The use of fluorogenic probes avoids the complications caused by detection of non-specific amplification. Becausenon-specific amplification is more of a problem at low target levels, fluorogenic probe assays tend to be moresensitive for detection of low amounts of target and have a greater dynamic range compared to assays using DNAbinding dyes. Another advantage of fluorogenic probes is that, on the ABI PRISM® 7700 system, the target and endogenous control (e.g., rRNA) amplification can be performed in the same tube. This is possible because target andcontrol probes can be labeled with distinguishable reporter dyes. This reduces the number of reactions that need tobe run and ensures that exactly the same amount of template is available for target and control amplification. Also, theinclusion of an in-tube internal positive control increases confidence in the results obtained for target quantitation.Detection on the ABI PRISM® 7700 system tends to give lower coefficients of variation than detection on theGeneAmp® 5700 system. Improved precision means that smaller differences in initial copy number can be distinguished.
Conclusions
Compared to endpoint quantitation methods, real-time PCR offers streamlined assay development, reproducibleresults, and a large dynamic range. Real-time PCR eliminates the need for competitive in-tube standards withidentical primer sets as targets. Thus, the process of creating quantitative assays is streamlined because the construction and characterization of such standards are no longer required. Real-time PCR now makes quantitationof DNA and RNA much more precise and reproducible because it relies on CT values determined during the exponential phase of PCR rather than endpoint. In addition, the use of CT values allows a larger dynamic range. Thisincreases throughput because it is no longer necessary to analyze dilutions of each sample in order to obtain accurateresults.The researcher now has a number of options for implementing real-time quantitation in his or her lab. Homogeneousdetection of PCR products can be done using double-stranded DNA binding dyes or fluorogenic probes.Detection of fluorescence during the thermal cycling process can be performed using either the GeneAmp® 5700or ABI PRISM® 7700 Sequence Detection Systems. Choosing among these options requires balancing the demandsof sensitivity, convenience, precision, and cost.
下载全文：http://www.bbioo.com/Soft/2007/1079.htm
References
1. Higuchi, R., Dollinger, G., Walsh, P. S., and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences.Biotechnology 10:413–417.
2. Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Kinetic PCR: Real time monitoring of DNA amplification reactions.Biotechnology 11:1026–1030.
3. Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. 1991. Detection of specific polymerase chain reaction product by utilizing the 5' to 3' exonuclease activity of Thermus aquaticus DNA polymerase. Proceedings of the National Academy of Sciences USA 88:7276–7280.
4. Gelfand, D. H., Holland, P. M., Saiki, R. K., and Watson, R. M. 1993. U. S. Patent 5,210,015.
5. Lee, L. G., Connell, C. R., and Bloch, W. 1993. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Research 21:3761–3766.
6. Livak, K. J., Flood, S. J. A., Marmaro, J., Giusti, W., and Deetz, K. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods and Applications 4:357–362.
7. Lyamichev, V., Brow, M. A. D., and Dahlberg, J. E. 1993. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260:778–783.
8. Nielsen, P.E. 1991. “Sequence-selective DNA recognition by synthetic ligands,” Bioconjugate Chemistry 2:1–12.
9. Searle, M.S., and Embrey, K. E. 1990. “Sequence-specific interaction of Hoescht 33258 with the minor groove of an adenine-tract DNA duplex studied in solution by 1H NMR spectroscopy,” Nucleic Acids Research 18:3753–3762.
10. Molecular Probes.
11. Wang, A. M., Doyle, M. V., and Mark, D. F. 1989. “Quantitation of mRNA by the polymerase chain reaction,” Proceedings of the National Academy of Sciences 86:9717–9721.
12. Becker-André, M., and Hahlbrock, K. 1989. “Absolute mRNA quantification using the polymerase chain reaction. A novel approach by a PCR aided transcript titration assay PATTY.,” Nucleic Acids Research 17:9437–9446.
13. Gilliand, G., Perrin, S., Blanchard, K., and Bunn, F. 1990. “Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction,” Proceedings of the National Academy of Sciences 87:2725–2729.
14. Piatak, M., Luk, K. C., Williams, B., and Lifson, J. D. 1993. “Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species,” BioTechniques 14:70–80.
15. ‘TaqMan® Universal PCR Master Mix’ Protocol, P/N 4304449, pp. 13–21.
16. User Bulletin No. 2, “Relative quantitation of gene expression,” P/N 4303859.
