Biology Reference
In-Depth Information
that cuts
outside
the region of known sequence to produce a fragment of
≈
3-4 kb. Southern-blot analyses may be necessary to identify restriction enzymes
that produce fragments of suitable size for circularization and amplification. If
an enzyme is used that cleaves within the core region, either the upstream or
downstream segment of DNA will be amplified.
Once the DNA has been digested, the ends of the fragment are ligated to
form a circular molecule. Ligation is performed with T4 DNA ligase in a dilute
DNA concentration to favor formation of monomeric circles. Amplifying the
flanking DNA outside the “core” region is carried out using primers oriented in
the
opposite direction
of the usual (
Silver 1991
). Primers for inverse PCR thus
are homologous to the ends of the core region so that DNA synthesis proceeds
across the
uncharacterized
region of the circle rather than across the character-
ized core region (
Figure 8.3
).
8.4.8 Long PCR or High-Fidelity PCR
In theory, standard PCR using a
Taq
DNA polymerase can amplify up to
≈
10 kb of
DNA sequence. In practice, amplification of such long sequences is difficult; most
amplifications are limited to 1-2 kb. However, large (up to 40 kb) DNA fragments
can be amplified with high fidelity and yield if two DNA polymerases are used
(
Barnes 1994; Cheng et al. 1994a,b; Lahr and Katz 2009
).
The effectiveness of Long PCR is due to the two DNA polymerases working
together. A polymerase such as
Pfu
has a 3
′
-exonuclease activity (which
Taq
does not have), which means that
Pfu
can remove any accidental mismatches
introduced into the growing DNA molecule. The theory is that one of the deter-
rents to truly long PCR products may be due to the incorporation by
Taq
of mis-
matched or damaged nucleotides every few kilobases, thus eliminating those
DNA molecules from further amplification. The incorporation of errors occurs
because
Taq
lacks a proofreading function. Adding a small amount of a proof-
reading polymerase such as
Pfu
to an excess of
Taq
provides
Taq
with a proof-
reading “helper.” Primer mismatchs are corrected and
Taq
is able to extend
primers for longer distances. Several combinations of DNA polymerases have
been used in long PCR;
Cheng et al. (1994a)
found that rTth (from
Thermus ther-
mophilus
) and Vent (from
Thermococcus litoralis
) polymerases were the most
reliable combination under their test conditions.
Several other modifications in the Long PCR protocol enhance the likelihood
of obtaining longer products. Long PCR typically uses long primers (at least
30nt), works best at a higher pH (8.8-9.0), uses a high rate of change in tem-
peratures (rapid cycling), and a longer synthesis interval.
