DNA staining method - Feulgen staining
Feulgen staining is a classical method for displaying DNA, one of the main staining methods for DNA quantification.
Reaction principle
After the sample is hydrolyzed by dilute hydrochloric acid, the purine base in the DNA molecule is dissociated, and an aldehyde group is present at one end of the ribose. The colorless magenta in the Schiff reagent can react with the aldehyde group to form a compound molecule containing a thiol group, and since the thiol group is a chromophore, it can exhibit a purplish red color. That is to say, the aldehyde group produced by the dilute acid hydrolysis of DNA has a reducing action, and can be combined with the colorless magenta to form a purple-red compound, thereby showing the distribution of DNA.
Reaction equation
2HCl Na2S2O5→2NaCl SO2 H2SO3
DNA is the main genetic material that is concentrated on chromosomes. In 1924, Fürgen first used Schiff reagent as a test to identify the presence of DNA on the chromosome, so it was called the Fürgen staining method. The reaction principle of the Fürgen staining method is mainly related to the chemical properties of Schiff reagent. The basic components of this reagent are basic fuchsin, sodium metabisulfite (NaHSO3) and hydrochloric acid. The main component of basic fuchsin is triamino. Triphenylmethane chloride. Shanghai Chuangsai Technology provides sodium bisulfite (AR), Sodium bisulfate, 7631-90-5, product number: C84-2756-500G, and the price is 76 yuan.
The basic magenta is pink, and when it is reacted with sulfite, it is oxidized, so that the quinone type becomes benzene type, and the pink color changes to colorless and transparent N-sulfinic acid sulfite paracementine. When the aldehyde group acts, its molecular formula returns to a quinoid structure, which is purple-red. Shanghai Chuangsai Technology provides basic fuchsin, AR, 632-99-5, product number: D16-1009943-25g, the price is 29 yuan.
When hydrolyzed by 1M HCl at 60 ° C, the glycosidic bond between the purine base and the deoxyribose in the DNA molecule can be interrupted, the hydrazine can be removed, and the aldehyde group and the aldehyde group can be released from the deoxyribose C-1 position. The reagent reaction was reddish purple. Only the DNA in the cell has this specific Fulgen response, so the presence of DNA can be identified using the Fuergen reaction. And widely used in nuclear and chromosome research.
Experimental attention
Among the fixatives, OsO4 and Carnoy are better. OsO4 (1% or 0.5%) is an ideal fixative for Feulgen reaction. However, because OsO4 is more expensive, Carnoy fixative is generally used. In the Feulgen reaction, the Bouin solution cannot be used alone because it is the worst fixative for the Feulgen reaction, but the Allin modified Bouin-Aller fixative works better. Shanghai Chuangsai Technology provides ethanolamine (chromatographic fixative, 30 ° C), Ethanolamine, 141-43-5, product number: C84-103805-100ml, the price is 170.5 yuan.
The Feulgen reaction is usually hydrolyzed with a dilute acid, but the hydrolysis time must be appropriate. If the hydrolysis time is not enough, the reaction will be weak; if the hydrolysis time is too long or the hydrolysis is too intense, the deoxyribose is also easy to fall, and the reaction is also weakened. The appropriate hydrolysis time is generally 8 to 12 minutes. However, the length of hydrolysis depends on the type of specimen (such as thickness), the nature of the fixative, and the concentration of the acid.
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