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

242 Khalturin, Kuznetsov, and Bosch
the annealing temperature should be decreased from 40°C down to the 35°C. Besides that,
the ratio between random 10-mer primer and tailing primer should be adjusted. Using
fluorescent TAMRA-dUTP in the reaction mix adds additional complexity to the DD-PCR
approach because it can be destroyed by frequent freeze-thaw cycles. PCR products
labeled with new TAMRA-dUTP are nearly 10 times brighter than those labeled using
TAMRA-dUTP that had undergone 3 to 4 freeze-thaw cycles. Because of that, TAMRAdUTP
should be always kept in 2- to 3-µL aliquots. Usually, it is difficult to estimate
the optimal concentration of TAMRA-dUTP for effective labeling. Therefore, a different
dTTP:TAMRA-dUTP ratio should be tried (for example 301, 1001, 2001). Unincorporated
TAMRA-dUTP results in the fluorescent cloud in the gel indicated by the asterisk
in Fig. 2.
4. One of the major variables during the DD-PCR experiment appears to be the fact that
thermal cyclers vary greatly in their ramping time and other technical details. In our
laboratory, we use two types of PCR machines: Omn-E (Hybaid) and RoboCycler
(Stratagene). The former has a Peltier-element with the ramping time of approx 2.5°C/s
(while cooling from 94° to 70°C), the latter nearly no ramping since a robotic arm transfers
the samples from one thermoblock to another. When examining the influence of the
ramping time on the DD-PCR and using the same temperature and time profiles in both
machines (as well as identical components of the PCR mix) we observed considerably more
fragments produced using the Omn-E thermal cycler in comparison with the Robocycler.
The difference is not caused by the destruction of TAMRA-dye because the same result is
observed in silver stained gels (7) as well as on those viewed by the fluorescent scanner.
It seems the rapid change of the temperature provided by the RoboCycler prevents the
efficient synthesis of PCR products when using standard PCR protocol. An increase of the
annealing time from 30 to 90 s may overcome that drawback.
Acknowledgments
The work in the laboratory is supported by grants from the DFG (to T.C.G.B.) and
by the Daimler-Benz Foundation (S.K.)
References
1. Liang, P., Averboukh, L., Keyomarsi, K., Sager, R., and Pardee, A. B. (1992) Differential
display and cloning of messenger RNAs from human breast cancer versus mammary
epithelial cells. Cancer Res. 52, 6966–6968.
2. Liang, P. and Pardee, A. B. (1992) Differential display of eukaryotic messenger RNA by
means of the polymerase chain reaction. Science 257, 967–971.
3. Martin, K. J., Kwan, C. P., O’Hare, M. J., Pardee, A. B., and Sager, R. (1998) Identification
and verification of differential display cDNAs using gene-specific primers and hybridization
arrays. BioTechniques 24, 1018–1026.
4. McClelland, M., Mathieu-Daude, F., and Welsh, J. (1995) RNA fingerprinting and differential
display using arbitrarily primed PCR (review). Trends Genet. 11, 242–246.
5. Rompf, R. and Kahl, G. (1997) mRNA differential display in agarose gels. BioTechniques
23, 28–32.
6. Bosch, T. C. G. and Lohmann, J. (1996) Non-radioactive differential display of messenger
RNA, in Fingerprinting Methods Based on Arbitrarily Primed PCR, Springer Lab Manual
(Micheli, M.R. and Bova, R., eds.), Springer Verlag, Heidelberg.
7. Lohmann, J., Schickle, H. P., and Bosch, T. C. G. (1995) REN, a rapid and efficient
method for non-radioactive differential display and isolation of mRNA. BioTechniques
18, 200–202.