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

176 Tellier et al.
13 min for the last 10 cycles (1). To minimize recombination events during PCR, one
should probably err on the side of longer elongation times.
8. The master mix is merely corrected for a 5 µL volume of template, taking into account the
amount of buffer carried from the first reaction.
9. For nested PCR, the primers of the second round must be internal to those of the first round
(although they can overlap). If the same primers were to be used, artifactual bands produced
by long PCR because of false priming would also get re-amplified. A second round of long
PCR with the same primers can be performed, but this necessitates purification of the first
round amplicon by agarose gel electrophoresis (9).
10. The overall sensitivity that can be achieved can be close to standard PCR: for the lambda
phage DNA, we have obtained an 11-kb amplicon from as few as 200 copies. When we
used a nested long PCR protocol we could obtain, at the end of the whole process, a 5-kb
amplicon starting from as few as 20 copies (1).
11. This protocol assumes that the RNA is diluted in 10 mM DTT and 5% (vol/vol) RNasin
(20–40 U/µL, Promega). If one uses RNA dissolved in RNase free water, increase the DTT
in the RT master mix to 2 µL, and decrease the amount of primer to 1.5 µL.
12. The primer for the reverse transcription must be a template-specific primer and can be one
of the primers used in the long PCR. Do not use random hexamers.
13. The optimal temperature for Superscript II is between 42 and 45°C (10), but it is active
up to 50°C. Although incubation at 50°C was less sensitive in our hands (1), for some
templates with strong secondary structures, it may be advantageous.
14. The treatment with RNase H and RNase T1 is not absolutely necessary, but we have found
that it increases the sensitivity of the long RT-PCR (1). Other authors have also reported
benefits from the use of RNase H (11–13). We have not tested separately the contributions
of RNase H and RNase T1, but the benefits of RNase T1 are expected to vary depending
on the amount of extraneous RNA present in the template.
References
1. Tellier, R., Bukh, J., Emerson, S. U., Miller, R. H., and Purcell, R. H. (1996) Long PCR and
its application to hepatitis viruses: Amplification of hepatitis A, hepatitis B and hepatitis C
virus genomes. J. Clin. Microbiol. 34, 3085–3091. (Erratum published in J. Clin. Microbiol.
1997;35:2713).
2. Duckmanton, L. M., Tellier, R., Liu, P., and Petric, M. (1998) Bovine torovirus: Sequencing
of the structural genes and expression of the nucleocapsid protein of Breda virus. Virus
Res. 58, 83–96.
3. Martino, T. A., Tellier, R., Petric, M., Irwin, D. M., Afshar, A., and Liu P. (1999) The
complete consensus sequence of coxsackievirus B6 and generation of infectious clones by
long RT-PCR. Virus Res. 64, 77–86.
4. Chen, B., Rigat, B., Curry, C., and Mahuran, D. J. (1999) Structure of the GM2A gene:
identification of an exon 2 nonsense mutation and a naturally occurring transcript with an
in-frame deletion of exon 2. Am. J. Hum. Genet. 65, 77–87.
5. 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.
6. Lawyer, F. C., Stoffel, S., Saiki, R. K., Chang, S. Y., Landre, P. A., Abramson, R. D., et al.
(1993) High-level expression, purification, and enzymatic characterization of full-length
Thermus aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease
activity. Genome Res. 2, 275–287.
7. Clontech (1998) User Manual for Advantage cDNA PCR kit and Advantage cDNA
Polymerase Mix.