Abstract
The chief attribute of the fluorogenic 5′ nuclease assay is that it is completely homogeneous. After mixing the sample and reaction components, the assay is run in a closed tube format with no postpolymerase chain reaction (PCR) processing steps. Results are obtained by simply measuring the fluorescence of the completed reactions. By eliminating post-PCR processing, allelic discrimination with fluorogenic probes reduces the time of analysis, eliminates the labor and supply costs of post-PCR steps, reduces the risk of crossover contamination, and minimizes sources of error. The assay has the sensitivity of PCR so that a minimum amount of genomic DNA is required. The use of endpoint fluorescence measurements maximizes throughput. Using a single ABI Prism® 7900HT Sequence Detection System and 24-h operation, it is possible to generate up to 250,000 SNP results per day. TaqMan® MGB probes and the entire operating system outlined below make it possible to quickly apply the 5′ nuclease assay to any allelic discrimination application where high throughput is of paramount concern.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Holland, P. M., Abramson, R. D., Watson, R., and Gelfand, D. H. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′ in place of 3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–7280.
Holland, P. M., Abramson, R. D., Watson, R., Will, S., Saiki, R. K., and Gelfand, D. H. (1992) Detection of specific polymerase chain reaction product by utilizing the 5′ in place of 3′ exonuclease activity of Thermus aquaticus DNA polymerase. Clin. Chem. 38, 462–463.
Lee, L. G., Connell, C. R., and Bloch, W. (1993) Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res. 21, 3761–3766.
Förster, V. T. (1948) Zwischenmolekulare energiewanderung und fluoresenz. Ann. Physics (Leipzig) 2, 55–75.
Livak, K. J., Flood, S. A. J., 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 Appl. 5, 357–362.
Livak, K. J., Marmaro, J., and Todd, J. A. (1995) Towards fully automated genome-wide polymorphism screening. Nat. Genet. 9, 341–342.
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.
Afonina, I., Zivarts, M., Kutyavin, I., Lukhtanov, E., Gamper, H., and Meyer, R. B. (1997) Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 25, 2657–2660.
Kutyavin, I. V., Lukhtanov, E. A., Gamper, H. B., and Meyer, R. B. (1997) Oligonucleotides with conjugated dihydropyrroloindole tripeptides: Base composition and backbone effects on hybridization. Nucleic Acids Res. 25, 3718–3723.
Kutyavin, I. V., Afonina, I. A., Mills, A., Gorn, V. V., Lukhtanov, E. A., Belousov, E. S., et al. (2000) ′’-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 28, 655–661.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc.
About this protocol
Cite this protocol
Livak, K.J. (2003). SNP Genotyping by the ′’-Nuclease Reaction. In: Kwok, PY. (eds) Single Nucleotide Polymorphisms. Methods in Molecular Biology™, vol 212. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-327-5:129
Download citation
DOI: https://doi.org/10.1385/1-59259-327-5:129
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-968-1
Online ISBN: 978-1-59259-327-9
eBook Packages: Springer Protocols