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

Long PCR 169
human papilloma virus (34), SV-40 (35), varicella-zoster virus (36), the proviruses
of the simian foamy virus of chimpanzees (SFVcpz) (37), human T-cell leukemia
virus 1 (HTLV-1) (38), and human immunodeficiency virus type 1 (HIV-1) (39–41)
and type 2 (HIV-2) (42).
Long RT-PCR for RNA viruses has also been successful. Large fragments of the viral
genome have been amplified for the tick-borne encephalitis virus (6), the Norwalk-like
viruses (12), the hepatitis C virus (HCV) (13), and the bovine torovirus (43). The near
full-length genome was amplified for HCV (8,14) and the full-length genome for the
potato virus Y (4), hepatitis A virus (HAV) (7,9), hepatitis E virus (44), poliovirus (11),
and coxsackie B2 virus (10,15).
For HAV, we have shown that RNA transcribed directly from the full-length amplicon
is infectious (7,9). Furthermore, long PCR has greatly facilitated the construction
of infectious clones or infectious cDNAs for HBV (33), SV-40 (35), SFVcpz (37),
HIV-1 (41), tick borne encephalitis virus (6), HAV (9), coxsackie B2 (15), coxsackie
B6 (45), HCV (46,47), and potato virus Y (4). These results confirm the high fidelity
of long PCR.
In addition to streamlining the cloning and sequencing of viral genomes, long PCR
presents other advantages for virology. For example, many viral sequences are toxic to
Escherichia coli, leading to selection bias in cloning procedures (48). This problem
can be circumvented to a great extent by long PCR because large amplicons can be
directly sequenced (or transcribed). Furthermore, direct sequencing of amplicons
provides a certain protection against DNA polymerase mistakes during PCR: Unless
these mistakes occur in the early cycles, at each position the majority of amplicons will
have the correct nucleotide and the sequencing will provide the correct result, whereas
cloning might select an amplicon with mistakes. However, one must remember
that there are circumstances, such as when encountering homopolymeric stretches,
tandem repeats, extreme AT or GC content, or strong secondary structures, in which
polymerases can produce systematic mistakes (49–52).
Long PCR is also of great interest for RNA viruses, which typically exist as
“quasispecies,” a mixed population containing several variants. Not only does direct
sequencing of the amplicon yield immediately the consensus sequence, but it has been
proposed that long PCR can be used to preserve and propagate a representative DNA
version of such a “quasispecies” (9,11). In fact, it has been shown that long PCR
preserves the distribution of variants of poliovirus (11) and HIV-1 (53). In the latter
study, however, a relatively high rate of recombination between templates was observed
after performing PCR (53). Recombination was measured by cloning and sequencing,
and thus this measurement is possibly affected by a selection bias from the cloning
process. Nonetheless, because of this observation, and pending further research on
this topic, the possibility of artifactual recombination should be kept in mind. Because
the recombination is thought to occur as the result of incomplete transcripts acting
as “mega primers” in subsequent PCR cycles (53,54), careful optimization of the
elongation time during PCR may minimize this problem. The use of polymerases with
high processivity may also diminish the occurrence of recombination (54).
References
1. Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high
yield from λ bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216–2220.