WO2018107879A1 - Silver staining kit for detecting dna in polyacrylamide gel, and use thereof - Google Patents

Abstract

Provided is a silver staining kit for detecting DNA in a polyacrylamide gel, the kit comprising reagent A, wherein the reagent is an aqueous solution, and in each litre of the aqueous solution, there is 3-5 mL of glacial acetic acid, 1-3 g of silver nitrate and 50-100 mL of ethanol; reagent B, wherein the reagent is an aqueous solution, and in each litre of the aqueous solution, there is 0.5-1 g of sodium chloride, 0.5-2.5 g of sodium sulphite, 0.1-0.3 g of carbohydrazide, 2-10 g of sodium carbonate and 5-10 g of glucose; and reagent C, wherein the reagent is an aqueous solution, and in each litre of the aqueous solution, there is 3-5 mL of glacial acetic acid.

Description

Silver staining kit for detecting DNA in polyacrylamide gel and application thereof Technical field

The present invention relates to the testing or analysis of materials by measuring the chemical or physical properties of materials, and in particular to silver staining reagents for detecting DNA in a polyacrylamide gel electrophoresis method.

Background technique

Silver staining of polyacrylamide gel is one of the commonly used methods for detecting DNA molecular markers. This method has high sensitivity and high resolution, and can distinguish 1 base difference [Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, 1989, 304-306] and identification of DNA as low as 27.8 pg [An Z, Xie L, Cheng H, Zhou Y, Zhang Q, He X, Huang HA silver Staining procedure for nucleic acids in polyacrylamide gels without fixation and pretreatment. Anal Biochem, 2009, 391: 77-79]. However, from the currently reported traditional classical silver staining method for detecting PCR amplification products [Bassam BJ, Caetano AG, Gresshoff PM. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem, 1991, 196(1): 80- 83) and modified silver dyeing method (Liang Q, Wen D, Xie J, Liu L, Wei Y, Wang Y, Shi SA rapid and effective method for silver staining of PCR products separated in polyacrylamide gels. Electrophoresis, 2014, 35 (17 ): 2520-2523] In view of the required reagents, formaldehyde is an essential reagent in all the silver dyeing methods, and the concentration is between 370 and 1480 mg/L [Guo Peiguo, Liu Wenjie, Li Haiyang, Wang Zhiliang, Xia Rock, Li Ronghua. A silver staining method for polyacrylamide gels for rapid and effective detection of SSR markers. Journal of Guangzhou University (Natural Science Edition), 2016, (4): 1-6].

As is known, formaldehyde is a highly irritating toxic gas that is easily soluble in water and is volatile at room temperature. Formaldehyde in the environment is a pollutant that seriously affects human health. Its main harm to the human body includes (1) stimulating effect: mainly in the stimulation of the skin mucosa, and can be combined with the amino group of the protein, and the severe respiratory tract irritation and edema, eye irritation and headache occur when inhaled at a high concentration; (2) sensitization: mainly in the direct contact with formaldehyde on the skin can cause allergic dermatitis, pigmentation, tissue cell necrosis; (3) mutagenic effect: is a genotoxic substance, is one of the potential strong mutagen, such as Can cause nasopharyngeal tumors, etc. (YAN Jinping. Formaldehyde and its harm to the human body. Chemical World, 2004 (10): 558-559). Because of its high toxicity, the World Health Organization (WHO) and the US Environmental Protection Agency (EPA) have listed formaldehyde as a potential dangerous carcinogen and an important environmental pollutant (Xu Xiangrong and Xu Zengkang. The harm of formaldehyde and its sanitary inspection method. Occupation And health, 2003, 19 (12): 47-49); in the list of priority control of toxic chemicals in China, formaldehyde ranks second. [Zhou Xuemei, Li Yuguang, Chen Lucing, Fu Huiqun, Jiang Sipeng, Wang Yong. A high-efficiency formaldehyde Study on separation and screening and degradation characteristics of degrading bacteria. Journal of Eco-Environment, 2015, 24(12): 2040-2044]. Correct For operators who use the silver staining method to detect PCR amplification products, long-term use and exposure to formaldehyde solution, the human body will inevitably inhale formaldehyde volatilized in the air, or contact with the skin to cause formaldehyde to invade the human body, thereby endangering health.

Summary of the invention

The technical problem to be solved by the present invention is to provide a silver staining kit for detecting DNA in a polyacrylamide gel, which can replace not only the existing DNA silver staining reagent but also formaldehyde which is harmful to the health of the operator. .

The technical solution of the present invention to solve the above problems is:

A silver staining kit for detecting DNA in a polyacrylamide gel, the kit comprising:

Reagent A: the reagent is an aqueous solution, and each liter of the aqueous solution has 3-5 mL of glacial acetic acid, 1-3 g of silver nitrate, and 50-100 mL of ethanol;

Reagent B: the reagent is an aqueous solution, and each liter of the aqueous solution has 0.5 to 1 g of sodium chloride, 0.5 to 2.5 g of sodium sulfite, 0.1 to 0.3 g of carbohydrazide, 2 to 10 g of sodium carbonate, and 5 to 10 g of glucose;

Reagent C: The reagent is an aqueous solution, and each liter of the aqueous solution has 3 to 5 mL of glacial acetic acid.

The above kit can be used to obtain a DNA polyacrylamide gel staining picture, and the specific method for obtaining the DNA polyacrylamide gel staining image comprises the following steps:

(1) taking the glue: removing the rubber plate after the PCR amplification product is subjected to polyacrylamide gel electrophoresis, gently peeling off the glass plate with the groove, and retaining another glass plate with the polyacrylamide gel attached thereto;

(2) Silver staining: a glass plate to which a polyacrylamide gel is attached is placed in a tray, and the side to which the polyacrylamide gel is attached is upward, and then the reagent A is added to be soaked; On the oscillator, shake at a frequency of 30 to 60 times per minute for 8 to 15 minutes, then take it out and rinse with water for 3 to 5 seconds.

(3) Color development: the silver-dyed glass plate is placed in the tray, and the side of the polyacrylamide gel is attached upward, and then the reagent B is added first, and then the tray is placed in the oscillator. On the top, oscillate at a frequency of 10 to 30 times per minute until the DNA band in the gel can be clearly observed;

(4) Termination: Place the colored glass plate in the tray and place the side of the polyacrylamide gel upward, then add the reagent C first, then place the tray on the shaker. , shake at a frequency of 30 to 60 times per minute for 1 to 2 minutes, rinse with water for 3 to 5 seconds;

(5) Obtaining a DNA polyacrylamide gel staining picture: The glass plate treated by the step (4) is air-dried, and a polyacrylamide gel picture is obtained by scanning with a scanner.

Compared with the prior art solutions, the kit of the invention has the following advantages:

1. The reagent in the kit of the present invention discards formaldehyde which is harmful to human health.

2. The minimum detectable limit of the kit of the present invention is 7.3 pg/μL, and the sensitivity is significantly higher than that of the prior art.

DRAWINGS

Figure 1 is a DNA polyacrylamide gel staining image of 30 copies of tobacco material PCR amplification products. In the figure, M is an electrophoresis band of DNA standards, and 1 to 30 are the first to 30th pieces of tobacco material PCR amplification products. Electrophoresis strips.

Fig. 2 is a DNA polyacrylamide gel staining picture of 30 PCR products of the Chinese cabbage material. In the figure, M is an electrophoresis band of DNA standards, and 1 to 30 are PCR amplification of the first to 30th core materials. Electrophoresis band of the product.

Fig. 3 is a DNA polyacrylamide gel staining image of 30 PCR products of the giant salamander material. In the figure, M is an electrophoresis band of DNA standards, and 1 to 30 is the first to 30th maggot material PCR. An electrophoresis band of the amplified product.

Figure 4 is a DNA polyacrylamide gel staining image of 66 parts of barley material PCR amplification products. In the figure, M is an electrophoresis band of DNA standards, and 1-66 is a PCR amplification product of 1 to 66 parts of barley material. Electrophoresis strips.

Fig. 5 is a DNA polyacrylamide gel staining picture of DL500 DNA standard. In the figure, lane 1 is an electrophoresis band of a DNA standard stock solution, and lanes 2 to 15 are electrophoresis bands of different concentration standard stock solutions, respectively.

detailed description

Example 1

First, establish a kit

1, preparation reagent A: 1 g of silver nitrate dissolved in 800L of deionized water, then add 50mL of ethanol and 3mL glacial acetic acid, and then add deionized water to a volume of 1L;

2, preparation reagent B: take 0.5g sodium chloride, 2.5g sodium sulfite, 0.1g carbohydrazide, 10g sodium carbonate and 8g glucose, add deionized water to a volume of 1L;

3. Preparation reagent C: Take 5 mL of glacial acetic acid, and add to deionized water to make up to 1 L.

2. DNA polyacrylamide gel staining image of tobacco leaf material

(1) PCR amplification: Take 30 samples of tobacco material DNA (DNA concentration of 10-20 ng/μL) as a DNA template, and then perform PCR amplification by the following methods using the following primers, respectively, to retain PCR of each tobacco material. Amplification products:

Forward primer: 5'-TCCAGCCTATTCCTTTCTTGTT-3' (SEQ ID No. 1),

Reverse primer: 5'-CCACAAGCAGTATTGGAGCA-3' (SEQ ID No. 2);

(2) Gel electrophoresis: PCR amplification products of each tobacco material were subjected to 6% polyacrylamide gel electrophoresis according to a conventional method to obtain a glass plate to which a polyacrylamide gel was attached;

(3) taking the glue: removing the rubber plate after the PCR amplification product is subjected to polyacrylamide gel electrophoresis, gently peeling off the glass plate with the groove, and retaining another glass plate with the polyacrylamide gel attached thereto;

(4) Silver staining: a glass plate to which a polyacrylamide gel is attached is placed in a tray, and the side to which the polyacrylamide gel is attached is upward, and then added to the reagent A in the above kit for soaking; The pot was placed on a shaker, shaken at a frequency of 30 times per minute for 15 minutes, taken out, and rinsed with water for 3 seconds;

(5) Color development: Place the silver-dyed glass plate in the tray and place it on the side with the polyacrylamide gel. Then, add the reagent B in the above kit, and then place the tray. On the oscillator, shake it at a frequency of 10 times per minute until it can clearly observe the DNA band in the gel;

(6) Termination: Place the colored glass plate in the tray and attach it to the side of the polyacrylamide gel. Then, add the reagent C in the above kit, and then place the tray in the oscillation. On the device, shake it at a frequency of 30 times per minute for 2 minutes, rinse with water for 3 seconds;

(7) Obtaining a polyacrylamide gel staining picture: The glass plate treated by the step (6) was air-dried, and a polyacrylamide gel picture as shown in Fig. 1 was obtained by scanning with a BenQ M800 scanner.

Example 2

First, establish a kit

1, preparation reagent A: take silver nitrate 3g dissolved in 800L double distilled water, then add 100mL ethanol and 5mL glacial acetic acid, and then add double distilled water to a volume of 1L;

2, preparation reagent B: take 1g sodium chloride, 0.5g sodium sulfite, 0.3g carbohydrazide, 2g sodium carbonate and 10g glucose, add double distilled water to a volume of 1L;

3. Preparation reagent C: Take 3 mL of glacial acetic acid, and add double distilled water to make up to 1 L.

Second, obtain the DNA polyacrylamide gel staining picture of the cabbage material

(1) PCR amplification: Take 30 copies of the DNA sample of the cabbage material (DNA concentration of 10-20 ng/μL) as a DNA template, and then perform PCR amplification by the following methods using the following primers, respectively, and retain each of the cabbage materials. PCR amplification products:

Forward primer: 5'-CTGCTCGCATTTTTTATCATAC-3' (SEQ ID No. 3),

Reverse primer: 5'-TACGCTTGGGAGAGAAAACTAT-3' (SEQ ID No. 4);

(2) Gel electrophoresis: PCR amplification products of each of the cabbage materials were subjected to 6% polyacrylamide gel electrophoresis according to a conventional method to obtain a glass plate to which a polyacrylamide gel was attached;

(3) taking the glue: removing the rubber plate after the PCR amplification product is subjected to polyacrylamide gel electrophoresis, gently peeling off the glass plate with the groove, and retaining another glass plate with the polyacrylamide gel attached thereto;

(4) Silver staining: a glass plate to which a polyacrylamide gel is attached is placed in a tray, and the side to which the polyacrylamide gel is attached is upward, and then added to the reagent A in the above kit for soaking; Place the basin on the shaker at 45 times per minute The frequency was shaken for 12 minutes, taken out, and rinsed with water for 4 seconds;

(5) Color development: Place the silver-dyed glass plate in the tray and place it on the side with the polyacrylamide gel. Then, add the reagent B in the above kit, and then place the tray. On the oscillator, shake it at a frequency of 20 times per minute until it can clearly observe the DNA band in the gel;

(6) Termination: Place the colored glass plate in the tray and attach it to the side of the polyacrylamide gel. Then, add the reagent C in the above kit, and then place the tray in the oscillation. On the device, shake at a frequency of 45 times per minute for 1.5 minutes, and rinse with water for 4 seconds;

(7) Obtaining a polyacrylamide gel staining picture: The glass plate treated by the step (6) was air-dried, and a polyacrylamide gel picture as shown in Fig. 2 was obtained by scanning with a BenQ M800 scanner.

Example 3

First, establish a kit

1. Preparation reagent A: 2 g of silver nitrate is dissolved in 800 L of deionized water, then 80 mL of ethanol and 4 mL of glacial acetic acid are added, and then double distilled water is added to make a volume of 1 L;

2, preparation reagent B: take 0.8g sodium chloride, 1.5g sodium sulfite, 0.2g carbohydrazide, 6g sodium carbonate and 5g glucose, add double distilled water to a volume of 1L;

3. Preparation reagent C: Take 4 mL of glacial acetic acid, and add double distilled water to make up to 1 L.

Second, the DNA polyacrylamide gel staining picture of the big cockroach material

(1) PCR amplification: Take 30 DNA samples of Daphnia material (DNA concentration: 10-20 ng/μL) as DNA template, and then perform PCR amplification by the following methods using the following primers, respectively, and retain each large sputum. PCR amplification products of ruthenium materials:

Forward primer: 5'-TAATACCAGCACCACCATTT-3' (SEQ ID No. 5),

Reverse primer: 5'-GCTTCATGCAATTAGAGCAG-3' (SEQ ID No. 6);

(2) Gel electrophoresis: 6% polyacrylamide gel electrophoresis of each PCR amplification product of the large sputum material is obtained by a conventional method to obtain a glass plate to which a polyacrylamide gel is attached;

(3) taking the glue: removing the rubber plate after the PCR amplification product is subjected to polyacrylamide gel electrophoresis, gently peeling off the glass plate with the groove, and retaining another glass plate with the polyacrylamide gel attached thereto;

(4) Silver staining: a glass plate to which a polyacrylamide gel is attached is placed in a tray, and the side to which the polyacrylamide gel is attached is upward, and then added to the reagent A in the above kit for soaking; The pot was placed on a shaker, shaken at a frequency of 60 times per minute for 15 minutes, then taken out, and rinsed with water for 5 seconds;

(5) Color development: the silver-dyed glass plate is placed in the tray, and the side of the polyacrylamide gel is attached thereto. Then, the reagent B in the above kit is first added, and then the tray is placed on the shaker, and shaken at a frequency of 30 times per minute until the DNA strip in the gel can be clearly observed;

(6) Termination: Place the colored glass plate in the tray and attach it to the side of the polyacrylamide gel. Then, add the reagent C in the above kit, and then place the tray in the oscillation. On the device, shake it at a frequency of 60 times per minute for 2 minutes, rinse with water for 5 seconds;

(7) Obtaining a polyacrylamide gel staining picture: The glass plate treated by the step (6) was air-dried, and a polyacrylamide gel picture as shown in Fig. 3 was obtained by scanning with a BenQ M800 scanner.

Example 4

First, establish a kit

1, preparation reagent A: take silver nitrate 2.5g dissolved in 800L deionized water, then add 60mL ethanol and 3.5mL glacial acetic acid, and then add double distilled water to a volume of 1L;

2, preparation reagent B: take 0.9g sodium chloride, 1g sodium sulfite, 0.25g carbohydrazide, 5g sodium carbonate and 9g glucose, add double distilled water to a volume of 1L;

3. Preparation reagent C: Take 3 mL of glacial acetic acid, and add double distilled water to make up to 1 L.

2. Obtaining DNA polyacrylamide gel staining picture of barley material

(1) PCR amplification: DNA samples of 66 parts of barley material (DNA concentration of 10-20 ng/μL) were used as DNA templates, and then PCR amplification was carried out by the following methods using the following primers, and PCR of 66 barley materials was retained. Amplification products:

Forward primer: 5'-GAAACCCATCATAGCAGC-3' (SEQ ID No. 7),

Reverse primer: 5'-AAACAGCAGCAAGAGGAG-3' (SEQ ID No. 8);

(2) Gel electrophoresis: PCR amplification products of each barley material were subjected to 6% polyacrylamide gel electrophoresis according to a conventional method to obtain a glass plate to which a polyacrylamide gel was attached;

(3) taking the glue: removing the rubber plate after the PCR amplification product is subjected to polyacrylamide gel electrophoresis, gently peeling off the glass plate with the groove, and retaining another glass plate with the polyacrylamide gel attached thereto;

(4) Silver staining: a glass plate to which a polyacrylamide gel is attached is placed in a tray, and the side to which the polyacrylamide gel is attached is upward, and then added to the reagent A in the above kit for soaking; The pot was placed on a shaker, shaken at a frequency of 60 times per minute for 15 minutes, then taken out, and rinsed with water for 5 seconds;

(5) Color development: Place the silver-dyed glass plate in the tray and place it on the side with the polyacrylamide gel. Then, add the reagent B in the above kit, and then place the tray. On the oscillator, shake it at a frequency of 30 times per minute until it can clearly observe the DNA band in the gel;

(6) Termination: Place the colored glass plate in the tray and attach it to the side of the polyacrylamide gel. Then, add the reagent C in the above kit, and then place the tray in the oscillation. On the device, shake it at a frequency of 60 times per minute for 2 minutes, rinse with water for 5 seconds;

(7) Obtaining a polyacrylamide gel staining picture: The glass plate treated by the step (6) was air-dried, and a polyacrylamide gel picture as shown in Fig. 4 was obtained by scanning with a BenQ M800 scanner.

Example 5

The kit of the above Example 2 was used in this experiment.

1, sample

Purchased the DL500 DNA standard from Takara Bio Inc., Japan, which has 7 DNA standard bands of 50 bp, 100 bp, 150 bp, 200 bp, 300 bp, 400 bp, and 500 bp, except for the 200 bp standard band. The DNA concentration was 30 ng/μL, and the rest were 10 ng/μL.

The DNA standards were sequentially diluted by a 2-fold gradient, that is, the DNA concentration of the diluted samples was 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 of the standard. , 1/256, 1/512, 1/1024, 1/2048, 1/4096, 1/8192, 1/16384, 1/32798.

2, experimental methods

(1) Separation of DNA standards by polyacrylamide gel electrophoresis

Select CBS MGV-216-33 double-sided triple-width vertical electrophoresis tank and electrophoresis apparatus. The size of the electrophoretic glass plate is 33cm wide × 16cm high. The DNA standard is separated by 6% polyacrylamide gel electrophoresis. The polyacrylamide gel consists of acrylamide and methylidene bisacrylamide in a mass ratio of 29:1. The gel thickness was 1.5 mm, and each sample hole was 2 mm wide; 1 μL of each sample was sampled from left to right in a 6% sample well according to the DNA concentration. The electrophoresis buffer was 0.5×TBE buffer [45 mM Tris-boric acid buffer (containing 1 mM EDTA); the preparation method was to weigh 5.4 g of Tris base and 2.75 g of boric acid, and to absorb 2 mL of 0.5 M EDTA (pH 8.0), and double steaming. The water is made up to 1L]; the electrophoresis conditions are: voltage 120V, electrophoresis time 100min.

(2) Silver stain detection DNA standard

1 Glue: remove the PCR product after polyacrylamide gel electrophoresis, gently remove the grooved glass plate, and retain another glass plate with polyacrylamide gel attached;

2 silver staining: the glass plate with the polyacrylamide gel attached is placed in the tray, and the side of the polyacrylamide gel is attached upward, and then the reagent A is added to soak; the tray is placed in the oscillation On the device, shake it at a frequency of 50 times per minute for 12 minutes, take it out, rinse with water for 3 seconds;

3 color development: the silver stained glass plate is placed in the tray, and the side with the polyacrylamide gel attached thereto, then the reagent B is added first, and then the tray is placed on the oscillator. Oscillation at a frequency of 15 times per minute until a clear observation can be made Remove the DNA band in the gel and rinse with double distilled water for 3 seconds;

4 termination: the colored glass plate is placed in the tray, and the side with the polyacrylamide gel attached thereto is upward, then the reagent C is added first, and then the tray is placed on the shaker, and then The frequency was shaken 30 times per minute for 1 minute, rinsed with water for 3 seconds, and placed on a glass rack to dry.

3. Experimental results

The dried gel was taken out, and the strip of the DNA standard was directly observed, and a polyacrylamide gel image as shown in Fig. 5 was obtained by scanning with a BenQ M800 scanner. In the figure, lane 1 is a sample of DNA standard stock, and lanes 2 to 15 are 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128 of the standard sample DNA concentration, respectively. , 1/256, 1/512, 1/1024, 1/2048, 1/4096, 1/819, 1/16,384 DNA concentration samples, wherein 200 bp of DNA is clearly discernible in lane 13, at which time the concentration of DNA is 1/2 standard stock ^12, indicated that the detection sensitivity DNA can reach 30ng / μL × 1/4096 = 7.3 × 10 -3 ng / μL = 7.3pg / μL; other standard DNA bands can be observed in lanes 11 or more, indicating that the detectable DNA sensitivity can be at least 10 ng / μL × 1 / 1024 = 9.8 × 10 ^-3 ng / μL = 9.8 pg / μL; the result is significantly higher than the An et al (2009 introduced in the background art) The highest sensitivity of detection of DNA by polyacrylamide silver staining using formaldehyde by Liang et al. (2014) is 27.8pg/μL and 97pg/μL, respectively.

Claims (2)

A silver staining kit for detecting DNA in a polyacrylamide gel, the kit comprising: Reagent A: the reagent is an aqueous solution, and each liter of the aqueous solution has 3-5 mL of glacial acetic acid, 1-3 g of silver nitrate, and 50-100 mL of ethanol; Reagent B: the reagent is an aqueous solution, and each liter of the aqueous solution has 0.5 to 1 g of sodium chloride, 0.5 to 2.5 g of sodium sulfite, 0.1 to 0.3 g of carbohydrazide, 2 to 10 g of sodium carbonate, and 5 to 10 g of glucose; Reagent C: The reagent is an aqueous solution, and each liter of the aqueous solution has 3 to 5 mL of glacial acetic acid. A method for obtaining a DNA polyacrylamide gel stained image, the method comprising the steps of: (1) taking the glue: removing the rubber plate after the PCR amplification product is subjected to polyacrylamide gel electrophoresis, gently peeling off the glass plate with the groove, and retaining another glass plate with the polyacrylamide gel attached thereto; (2) Silver staining: a glass plate to which a polyacrylamide gel is attached is placed in a tray, and the side to which the polyacrylamide gel is attached is upward, and then the reagent A according to claim 1 is added; Place the tray on the shaker, shake it at a frequency of 30 to 60 times per minute for 8 to 15 minutes, then take it out and rinse it with water for 3 to 5 seconds. (3) Color development: the silver-dyed glass plate is placed in the tray, and the side to which the polyacrylamide gel is attached is upward, and then the reagent B according to claim 1 is added first, and then the tray is placed. On the oscillator, shake at a frequency of 10 to 30 times per minute until the DNA band in the gel can be clearly observed; (4) Termination: the colored glass plate is placed in the tray, and the side to which the polyacrylamide gel is attached is upward, and then the reagent C according to claim 1 is added first, and then the tray is placed. On the oscillator, shake it at a frequency of 30 to 60 times per minute for 1 to 2 minutes, and rinse with water for 3 to 5 seconds. (5) Obtaining a polyacrylamide gel staining picture: The glass plate treated by the step (4) is air-dried, and a polyacrylamide gel picture is obtained by scanning with a scanner.

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