Estimation of DNA by the Diphenylamine Method

Principle
When DNA is treated with diphenylamine under acidic conditions, a blue compound is formed with the sharp observation maximum at 595 n. This reaction is produced by 2 deoxypentose in general and is not specific for DNA. In acid solution the straight from the deoxypentose is converted to the highly reactive b-hydroxy lerulin aldehyde, which reacts with diphenylamine to produce a blue complex. In DNA, only the deoxyribose of purine nucleotide reacts so that the value obtained represents one half of the total deoxyribose produced.

Materials

• Commercial sample of DNA – 10 mg.
• Buffer saline 0.15 mL/L NaCl, 0.15 mL/L sodium citrate made up to 500 mL.
• Diphenyl amine reagent: dissolved in 10 gms of pure diphenyl amine in 1 liter of glacial acetic acid and add 25 mL of concentrated H₂SO₄. This solution must be prepared fresh.
• Boiling water bath

Procedure
Dissolve 10 gms of nucleic acid in 50 mL of buffer saline. Remove 2 mL and add 4 mL of diphenylamine. Remove 2 mL and add 4 mL of DPA reagent. Heat on a boiling water bath for 10 nm. Cool and read the extension at 595 nm. Read the test and standard against nucleic acid and the commercial sample for DNA.

Result:
Concentration of DNA present in the given sample ............... mg/2 mL.