Dear Santosh,

The principle underlying estimation of DNA using diphenylamine is the reaction of diphenylamine with deoxyribose sugar producing blue-coloured complex. The DNA sample is boiled under extremely acidic conditions; this causes depurination of the DNA followed by dehydration of deoxyribose sugar into a highly reactive ω-hydroxylevulinylaldehyde. The reaction is not specific for DNA and is given by 2-deoxypentoses, in general. The ω-hydroxylevulinylaldehyde, under acidic conditions, reacts with diphenylamine to produce a blue-coloured complex that absorbs at 595 nm. The mechanism of reaction of deoxyribose sugar with diphenylamine is shown in attached figure. As the sugar linked to only purine residues participates in the reaction, the readout is only from 50% of the total number of nucleotides. As this holds true for both the known standard and the given unknown sample, the concentration of the unknown sample can be directly calculated from the standard graph.

 CALORIMETRIC METHOD

Principle-The method depends on the production of a pink color by the reaction of cysteine and sulfuric acid with DNA. Since pentoses do not yield a color reaction with cysteine and sulfuric acid, yeast nucleic acid is unreactive. Therefore, despite the presence of ribonucleic acid (RNA), a direct estimation of desoxyribonucleic acid can be made.

 Reagents

1.        5 % cysteine hydrochloride  solut,ion in water.

2.       70 % sulfuric acid. 3

3.       DNA standard. 0.05 % solution of sodium desoxyribose nucleate’ in water solution kept at 40. The N:P ratio of the sodium salt was 1: 1.66 (theoretical, 1: 1.69).

4.       RNA standard. 0.05 % of sodium ribose nucleate 2 in water solution kept at 40 The N: P ratio of the sodium salt was 1: 1.69 (theoretical, 1 : 1.69).

Please find attached herewith fig and pdf file

Regards,

Prem Baboo

There are several methods to establish the concentration of a solution of nucleic acids, including spectrophotometric quantification and UV fluorescence in presence of a DNA dye.

https://en.wikipedia.org/wiki/Nucleic_acid_quantitation

which method you are focusing

It's quite simple if you read the absorbance of you sample at 260 nm.