A simple and efficient method for the quantitative analysis of thymine dimers in cyanobacteria, phytoplankton and macroalgae

Cyclobutane dimers are the most common DNA lesions after exposure of cells to UV-B radiation. A quantitative method was developed to determine the fre quency of thymine dimers in aquatic primary producers such as cyanobacteria , phytoplankton and macroalgae to study the effects of UV radiation. Genomi c DNA was extracted and purified by using standard biochemical and molecula r biology techniques. DNA was transferred to a nylon membrane in a slot or dot blot and incubated with a primary antibody (anti thymine dimer KTM53) a gainst thymine dimers. The secondary antibody was an anti-mouse IgG (Fab sp ecific) peroxidase conjugate. The blots were quantified in a Kodak Digital Science Image Station 440 CF by the chemiluminescence method. The calibrati on of the method was achieved by using the plasmid pBSK with known DNA sequ ence, length and number of adjacent thymine pairs. This method permits the measurement of low as well as high levels of DNA lesions in nanogram quanti ties of DNA. This method can be used for cultured as well as naturally occu rring organisms.