The use of streptavidin-coated magnetic beads and biotinylated antibodies to investigate induction and repair of DNA damage: analysis of repair patches in specific sequences of uv-irradiated human fibroblasts.
A new immunoextraction method using biotinylated antibodies and streptavidin-coated magnetic beads has been developed and applied to study the repair of uv-induced DNA damage in specific DNA sequences. uv-irradiated cells were allowed to carry out DNA repair for various time intervals in the presence of 5-bromodeoxyuridine (BrdU). Purified and restricted DNA was subjected to an immunoextraction method employing an anti-BrdU antibody (alpha Brdu), biotinylated goat antimouse antibodies (G alpha Mbio), and streptavidin-coated polymeric magnetic beads. Separation of BrdU containing DNA was achieved by using a magnetic device. This extraction procedure resulted in two fractions of DNA, i.e., BrdU-containing and non-BrdU-containing DNA. Both fractions were blotted on filters and subsequently hybridized with specific DNA probes to determine the relative amount of defined fragments in the two fractions of DNA. Repair experiments using normal primary human fibroblasts showed no difference in the incorporation of repair label in the active adenosine deaminase gene and the inactive 754 locus during the first 4 h following uv irradiation. After longer repair times the active gene incorporated more repair label than the inactive gene, consistent with the known preferential repair of cyclobutane pyrimidine dimers from active housekeeping genes.
Kalle
W H
WH
MGC-Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, The Netherlands.
Hazekamp-van Dokkum
A M
AM
Lohman
P H
PH
Natarajan
A T
AT
van Zeeland
A A
AA
Mullenders
L H
LH
D000906:Antibodies; D001426:Bacterial Proteins; D001710:Biotin; D001973:Bromodeoxyuridine / Q000276:immunology; D004247:DNA / Q000302:isolation & purification / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000528:radiation effects*; D005069:Evaluation Studies as Topic; D005347:Fibroblasts / Q000528:radiation effects; D006801:Humans; D008280:Magnetics; D019809:Streptavidin; D014466:Ultraviolet Rays
D000906:Antibodies; D001426:Bacterial Proteins; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D001973:Bromodeoxyuridine