TUNEL Assay: An Overview of Techniques

The study of DNA damage holds a wide interest within both basic and applied fields of research. Elucidating the mechanisms involved in the generation of DNA damage, and the consequences of this damage, will have an enormous impact on multiple fields of scientific research and will ultimately lead to a better understanding of human disease. One of the most widely used methods for detecting DNA damage in situ is TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining (1 ). TUNEL staining was initially described as a method for staining cells that have undergone programmed cell death, or apoptosis, and exhibit the biochemical hallmark of apoptosis—internucleosomal DNA fragmentation (2 –6 ). TUNEL staining relies on the ability of the enzyme terminal deoxynucleotidyl transferase to incorporate labeled dUTP into free 3′-hydroxyl termini generated by the fragmentation of genomic DNA into low molecular weight double-stranded DNA and high molecular weight single stranded DNA (1 ). While TUNEL staining has nearly universally been adopted as the method of choice for detecting apoptosis in situ , it should be recognized that TUNEL staining is not limited to the detection of apoptotic cells. TUNEL staining may also be used to detect DNA damage associated with non-apoptotic events such as necrotic cell death induced by exposure to toxic compounds and other insults (7 ), and TUNEL staining has also been reported to stain cells undergoing active DNA repair (8 ). Therefore TUNEL staining may be considered generally as a method for the detection of DNA damage (DNA fragmentation), and under the appropriate circumstances, more specifically as a method for identifying apoptotic cells.