John M. S. Bartlett.pdf - Bio-Nica.info

Long PCR Methodology 175
2. Aliquots (48 µL) of the master mix are placed in thin-wall 0.5-mL PCR tubes and overlaid
with 40 µL of mineral oil.
3. Finally, 2 µL of the RT reaction is added. We neglect the contribution of the 2 µL of RT
reaction to primer and dNTP concentrations, etc.
4. Notes
1. The KlenTaq Advantage polymerase mix also contains anti-Taq antibody, which ensures
a “hot start” to the PCR. We have not found it necessary to include an initial prolonged
denaturation step, so the way we are using the polymerase mix is in fact “time-release
PCR.”
2. The Clontech buffer is Tricine-based, and therefore is not subject to the same drop in pH
at high temperatures that is seen with Tris-based buffers (8). The buffer already contains
Mg 2+ ; we have not found it necessary to modify the concentration of Mg 2+ for any of the
templates we have amplified. The buffer does contain KOAc instead of KCl, the latter
having been shown to decrease the processivity of DNA polymerases (8).
3. Primers: 10 pmol of each primer are used in the PCR. Because long PCR in general uses
a higher annealing temperature than does standard PCR, a requirement of primer design is
that the Tm must be high enough to hybridize at that temperature. Whereas 20 to 25 mers
with a sufficiently high GC content will work, we have also often used primers of 30 to 40
bp without problems, and when incorporating promoters and cloning sites, primers of up
to ~60 bp. Barnes (5) has also reported the use of amplicons of a few hundred bp as “mega
primers.” Primer design should include consideration of secondary structures, dimers, etc.,
but because of the high temperatures throughout the PCR cycle and the “hot start,” there
is in fact greater tolerance for weak secondary structures than with many “standard” PCR
protocols. Because of the high annealing temperature, however, there is a low tolerance
for mismatches between the primers and the template.
4. Long PCRs usually require rapid temperature transition (however, some templates are more
tolerant than others), and several parameters are optimized toward this goal, including the
small reaction volume (50 µL) and the small volume of oil. Because different volumes lead
to different thermal capacity, great consistency is required for optimization. We use the
Stratagene thin-wall tubes; we have found that some other brands may require, for some
templates, different parameters: again, consistency is the essential element.
5. Long PCR requires a thermal cycler with fast temperature transitions; depending on the
template and the size of the target for amplification, not all thermal cyclers will allow
successful amplification (5). In any case, cycling parameters must be determined for each
different model of thermal cycler.
6. We usually use 35 cycles of PCR amplification. If greater yield or sensitivity is required,
consider using nested PCR.
7. The elongation time is usually the only parameter to adjust in the protocol and depends on the
template and the size of the targeted amplicon. Elongation times shorter or longer than the
optimal time can result in sensitivity loss (or even negative results). Extension times that
are too long are often associated with a “smearing” of the reaction product when it is
electrophoresed on agarose gels. Optimal elongation time is best determined by using a
serial dilution of the template (since suboptimal times may give positive reactions but with
a loss of a few logs in sensitivity). The initial time should be chosen between 1 and 2 min
per kb. Like other authors (8), we have found that step-wise increase of the elongation
time can be beneficial. Examples of elongation times for various templates can be found
in Barnes (5) and Tellier et al. (1). For example, to obtain amplicons from the tobacco
mosaic virus (6.2 kb), HAV (7.5 kb), and HCV (9.25 kb), we found we could use the same
elongation times: 9 min 45 s for the first 15 cycles, 11 min for the next 10 cycles, and