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The Roche Lightcycler 480 instrument housed in the Agricultural Sciences building, Faculty of Natural and Agricultural Sciences at the University of Pretoria is a national facility with the aim to increase the capacity for quantitative real-time PCR both regionally and nationally. Use of the instrument is therefore not limited to academic researchers in Pretoria, but also to academic and commercial researchers throughout South Africa. Training workshops are provided by Roche at least twice per year, highlighting the principles of the real-time PCR techniques, the capabilities of the instrument, experimental design strategies, as well as providing hand-on training on the operation of the instrument.
Funding for the instrument was provided in partnership with the National Research Foundation on the National Equipment Programme for 2005. Additional contributions were made by stakeholders at the University of Pretoria and industrial partners.
University of Pretoria contributors:
- Prof Robin Crewe - Vice-Principal: Research & Postgraduate studies
- Prof Anton Stroh - Dean: Faculty of Natural and Agricultural Sciences
- Prof Dave Berger - Department of Botany
- Prof Zander Myburg - Department of Genetics
- Prof Jacques Theron - Department of Microbiology and Plant Pathology
- Dr Emma Steenkamp - Department of Microbiology and Plant Pathology
- Dr Altus Viljoen - Department of Microbiology and Plant Pathology
- Prof Mike Wingfield - Forestry and Agricultural Biotechnology Institute
- Dr Rachel Chikwamba - Forestry and Agricultural Biotechnology Institute
External contributors:
- Sappi Ltd
- Mondi South Africa Ltd
The LightCyclerŽ 480 instrument is a rapid, thermal blockcycler capable of performing a 40-cycle, 96- or 384-well quantitative PCR run in less than 40 minutes with extraordinary well-to-well temperature homogeneity and maximized inter-well, inter-cycle reproducibility. The special arrangement of optical components ensures the uniform collection of signals across the plate and makes analysis independent of the sample position on the plate. All current probe formats are supported (e.g., SYBR Green I, Hydrolysis Probes, HybProbe Probes), and the instrument is ideal for fast and precise qualitative or quantitative detection of nucleic acids, genotyping, and mutation analysis. The ability to freely combine five excitation and six emission filters permits analysis of signals from multiple dyes (e.g., LightCyclerŽ Red 610, 640, LightCyclerŽ CYAN 500, FAM, VIC, HEX, Cy5, and Fluorescein) in monocolor as well as in multiplex assays. The instrument's software enables highly flexible and extensive data analysis for different scientific needs.
Applications of the Lightcycler 480 instrument include:
- High-resolution melting curve analysis
- High-throughput gene expression and genotyping analysis
- High-resolution and high-throughput multiplex PCR analysis
For more technical information visit the LightCyclerŽ website.
For information about using the LightCyclerŽ 480 instrument at the University of Pretoria, please contact Martin Ranik or Nicky Creux. The instrument is managed by Prof Dave Berger, Botany Dept, FABI, UP and Prof Zander Myburg, Genetics Dept, FABI, UP.
Examples of postgraduate student degrees and publications that have succesfully employed quantitative RT-PCR at UP are:
- Dr. B Crampton (CSIR and PhD Botany UP, 2006); Elucidation of defence response mechanisms in pearl millet
- Michele Victor MSc Genetics cum laude
- Liesel Burger - MSc (Microbiology)
- Wayne A. Barnes - BSc (Hons) (Microbiology)
- Ntsane Moleleki - PhD (Genetics)
- Mauricio Marin - PhD (Genetics)
- Almuth Hammerbacher - MSc (Genetics)
- Moleleki N, Preisig O, Wingfield MJ, Crous PW and Wingfield BD (2002). PCR-RFLP and sequence data delineate several Diaporthe species associated with stone and pome fruit trees in South Africa. European Journal of Plant Pathology 108, 909-912.
- Moleleki N., van Heerden S, Wingfield MJ, Wingfield MJ and Preisig O (2003). Transfection of Diaporthe perjuncta with Diaporthe RNA virus (DaRV). Applied and Environmental Microbiology 69, 3952-3956.
- Marin M, Preisig O, Wingfield BD, Kirisits T, Yamoka Y and Wingfield MJ (2005). Phenotypic and DNA sequence data comparisons reveal three discrete species in the Ceratocystis polonica species complex. Mycological Research 109, 1137-1148.
- Marin M, Castro B, Gaitan A, Preisig O, Wingfield BD and Wingfield MJ (2003). Relationships of Ceratocystis fimbriata isolates from Colombian coffee-growing regions based on molecular data and pathogenicity.
Journal of Phytopathology 151, 395-405.
- Van den Berg N, Berger DK, Hein I, Birch PRJ, Wingfield MJ and Viljoen A (2007) Tolerance in banana to Fusarium wilt is associated with early up-regulation of cell wall-strengthening genes in the roots, Molecular Plant Pathology, 8(3) 333-341.
- Dr. Noelani Van Den Berg (PhD Plant Pathology, UP, 2006): The identification of banana genes associated with tolerance against Fusarium oxysporum f.sp. cubense subtropical race 4
Zander Myburg,
Tel: +27 12 420 4945
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