Terminal deoxynucleotidyl transferase

Terminal Deoxynucleotidyl Transferase

Terminal deoxynucleotidyl transferase , also known as DNA nucleotidylexotransferase or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells...

 dUTP nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids.

Method

TUNEL is a common method for detecting DNA

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 fragmentation that results from apoptotic

Apoptosis

Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

 signaling cascades. The assay

Assay

An assay is a procedure in molecular biology for testing or measuring the activity of a drug or biochemical in an organism or organic sample. A quantitative assay may also measure the amount of a substance in a sample. Bioassays and immunoassays are among the many varieties of specialized...

 relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase

Terminal Deoxynucleotidyl Transferase

Terminal deoxynucleotidyl transferase , also known as DNA nucleotidylexotransferase or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells...

 or TdT, an enzyme

Enzyme

Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

 that will catalyze the addition of dUTPs that are secondarily labeled with a marker

Marker

Marker may refer to:* Marker , a morpheme that indicates some grammatical function* Marker , a special-purpose computer* A set of sewing patterns tightly arranged within a rectangle that is placed over cloth to be cut...

. It may also label cells that have suffered severe DNA damage.

History

Originally described in the paper by Garvrieli, 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

Necrosis

Necrosis is the premature death of cells in living tissue. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma. This is in contrast to apoptosis, which is a naturally occurring cause of cellular death...

 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

Apoptosis

Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

.. 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. 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.