TUNEL is a method for detecting
apoptoticDNA fragmentation, widely used to identify and quantify apoptotic cells, or to detect excessive DNA breakage in individual cells.[3] The
assay relies on the use of
terminal deoxynucleotidyl transferase (TdT), an
enzyme that
catalyzes attachment of deoxynucleotides, tagged with a
fluorochrome or another
marker, to 3'-hydroxyl termini of DNA double strand breaks. It may also label cells having DNA damage by other means than in the course of apoptosis.
History
The fluorochrome-based TUNEL assay applicable for
flow cytometry, combining the detection of DNA strand breaks with respect to the
cell cycle-phase position, was originally developed by Gorczyca et al.[4] Concurrently, the avidin-peroxidase labeling assay applicable for
light absorption microscope was described by Gavrieli et al.[5] Since 1992 the TUNEL has become one of the main methods for detecting apoptotic programmed cell death.[6] However, for years there has been a debate about its accuracy, due to problems in the original assay which caused
necrotic cells to be inappropriately labeled as apoptotic.[7] The method has subsequently been improved dramatically and if performed correctly should only identify cells in the last phase of
apoptosis.[8][9] New methods incorporate the dUTPs modified by fluorophores or haptens, including
biotin or
bromine, which can be detected directly in the case of a fluorescently-modified
nucleotide (i.e., fluorescein-dUTP), or indirectly with streptavidin or
antibodies, if biotin-dUTP or BrdUTP are used, respectively. The most sensitive of them is the method utilizing incorporation of BrdUTP by
TdT followed by immunocytochemical detection of
BrdU.[10]
References
^Gorczyca W, Traganos F, Jesionowska H, Darzynkiewicz Z (1993). "Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells. Analogy to apoptosis of somatic cells". Exp Cell Res. 207 (1): 202–205.
doi:
10.1006/excr.1993.1182.
PMID8391465.
^Gorczyca W, Bruno S, Darzynkiewicz RJ, Gong J, Darzynkiewicz Z (1992). "DNA strand breaks occurring during apoptosis: Their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors". International Journal of Oncology. 1 (6): 639–648.
doi:
10.3892/ijo.1.6.639.
PMID21584593.
^Negoescu A, Guillermet C, Lorimier P, Brambilla E, Labat-Moleur F (1998). "Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity". Biomed Pharmacother. 52 (6): 252–8.
doi:
10.1016/S0753-3322(98)80010-3.
PMID9755824.