Taq Polymerase 393.2.2. Primer
In theory, at least a 16 bp of primer are necessary for an unambigu-
ous hybridization to a given stretch of DNA in the human genome (3
× 109 bp). In practice, primers 20-25 nucleotides long are used in PCR
experiments, with the G-C content varying from 40-60% in order to
ensure specific amplification. Primers shorter than 20 nucleotides are
used successfully for allele-specific hybridization (19). Except for
this purpose, utilization of shorter primers should be avoided, since
they might detach from the template at the high temperature where
polymerization is effective.
To determine the approximate melting temperatures of a primer-
DNA duplex, the equation:
Tm [°CI = 2[T + A] + 4[G + C]
can be used (20). More exact numbers can be calculated by applying
algorithms (21) that take the occurrence of intramolecular interac-
tions, e.g., hairpin structures, into account.
In established oligodeoxynucleotide synthesis, a product yield of
more than 80% for short primers can be expected (20). Therefore, a
purification of the crude synthesis products is most often not required.
Depending on circumstances, which demand purification of the crude
products, e.g., an oligodeoxynucleotide has to be labeled prior to the
PCR, two methods are suitable for purification: (1) When little amounts
of primers are needed, denaturing PAGE with 7M urea followed by
detection by shadow casting (22) is a convenient method for purifica-
tion. Several oligodeoxynucleotides can be purified in parallel. Since
urea is an inhibitor of Taq polymerase, it must be removed by chro-
matographic techniques after elution from the gel slice. (2) Chromato-
graphic procedures are a better choice for the purifications of large
quantities of oligodeoxynucleotides. Reversed-phase HPLC techniques
and ion-exchange chromatography are commonly used (23).
Primers should always be stored lyophilized in aliquots at-20"C. It
is convenient to prepare 10X stock solutions with 1-10 gM concentra-
tions of the primer. A primer concentration of 0.1-1 laM is optimal for
the PCR. An increase in the oligodeoxynucleotide concentration will
produce a rise in nonspecific bands. One example of the production of
an unspecific band is owing to partial primer duplex formation and poly-
merization and leads to a product of less than the length of the two primers.