38 Landgraf and Wolfesfrom this limit, the yield of the reaction is restricted by too low enzyme
concentration or by competitive reannealing of the fragments (12).
For the amplification of long fragments, the reaction efficiency is very
dependent on the fragment length: The efficiency of 1.9 for a fragment
of 1000 nucleotides declines to 1.4 for a fragment of 7 kb (15).
3.2. Application ofTaq Polymerase in the PCR
The application of Taq polymerase in the PCR requires certain pre-
requisites to be fulfilled.
3.2.1. Buffers
The buffer used most commonly in the PCR consists of 10 mMTris-
HC1, pH 8.4, 50 mM KC1, 1.5-2.5 mM MgC12, and 0.01% gelatin as
stabilizer (2). The Mg 2÷ ion concentration influences predominantly
the specificity of the PCR. With increasing Mg 2÷ concentrations, the
amplification of unspecific sequences increases. At high dNTP con-
centrations, it should also be taken into account that the concentration
of free ions is reduced as a result of Mg 2÷ binding to dNTPs and to
DNA. It was reported that, in an incorporation experiment, high dNTP
concentrations (4-6 mM) were inhibitory (8).
A different widely used buffer system is composed of 67 mM Tris-
HC1, pH 8.8, 6.7 mMMgCI 2, 16.6 mM [NH412SO4, 10 mM 13-mercapto-
ethanol, and 0.02% bovine serum albumin (BSA) or gelatine. In principle,
gelatine should be given preference over BSA, because BSA is likely
to denature. On the other hand, in some protocols the use of stabilizing
proteins is totally abandoned (8). Sometimes 10% dimethylsulfoxide
(DMSO) is added to the buffers mentioned above (10).
The Michaelis Menten constant K m of Taq polymerase for each
dNTP has a value of 10-15 laM (16). A normal PCR experiment con-
tains 50-200 1aM of each dNTP. This concentration is sufficient to
amplify even large fragments to microgram yields without any limi-
tation of dNTP substrate. Even after 60 cycles, a pool of about 50%
dNTPs is still available (8). Sometimes the dNTP concentration is
lowered to 10 kVk/in order to achieve an optimum incorporation of
radioactive labeled deoxynucleotides (17). Modified nucleotides, e.g.,
deoxynucleoside-t~-thiotriphosphates (dNTPaS) (18), 7-deaza-2'-
deoxyguanosine-5'-triphosphate d(cTGTP) (8), 5-Biotin (11)-2'
deoxyuridine-5'- triphosphate (Bio 1 1 dUTP) (16), are incorporated at
high rates by Taq polymerase.