In situ ligation simplified: using PCR fragments for detection of double-strand DNA breaks in tissue sections.
The simplified in situ ligation procedure is described. All reagents for the assay can be easily obtained in any molecular or cell biology laboratory. The technique uses ligation of double-stranded, PCR-derived DNA fragments labeled with digoxigenin or fluorophores for highly selective detection of apoptotic cells in paraffin-embedded tissue sections. Two types of DNA fragments prepared by PCR are employed. The fragment synthesized by Taq polymerase contains single-base 3' overhangs, whereas the Pfu polymerase-made fragment is blunt ended. Both fragments can be used as specific, sensitive and cost-effective DNA damage probes. After ligation to apoptotic nuclei in tissue sections, they indicate the presence of double-strand DNA breaks with single-base 3' overhangs as well as blunt ends.
Departments of Neurosurgery and Molecular & Cellular Biology, Baylor College of Medicine, and Michael E DeBakey VA Medical Center, Houston, TX, USA. vdidenko@bcm.edu
Vladimir V
VV
Didenko
D000818:Animals; D053903:DNA Breaks, Double-Stranded; D015342:DNA Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*
D015342:DNA Probes