96 Grosse and Mannsresearch, still nothing is known about the biological function of TdT.
Nevertheless, TdT is an important marker for the diagnosis and clas-
sification of pre-B-cell and pre-T-cell leukemias and differential diag-
nosis of myeloid leukemias. The use of TdT as a marker in leukemia
diagnosis is the subject of two excellent reviews (2,3).
1.3. Applications
Because of its terminal addition properties, TdT has been widely
employed for the production of synthetic homo- and heteropolymers.
A wide variety of polymers can be synthesized from derivatives of dNTPs,
which areN-acetylated orN-alkylated.Adetailed discussion of the mecha-
nism of these reactions, the statistics of polymerization, inhibitors,
and practical synthetic applications has been provided by Bollum (4).
Homopolymeric tailing of linear duplex DNA for in vitro genetic
recombination is the most common application today. In a key experi-
ment of genetic engineering, linear bacteriophage P22 DNA was tailed
with oligo(dA), and another set of linear P22 was tailed with the comple-
mentary oligo(dT). After annealing, the dA/dT-tailed recombinant
molecule was ligated to give covalently closed dimeric circles (5).
Details of the tailing reaction were recently review~
Another application utilizes the limited addition of rNTPs (8),
ddNTPs (9), and cordycepin triphosphate (10,11) for terminal addi-
tions. Since these substrates lead to chain terminations, 3' ends can be
extended in a controlled manner. This has been exploited for the radio-
active labeling of the 3'-hydroxyl ends of single- and double-stranded
DNA. A modification of this method is the 3' labeling of DNA primers
by biotin- 11-dUTP (12,13) or fluorescent succinylfluorescein-labeled
dideoxnucleoside triphosphates (14). TdT has also been used to elon-
gate primers by only one phosphorothioate deoxynucleotide. The result-
ing extension products were purified by means of a mercury beaded column.
This approach has been used for the introduction of mispaired primer ends
for site-directed mutagenesis protocols (6).
- The Enzyme
2.1. Structure
TdT is a monomeric enzyme with a mol mass of about 60,000 Da,
whose eDNA has been cloned and expressed in various organisms
(see, e.g., [15]). Dependent on the biological source, the enzyme con-