banding method that can simultaneously observe variations in the NOR region and chromosome banding, thereby improving the accuracy of NOR region variation judgment.

[0006] To achieve the above objectives, the present invention provides a chromosome NG banding method, comprising 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 into a test tube, mix them quickly and then drop them onto the glass slide to be stained; 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: Banding, Giemsa banding staining, prepare 6 staining jars, immerse in the stain multiple times in a predetermined order, dry and develop color, the nucleolus region should be stained black and the chromosomes should be purple.

[0007] Furthermore, 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 A can be used stably for 2 weeks. Solution A needs to be stored in a capped brown glass bottle and protected from light.

[0008] Furthermore, solution B is a silver nitrate solution, which 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.

[0009] Furthermore, the six dyeing vats include vat a, vat b, vat c, vat d, vat e, and vat f. Vat a contains 200 ml of trypsin working solution, vats b and c each contain 200 ml of trypsin buffer, vat d contains 200 ml of Giemsa working solution, vat e contains 200 ml of acetone working solution, and vat f contains 200 ml of distilled water.

[0010] Furthermore, the trypsin buffer was prepared by adding 36g NaCl, 15.52g Na2HPO4-12H2O, and 0.96g KH2PO4 to 4000ml of pure water.

[0011] Furthermore, the pancreatic enzyme working solution was prepared by mixing 20 ml of pancreatic enzyme stock solution with 180 ml of pancreatic enzyme buffer solution, and the pancreatic enzyme stock solution was prepared by adding 0.25 g of pancreatic enzyme to 100 ml of pure water.

[0012] Furthermore, the Giemsa working solution was prepared by mixing 10 ml of Giemsa stock solution with 190 ml of Giemsa buffer, and the Giemsa buffer was prepared by adding 17 g of KH2PO4 and 2.91 g of NaOH to 2.5 L of pure water.

[0013] Furthermore, the acetone working solution is obtained by adding 1 ml of acetone to 199 ml of pure water.

[0014] Furthermore, step S5 also includes the following sub-steps: 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 in distilled water in dyeing vat f, remove them, drain the water, and place them in a slide drying machine to dry.

[0015] The present invention provides a chromosome NG banding method that combines the characteristics of NOR banding and G banding, allowing simultaneous observation of variations in the NOR region and chromosome banding patterns, thereby improving the accuracy of NOR region variation judgment; and by optimizing each experimental step, it overcomes the technical bias in the prior art that cannot perform NOR banding and G banding simultaneously. Attached Figure Description

[0016] The invention will now be further described and explained with reference to the accompanying drawings.

[0017] Figure 1 This is the preferred embodiment of the present invention for chromosome NG banding.

[0018] Figure 2 It is a proportional NOR display band.

[0019] Figure 3 It is the G-band in existing technology.

[0020] Figure 4 It is a NOR display band in existing technology. Detailed Implementation

[0021] The technical solution of the present invention will be more clearly and completely explained below with reference to the accompanying drawings and through the description of preferred embodiments of the present invention.

[0022] The reagents used in a preferred embodiment of the present invention for a chromosome NG banding method include: 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.

[0023] 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.

[0024] Pancreatic enzyme stock solution: prepared by adding 0.25g of pancreatic enzyme to 100ml of pure water.

[0025] Trypsin buffer: 36g NaCl, 15.52g Na2HPO4-12H2O, and 0.96g KH2PO4 are added to 4000ml of pure water.

[0026] Pancreatic enzyme working solution: prepared by mixing 20 ml of pancreatic enzyme stock solution with 180 ml of pancreatic enzyme buffer solution.

[0027] Giemsa buffer solution: prepared by adding 17g KH2PO4 and 2.91g NaOH to 2.5L of pure water.

[0028] Giemsa working solution: prepared by mixing 10 ml of Giemsa stock solution with 190 ml of Giemsa buffer solution.

[0029] Acetone working solution: 1 ml of acetone is added to 199 ml of pure water.

[0030] The chromosome NG banding method of this embodiment 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 into a test tube, mix them quickly and then drop them onto the glass slide to be stained; 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: Banding and Giemsa staining. Prepare six staining jars: jar a, jar b, jar c, jar d, jar e, and jar f. Jar a contains 200 ml of trypsin working solution; jars b and c each contain 200 ml of trypsin buffer; jar d contains 200 ml of Giemsa working solution; jar e contains 200 ml of acetone working solution; and jar f contains 200 ml of distilled water. Repeat staining in the order of a, f, and f.

[0031] 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 in distilled water in dyeing vat f, remove them, drain the water, and place them in a slide drying machine to dry.

[0032] The pancreatic enzyme treatment time can be adjusted appropriately based on the banding pattern. The nucleolar region should be stained black, and the chromosomes should be stained purple.

[0033] The banding pattern obtained after implementing this embodiment is as follows: Figure 1 As shown, it can identify the variation in the NORs region, and at the same time, it can also identify chromosome banding, thus determining the variation in chromosomes of groups D and G.

[0034] Comparative example: For the same sample, NOR banding was performed, as shown in the NOR banding diagram. Figure 2 As shown, conventional NOR banding can identify variations in NOR regions, but it cannot accurately identify chromosome banding patterns, and therefore cannot identify chromosomes in groups D and G.

[0035] The above-described specific embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Various modifications, substitutions, and improvements made by those skilled in the art to the technical solutions of the present invention based on the provided textual description and drawings, without departing from the design concept and spirit of the present invention, should all fall within the scope of protection of the present invention. The scope of protection of the present invention 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 into a test tube, mix them quickly and then drop them onto the glass slide to be stained; 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: Banding, Giemsa banding staining, prepare 6 staining jars, immerse in the stain multiple times in a predetermined order, dry and develop color, the nucleolus region should be stained black and the chromosomes should be purple.

2. The chromosome NG banding method according to claim 1, characterized in that, 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 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 a silver nitrate solution, which 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.

4. The chromosome NG banding method according to claim 1, characterized in that, The six dyeing vats include vat a, vat b, vat c, vat d, vat e, and vat f. Vat a contains 200 ml of trypsin working solution, vats b and c each contain 200 ml of trypsin buffer, vat d contains 200 ml of Giemsa working solution, vat e contains 200 ml of acetone working solution, and vat f contains 200 ml of distilled water.

5. The chromosome NG banding method according to claim 4, characterized in that, The trypsin buffer solution was prepared by adding 36g NaCl, 15.52g Na2HPO4-12H2O, and 0.96g KH2PO4 to 4000ml of pure water.

6. The chromosome NG banding method according to claim 5, characterized in that, The pancreatic enzyme working solution 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.

7. The chromosome NG banding method according to claim 6, characterized in that, The Giemsa working solution was prepared by mixing 10 ml of Giemsa stock solution with 190 ml of Giemsa buffer solution. The Giemsa buffer solution was prepared by adding 17 g of KH2PO4 and 2.91 g of NaOH to 2.5 L of pure water.

8. The chromosome NG banding method according to claim 7, characterized in that, The acetone working solution is obtained by adding 1 ml of acetone to 199 ml of pure water.

9. The chromosome NG banding method according to claim 4, characterized in that, Step S5 also includes the following sub-steps: 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 the water; S506: Wash the glass slides in distilled water in dyeing vat f, remove them, drain the water, and place them in a slide drying machine to dry.

Images