Estimation of DNA
Estimation of DNA is possible by a number of methods based on the physical or chemical property of the nucleic acid. A convenient and easy colorimetric method is available on the basis of quantitative reaction of deoxysugar with diphenylamine reagent.
Under extreme acid conditions, DNA is initially depurinated quantitatively followed by the dehydration of sugar to-hydroxylevulinylaldehyde. This aldehyde condenses, in acidic medium, with diphenytumine to produce a deep-blue colored condensation products with absorption maximum at 595nm.
» DNA Standard (0.5mg/mL)
» Saline Citrate (0.15M NaCl, 0.015 M Na3 Citrate) Solution
» Diphenylamine Reagent
Mix 5g fresh or recrystallized diphenylamine, 500mL glacial acetic acid and 13.75mL cone. H2SO4. Stable for six months at 2°C; warm to room temperature and swirl to remix before use.
This method is commonly applied for samples of 50—500|Mg DNA. Burton has introduced a modified diphenylamine reagent containing acetaldehyde; in this reagent, samples are heated at 30°C for several hours for developing color.
1. Burton, K (1956) BiochemJ 62 315.
2. Ashwell, G (1957) In: Methods in Enzymol 3 (Eds Colowick, S P Kaplan, N O) Academic Press New York p 99.
3. Sadasivam, S, Radhashanmugasundaram and Shanmugasundaram, E R B (1915) Arogya – J Health Sci 1 125.
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