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WO2018107879A1 - Kit de coloration à l'argent permettant la détection de l'adn dans un gel de polyacrylamide et son utilisation - Google Patents

Kit de coloration à l'argent permettant la détection de l'adn dans un gel de polyacrylamide et son utilisation Download PDF

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Publication number
WO2018107879A1
WO2018107879A1 PCT/CN2017/105452 CN2017105452W WO2018107879A1 WO 2018107879 A1 WO2018107879 A1 WO 2018107879A1 CN 2017105452 W CN2017105452 W CN 2017105452W WO 2018107879 A1 WO2018107879 A1 WO 2018107879A1
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WIPO (PCT)
Prior art keywords
polyacrylamide gel
reagent
glass plate
dna
tray
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Ceased
Application number
PCT/CN2017/105452
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English (en)
Chinese (zh)
Inventor
郭培国
李荣华
刘文杰
夏岩石
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Guangzhou University
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Guangzhou University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/302—Stain compositions

Definitions

  • 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.
  • 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].
  • 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.
  • 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. .
  • a silver staining kit for detecting DNA in a polyacrylamide gel 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:
  • 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;
  • the kit of the invention has the following advantages:
  • the reagent in the kit of the present invention discards formaldehyde which is harmful to human health.
  • the minimum detectable limit of the kit of the present invention is 7.3 pg/ ⁇ L, and the sensitivity is significantly higher than that of the prior art.
  • Figure 1 is a DNA polyacrylamide gel staining image of 30 copies of tobacco material PCR amplification products.
  • 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.
  • M is an electrophoresis band of DNA standards
  • 1 to 30 are PCR amplification of the first to 30th core materials.
  • Fig. 3 is a DNA polyacrylamide gel staining image of 30 PCR products of the giant salamander material.
  • M is an electrophoresis band of DNA standards
  • 1 to 30 is the first to 30th maggot material PCR.
  • Figure 4 is a DNA polyacrylamide gel staining image of 66 parts of barley material PCR amplification products.
  • M is an electrophoresis band of DNA standards
  • 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.
  • lane 1 is an electrophoresis band of a DNA standard stock solution
  • lanes 2 to 15 are electrophoresis bands of different concentration standard stock solutions, respectively.
  • 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;
  • 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;
  • Preparation reagent C Take 5 mL of glacial acetic acid, and add to deionized water to make up to 1 L.
  • PCR amplification Take 30 samples of tobacco material DNA (DNA concentration of 10-20 ng/ ⁇ L) 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:
  • Reverse primer 5'-CCACAAGCAGTATTGGAGCA-3' (SEQ ID No. 2);
  • 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;
  • 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;
  • 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;
  • Preparation reagent C Take 3 mL of glacial acetic acid, and add double distilled water to make up to 1 L.
  • PCR amplification Take 30 copies of the DNA sample of the cabbage material (DNA concentration of 10-20 ng/ ⁇ L) 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:
  • Reverse primer 5'-TACGCTTGGGAGAAAACTAT-3' (SEQ ID No. 4);
  • 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;
  • 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;
  • 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;
  • Preparation reagent C Take 4 mL of glacial acetic acid, and add double distilled water to make up to 1 L.
  • PCR amplification Take 30 DNA samples of Daphnia material (DNA concentration: 10-20 ng/ ⁇ L) 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 Take 30 DNA samples of Daphnia material (DNA concentration: 10-20 ng/ ⁇ L) as DNA template, and then perform PCR amplification by the following methods using the following primers, respectively, and retain each large sputum.
  • Reverse primer 5'-GCTTCATGCAATTAGAGCAG-3' (SEQ ID No. 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;
  • 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;
  • 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;
  • Preparation reagent C Take 3 mL of glacial acetic acid, and add double distilled water to make up to 1 L.
  • PCR amplification DNA samples of 66 parts of barley material (DNA concentration of 10-20 ng/ ⁇ L) 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:
  • Reverse primer 5'-AAACAGCAGCAAGAGGAG-3' (SEQ ID No. 8);
  • 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;
  • 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.
  • 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 ⁇ L 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.
  • Glue remove the PCR product after polyacrylamide gel electrophoresis, gently remove the grooved glass plate, and retain another glass plate with polyacrylamide gel attached;
  • 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;
  • 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.
  • lane 1 is a sample of DNA standard stock
  • 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.

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  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
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  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un kit de coloration à l'argent permettant la détection de l'ADN dans un gel de polyacrylamide, le kit comprenant un réactif A, ledit réactif constituant une solution aqueuse et chaque litre de la solution aqueuse contenant 3 à 5 ml d'acide acétique glacial, 1 à 3 g de nitrate d'argent et 50 à 100 ml d'éthanol; un réactif B, ledit réactif constituant une solution aqueuse et chaque litre de la solution aqueuse contenant 0,5 à 1 g de chlorure de sodium, 0,5 à 2,5 g de sulfite de sodium, 0,1 à 0,3 g de carbohydrazide, 2 à 10 g de carbonate de sodium et 5 à 10 g de glucose; et un réactif C, ledit réactif constituant une solution aqueuse et chaque litre de la solution aqueuse contenant 3 à 5 ml d'acide acétique glacial.
PCT/CN2017/105452 2016-12-13 2017-10-10 Kit de coloration à l'argent permettant la détection de l'adn dans un gel de polyacrylamide et son utilisation Ceased WO2018107879A1 (fr)

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CN201611144993.5A CN106596232B (zh) 2016-12-13 2016-12-13 一种检测聚丙烯酰胺凝胶中dna的银染试剂盒及其应用
CN201611144993.5 2016-12-13

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Cited By (1)

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CN116840328A (zh) * 2023-06-29 2023-10-03 济南大学 一种用聚丙烯酰胺凝胶电泳分析糖胺聚糖药物的方法

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CN106596232B (zh) * 2016-12-13 2019-04-30 广州大学 一种检测聚丙烯酰胺凝胶中dna的银染试剂盒及其应用

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