Original paper

Optimization of nucleic acids assay in green and blue-green algae: Extraction procedures and the light-activated diphenylamine reaction for DNA

Zachleder, Vilém

Algological Studies/Archiv für Hydrobiologie, Supplement Volumes No. 67 (1984), p. 313 - 328

11 references

published: Jun 20, 1984

DOI: 10.1127/algol_stud/67/1984/313

BibTeX file

ArtNo. ESP142009500020, Price: 29.00 €

Download preview PDF Buy as PDF

Abstract

A light-activated reaction of diphenylamine with DNA was adapted for the determination of DNA content in three species of green algae (Scenedesmus, Chlorella and Chlamydomonas) and one species of blue-green algae (Synechococcus elongatus). The shortest colour development of diphenylamine reagent with DNA was found to be 4-6 hrs at 40°C in 1N PCA, if the samples were illuminated from both sides by fluorescent lamps. Optimal conditions for hydrolysis of nucleic acids and colour reaction were examined. The extractability of RNA measured as absorbance at 260 nm was found to be very rapid (complete in about half an hour) and not dependent on algal species or their growth conditions. The extractability of DNA from cells differed markedly in various species of algae and even in one species according to both the age of the cells, and the irradiance and light quality under which they were grown. Therefore, it is recommended that the effects of duration and temperature of hydrolysis should be examined before cells grown under different conditions are analyzed. The concentration of 0.5 N perchloric acid was found to be most suitable for the hydrolysis. To give an optimal yield of colour after the diphenylamine photoactivated reaction, duration of hydrolysis should be 5 hrs at 60°C for Chlamydomonas geitleri, 3 hrs at 60°C or 2 hrs at 70°C for Scenedesmus quadricauda and Chlorella vulgaris and 5 hrs at 70°C for Synechococcus elongatus. These values were found suitable for algae grown under a wide range of irradiances in white light and optimal temperature.

Keywords

Synechococcus • Chlamydomonas • Chlorella • Scenedesmus • DNA assay • diphenylamine reaction • light activation • hydrolysis • extractability