Background

The QuantStudio^TM 12K Flex system is a comprehensive real-time PCR-based platform for conducting large-scale genotyping and gene expression studies. The QuantStudio^TM system at the University of Pretoria facility includes two interchangeable blocks that can accommodate 384-well and OpenArray® plates. The OpenArray® system is combined with pre-designed or custom TaqMan® Assays to generate more than 12,000 data points per run.

Applications on the QuantStudio^TM include:

• Large-scale gene expression analysis
• SNP genotyping
• microRNA analysis
• digital PCR

Funding for the instrument was obtained from the NRF Equipment Platform (NEP) Grant. The platform supports genotyping and functional genomics projects on a national level. Therefore, use of the instrument is available to researchers throughout South Africa.

Services and pricing information:

For more information on the QuantStudio^TM 12K Flex Real-time PCR facility, visit the Forest Molecular Genetics Facilities page or download this brochure. For any queries or technical support please contact Jane Bredenkamp () or follow the link to the Life Technologies website.

Location:

The QuantStudio^TM 12K Flex Real-time PCR instrument can be found in room 6-28 in the Natural and Agricultural Sciences Building at the University of Pretoria.

Contact information:

Ms Jane Bredenkamp

Tel: +27 12 420 4329

 

Dr Sanushka Naidoo

Tel: +27 12 420 4974

 

The Plant to Food Research group of Prof Lise Korsten in the Department of Microbiology and Plant Pathology, University of Pretoria, provides a microbial diagnostic service using the MALDI-TOF Biotyper.  The service is available for research purposes to all students and staff of the University of Pretoria as well as collaborators and other research institutions.

The MALDI Biotyper identifies microorganisms using MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) Mass Spectrometry measuring the unique protein fingerprint of an organism. Specifically, the MALDI Biotyper measures highly abundant proteins that are found in all microorganisms. The characteristic patterns of these proteins are used to reliably identify a particular microorganism by matching the respective patterns with an extensive open database to determine the identity of the microorganism down to species level. The current data base is however still being developed and a wide range of environmental, agricultural and food associated microorganisms are still not in the system.  As part of our research programme we plan to build this unique database for South Africa.   We therefore would like to request that selected important microorganisms be used to expand the database. 

To proceed to the MALDI-TOF Diagnostic Service website click here.

Contact information:

Ms Amelita Lombard
E- or fax to
T- 012 420 4588.

 

 

The Illumina BeadXpress® SNP genotyping facility is situated in the Agricultural Sciences building, at the University of Pretoria. This facility provides a research service for academic and commercial researchers in South Africa. The Illumina BeadXpress® system (Link to Illumina website: http://support.illumina.com/array/array_instruments/beadxpress.ilmn  ) is a platform for medium-throughput SNP genotyping using VeraCode® GoldenGate genotyping technology. Custom designed multiplex kits incorporating 48, 96, 144, 192 or 384 SNPs are designed through Illumina, consisting of 480 reactions. DNA samples are prepared by the user and all laboratory work and preliminary data analysis for quality control purposes is performed by trained technicians at the facility. Raw data is then provided to the user for analysis using the Illumina GenomeStudio® software.

Contact information:

Melissa Reynolds () / 012-420 6874

Prof Zander Myburg () / 012-420 4945

For information regarding SNP design and kit orders, please contact Shirley Ferris () at Whitehead Scientific. All bookings should be made before ordering the kit from Illumina.

High-throughput DNA Fingerprinting and Genotyping Research Service

The DNA Marker Analysis Facility is based in the Forest Molecular Genetics Laboratory at the University of Pretoria, and is supported by a team of technicians and geneticists. The facility offers high-throughput plant DNA isolation, fingerprinting and  genotyping research services focused on commercially grown tree species and occasionally other plant species on request.

The following research capacity is available:

• DNA isolation from a range of plant tissues including leaves, pine needles, xylem scrapings, wood and bark or cambium cores.
• Microsatellite DNA fingerprinting for most Eucalyptus   Pinus species grown in South Africa, using 10-20 microsatellite markers
• Individual identification, clonal verification and parentage analysis for both Eucalyptus and pine species.
• Species discrimination for  Eucalyptus (still under development)

 

Contact information:

Melissa Reynolds () / 012-420 6874

Prof Zander Myburg () / 012-420 4945

High-throughput quantitative real-time PCR facility

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. For more information on the next workshop, please contact Ms Ronishree Naidoo

The LightCycler® 480 instrument is a rapid, thermal blockcycler capable of performing a 40-cycle, 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., LightCycler® Red 610, 640, LightCycler® 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 LightCycler® website.

For information about using the LightCycler® 480 instrument at the University of Pretoria, please contact Ms Ronishree Naidoo. The instrument is managed by Prof Dave Berger, Botany Dept, FABI, UP and Prof Zander Myburg, Genetics Dept, FABI, UP.

Acknowledegements:

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

 

Contact information:

Ms Ronishree Naidoo

T- +27 12 420 4329

 

Prof Zander Myburg

T- +27 12 420 4945

Microarray technology has established itself as the central platform for functional genomics, with DNA oligonucleotide arrays the preferred method for large-scale expression measurement. Such expression data are needed to complement the increasing availability of EST and genome sequence information from many organisms.  Expression levels in a number of different tissues or situations provide a first step toward a functional characterisation of new entities revealed by DNA sequencing.
DNA microarray analysis is based on a hybridisation technique in which DNA fragments or oligonucleotides, corresponding to different genes or complementary DNA (cDNA) molecules, are immobilised on a solid support in an fixed and ordered layout. Mixtures of DNA or RNA isolated from biological sources are labeled enzymatically by the incorporation of nucleotides bearing fluorescent reporter tags. The labeled solution is hybridised to the complementary sequences (probes) on the chip surface. Since each probe element is chemically homogeneous and occupies a known location, the identity and quantity of each component in the fluorescent mixture can be ascertained by measuring the fluorescence intensity at each position in the array.

To avoid confusion, the terminology used is “probe” for the known printed DNA fragments immobilised on the solid support and “target” for the unknown fluorescently labeled DNA that is to hybridise with the probes.

The Facility

Since July 2002, the ACGT Microarray Facility has been providing a core service to biological researchers in South Africa. Managed by the ACGT, the Microarray Facility is located in the laboratory of Professor Dave Berger of the Department of Plant Science at the University of Pretoria, while the operation and technical maintenance is the responsibility of Nicky Olivier. This facility also forms a functional part of the Forestry and Agricultural Biotechnology Institute (FABI).

The Projects

The ACGT Microarray Facility has supported projects undertaken by scientists from all over South Africa and even beyond our borders. Most researchers using the Facility are from partner institutions of the ACGT, i.e. the University of Pretoria, the CSIR, the University of the Witwatersrand and the University of Johannesburg. Projects in these institutions mostly focus on genotyping, expression analyses and diagnostics in plants (Arabidopsis, Eucalyptus, cereals and banana), as well as various fungal species. Medical studies are in progress at the Medical Schools of the University of Pretoria, the University of the Witwatersrand and the University of KwaZulu Natal, while Veterinary Departments making use of the facility include the Veterinary Faculty at Onderstepoort and the International Livestock Research Institute (ILRI) in Nairobi, Kenya.

While many of the current projects are plant-related, the Facility is open to researchers from any field of biological science, both locally and abroad.

The Instruments

The major equipment available in the ACGT Microarray Facility includes a Molecular Dynamics Generation III Spotter, Molecular Devices Genpix 400B Scanner and Agilent Hybridisation Oven.

The Generation III Spotter from Molecular Dynamics is used for printing custom microarray slides. The print head consists of 12 stainless steel capillary pens that each load about 200nl of sample at a time. This is allows the printing of 36 slides in duplicate without having to reload from the sample plate. A maximum of 10 368 unique spots can be printed per slide (from 27 x 384-well spotting plates) or 4 992 samples printed in duplicate (13 x 384-well spotting plates).

 

The Molecular Devices 4000B scanner with GenePix Pro 6 software is used for the imaging and quantification of the hybridisation results. It has dual lasers and filters for detecting emission wavelengths at 532nm (Cy3) and 635nm (Cy5) in one scan, with resolution down to 5μm.

The Facility can assist with two different hardware technologies for hybridizations:

The Agilent Hybridization Oven and Agilent Hybridization Chambers are used with glass slides. Specific backing slides can compartmentalize different samples onto the number of arrays present on the slides. This is especially useful for Agilent arrays which can contain up to 8 distinct arrays on one slide. Since there is a single area backing slide version available, single area spotted array hybridizations can also be processed using this system. When assembled correctly the slide/backing slide contains an air bubble that mixes the hybridization solution during rotation in the Hybridization Oven, ensuring more efficient and even hybridizations.

A locally manufactured hybridisation chamber, referred to as the Hyb-up (pictured here), is used for performing static hybridizations in a water bath.

 

At present the Facility does not offer a hybridisation service and this should either be carried out in each investigator’s laboratory, or the Facility provides lab space and access to equipment for interested researchers.

Data Analysis

In partnership with BioPAD and the Bioinformatics and Computational Biology Unit of the University of Pretoria, the Facility established expertise in the analysis of microarray expression data using the limma software component of the freely available R Bioconductor package. A statistician is available to assist with experimental design and the evaluation of the statistical significance of proposed studies.

Workshop

In 2005 and 2006 the Facility, in partnership with BioPAD, presented Microarray Laboratory Workshops to provide interested researchers with experience in the experimental design, practical laboratory protocols and data analysis of microarray experiments. A less intensive laboratory course for a limited number of participants (called the Starter Kit) is presented at irregular intervals, but has the same aim in mind as the BioPAD subsidized workshops. In addition, the Facility presents an annual course in data analysis to researchers with some experience with microarrays and who might have data to analyze. This workshop gives a more intensive exposure to data analysis using the limma software package, but also covers clustering and data management issues amongst others.

Price list (2012)

Affiliation	Spotting (per slide)	Scanning and hybridisation (per slide)	Data analysis
ACGT	R450 – R550	R100	Free
Non-ACGT (Academic)	R450 – R550	R220	Free
External	R600 – R750	R350	R350/h

Links:

ACGT Microarracy Facility- www.microarray.up.ac.za

Forestry and Agricultural Biotechnology Institute (FABI)- http://www.fabinet.up.ac.za/

Contact information:

Prof Dave Berger

Tel: +27 12 420 4634

Fax: +27 12 420 6668

E-mail: http://www.fabinet.up.ac.za/people/email/p2596725

 

Nicky Olivier

Tel: +27 12 420 4239

Fax: +27 12 420 666

E-mail: http://www.fabinet.up.ac.za/people/email/p2516624

The Central Laboratory for Microscopy and Microanalysis at the University of Pretoria, situated in the Natural Sciences Building II, is operated as an independent laboratory and provides a range of services to students and staff members from all faculties as well as to users outside the University. All the facilities at the Unit are available at all times to users through prior arrangements.

The facility has various SEM-, TEM microscopes, Fluorescent microscopes and Confocal microscopes, which can be used for various research areas and are all fitted with Ultra-high resolution digital cameras.

The Unit offers various short courses on basic techniques in microscopy and also offers their services in the planning of experiments, preparation of samples and the use of the microscopes.

Contact information

Allan Hall, Andre Botha, or Chris van der Merwe, Tel: +27 12 420 2075

The ACGT Bioinformatics Facility, formally known as the Bioinformatics and Computational Biology Unit, was established in 2003. The unit is situated in the FABI Square Building on the University of Pretoria Main campus. The unit is involved in under-graduate training, post-graduate training, research, support and services.

Staff Members

Prof Fourie Joubert (Associate Professor)
Dr Oleg Reva (Senior Lecturer)
Mr Oliver Bezuidt (Temporary Techmical Assistant)

Undergraduate Training

The unit currently offers two courses on 3rd-year level, being BIF 310 and BIF 320. These courses are part of the curriculum for the BSc IT (Bioinformatics) degree, and are electives for students in the School of Biological Sciences.

Post-graduate Training

The unit offers BSc Hons, MSc and PhD degrees in Bioinformatics. The BSc Hons degree consists of coursework together with a small research project. The MSc and PhD degrees are research-based. All degrees are offered only on a full-time basis.

Research

Research programmes include:

Malaria drug target selection.
Bacterial genomics, genome linguistics and metagenomics.
High-throughput sequencing related to genomics and transcriptomics.

Support and Services

The unit provides support and services to the University and scientific community on an ad hoc and a collaborative basis.

Infrastructure

The Unit houses a large collection of computational infrastructure, including:

A 25x PC training laboratory
A 64x node Linux cluster
A 24x processor Linux Dell server with 256GB of RAM
Various 8x processor servers
Around 20 TB of storage space
A Robot backup system
Gigabit ethernet and DDR Infiniband networks

Contact information

Dr Fourie Joubert, Tel: +27 12 420 5828, Fax: +27 12 420 5800

The ACGT DNA Sequence Facility is a core facility within the Department of Genetics at the University of Pretoria, situated in the FABI Square and Informatics Building on the campus. The facility provides DNA sequencing and fragment analysis services to researches within this faculty, such as Microsatellite and AFLP analysis. The facility has recently acquired an ABI 3500xl. The instruments was funded by the National Research Foundation under the Research Infrastructure and  Support Programme: National Equipment Programme (2011/2012- Grant holder: Prof P. Bloomer) and is jointly supported by the University of Pretoria.

With the sequencing of the human genome and other advances in genetic research, genetics is one of the fastest growing research areas in Africa and globally. Two indispensable tools in a geneticist’s kit are DNA sequencing and DNA synthesis. Both are an every-day need for most laboratories working in human, animal, plant, microbial or viral genetics.

The basics

DNA sequencing is the analysis of the nucleotide sequence of a given piece of DNA. The knowledge about the nucleotide sequence of genes is a prerequisite for any molecular approach in basic as well as applied genetics. For scientists in Africa to compete globally in genetics research, DNA synthesis and sequencing services need to be both prompt and cost-effective.

Contact information:

T:+27 12 420 5804/5

Fax: +27 12 362 5327  12 841 2388