98 Grosse and Manns2(N-morpholino)ethane sulfonic acid (MES) has only one-quarter the
activity of TdT in cacodylate. With Tris-HCl, only 2% of maximal
activity was observed (19). We found that 100 mM Tris-acetate, pH
7.2, is an attractive alternative to cacodylate buffers, giving nearly the
same activity as Chelex-treated potassium cacodylate.
2.3. Divalent Cations
The polymerization reaction requires the presence of a divalent cation,
with an order of efficiency of Mg 2÷ > Zn 2÷ > Co 2÷ > Mn 2÷ (20) for the
elongation of oligonucleotide primers with dAMP. dGTP is also opti-
mally added in the presence of Mg2+; on the other hand, the polymeri-
zation of pyrimidines is best in C02+-containing buffers (18). In genetic
engineering, most tailing reactions are performed in the presence of
either Co 2÷ for pyrimidine additions or Mn 2÷ for purine additions. The
frequent use of Mn 2÷ for purine additions is based on the finding that
Mn 2÷ permits TdT to extend duplex termini (6), and that contaminat-
ing nucleases are less active or even inactive in the presence of Mn 2÷.
2.4. Parameters Influencing the Reaction
TdT binds its substrates with rather high K m values of 10O laM for
dATP and dGTP, 500 ~M for dTTP and dCTP, and 1 gM for oligo-
nucleotide primers and up to 1 mM for homopolymer primer ends (18).
Thus, for optimal reaction rates, all the substrates should be present in
highly concentrated solutions. This is most readily achieved by work-
ing in small volumes. The number of nucleotides added to a distinct
primer molecule will in principle be determined by the ratio of Mol
dNTPs:Mol Y-OH-termini in the reaction mixture. Thus, if there is a
100-fold molar excess of dNTP over primer ends, nearly 100 nucle-
otides will be incorporated per primer with a distribution of products
that ideally obeys the Poisson distribution (for details, see ref. 4). Such
a behavior is based on the assumption that all primer ends are equally
utilized by the enzyme, which is only true for the homopolymeric
extension of an isohomopolymeric primer, e.g., (dC)-tailing of (dC) 10.
On the other hand, tailing of restriction fragments is far from being ideal,
mainly because differences in primer binding exist that are dependent on
the availability of the 3'-OH group and the nucleotide composition of the
primer termini. Assume a blunt-ended DNA that should become extended
by an oligo(dC) tail. The very first nucleotide incorporation step at the
blunt end will be rather slow, because of the poor availability of a free,