Terminal deoxynucleotidyl transferase dUTP nick end labeling is a method for
detecting DNA fragmentation by labeling the terminal end of nucleic acids.
Method TUNEL is a common method for detecting
DNA fragmentation that results from apoptotic signaling cascades. The assay
relies on the presence of nicks in the DNA which can be identified by terminal
deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition
of dUTPs that are secondarily labeled with a marker. It may also label cells
that have suffered severe DNA damage. History
Originally described in the paper by Gavrieli, Sherman, and Ben-Sasson in
1992, TUNEL has become one of the main methods for detecting apoptotic
programmed cell death. 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. The method has subsequently
been improved dramatically and if performed correctly should only identify
cells in the last phase of apoptosis. 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, or
indirectly with streptavidin or antibodies, if biotin-dUTP or BrdUTP are
used, respectively. Often at late stages of apoptosis, adherent cells are known
to detach or “pop” off. For a reliable and reproducible TUNEL imaging assay,
the modified nucleotide must not only be an acceptable substrate for TdT, but the
detection method must also be sensitive without bringing about any additional
loss of cells from the sample. References
^ Gavrieli Y, Sherman Y, Ben-Sasson SA. "Identification of programmed cell death
in situ via specific labeling of nuclear DNA fragmentation". J Cell Biol 119:
493–501. doi:10.1083/jcb.119.3.493. PMC 2289665. PMID 1400587.
^ Grasl-Kraupp B, Ruttkay-Nedecky B, Koudelka H, Bukowska K, Bursch W,
Schulte-Hermann R. "In situ detection of fragmented DNA fails to discriminate
among apoptosis, necrosis, and autolytic cell death: a cautionary note".
Hepatology 21: 1465–8. doi:10.1002/hep.1840210534. PMID
7737654. ^ Negoescu A, Lorimier P, Labat-Moleur
F, Drouet C, Robert C, Guillermet C, Brambilla C, Brambilla E. "In situ
apoptotic cell labeling by the TUNEL method: improvement and evaluation on
cell preparations". J Histochem Cytochem 44: 959–68. doi:10.1177/44.9.8773561.
PMID 8773561. ^ Negoescu A, Guillermet C, Lorimier P,
Brambilla E, Labat-Moleur F. "Importance of DNA fragmentation in apoptosis with
regard to TUNEL specificity". Biomed Pharmacother 52: 252–8.
doi:10.1016/S0753-3322(98)80010-3. PMID 9755824.
External links TUNEL at the US National Library of
Medicine Medical Subject Headings