A method for assessing strand breaks in DNA.
A simple method has been developed to assess strand breaks in extracted DNA. The method uses the enzyme terminal deoxynucleotidyl transferase (TDT) to incorporate labeled deoxycytidine triphosphate (dCTP) in the presence of dideoxy-CTP (ddCTP) which is added to ensure that the reaction goes to completion. Following development of the method, the extent of DNA degradation in 21 blood or bone marrow samples, which had varying degrees of DNA degradation, was measured by the TDT assay, by gel electrophoresis, or by a laborious PCR-based method which quantifies the number of amplifiable N-ras targets in a sample. The TDT assay was more sensitive at detecting strand breaks than electrophoresis and there was good correlation between the results of the TDT assay and the N-ras assay. The TDT assay was also used to demonstrate the development of strand breaks during induced apoptosis. The TDT assay is thus a simple and semiquantitative method to study strand breaks produced by DNA damage.
Peng
L
L
Department of Hematology, Flinders University of South Australia, Bedford Park, South Australia, 5042, Australia.
Brisco
M J
MJ
Morley
A A
AA
D017209:Apoptosis / Q000502:physiology; D001854:Bone Marrow Cells / Q000378:metabolism; D004247:DNA / Q000097:blood; D004249:DNA Damage / Q000235:genetics*; D053938:DNA Fragmentation / Q000502:physiology; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides; D004587:Electrophoresis, Agar Gel / Q000379:methods; D011905:Genes, ras / Q000235:genetics; D008214:Lymphocytes / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods
D003845:Deoxycytosine Nucleotides; D003854:Deoxyribonucleotides; D010761:Phosphorus Radioisotopes; C024107:2'-deoxycytidine 5'-triphosphate; D004247:DNA; D004253:DNA Nucleotidylexotransferase