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Microarray technology is rapidly becoming a central platform
for functional genomics, with DNA arrays having become the preferred
method for large-scale expression measurement. Such expression data are
needed in view of the large amounts of sequence information available:
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 microscopic spots, consisting of DNA fragments or
oligonucleotides corresponding to different genes or complementary DNA
(cDNA) molecules are immobilised on a solid support. Mixtures of DNA or
RNA isolated from biological sources are labelled enzymatically by
incorporating nucleotides bearing fluorescent reporter tags, and
hybridised to components of the fluorescent sample (probe) and a
complementary sequence (target) on the chip surface. Each target
element is chemically homogeneous and occupies a known location -
therefore the
identity and quantity of each component in the fluorescent mixture can
be ascertained by measuring the fluorescence intensity at each position
in the microarray.
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 Botany at the
University of Pretoria. This facility also forms a functional part of
FABI. Operation and technical maintenance is the responsibility of
Danie Theron.
Microarrays have supported projects undertaken by scientists
from the University of Pretoria and the CSIR, while researchers at the
Forestry and Agricultural Biotechnology Institute (FABI) have used the
Facility for genotyping and expression analyses of plants including Arabidopsis,
Eucalyptus, various cereals and banana. More
recently the Facility is being used for projects in the UP medical
faculty and veterinary research unit at Onderstepoort, while the
University of Wits in Gauteng and International Lviestock Research
Institute (ILRI) in Nairobi are also making use of the services.
While may of the current projects are plant-related, facility
is open to researchers from any field of biological science, locally
and abroad.
The equipment available in the ACGT Microarray Facility
include a Spotter and a Scanner.
The GenIII Spotter from Molecular Dynamics is used for
printing 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 9 216 spots can be printed per
slide (12 x 384-well plates) consisting of 4 608 target samples printed
in duplicate.
To avoid confusion, the terminology used is "target" for the
known printed DNA fragments immobilised on glass slides and "probe" for
the unknown fluorescently labelled DNA that is to hybridise with the
target. View the result of a recent example of a
quality control run to test the performance of the system.
The Axon 4000B scanner with GenePix software is used for the
imaging of the hybridisation results. It has dual filters for detecting
emission wavelengths of 532nm (Cy3) and 635nm (Cy5) simultaneously.
A locally manufactured hybridisation chamber, referred to as
the Hyp-up, is used for performing the hybridisations in a water bath.
Hybridisations do not form part of the current service are should be
carried out in each investigator's laboratory.
| 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 |
Nicky
Olivier,
Tel: +27 12 420 4239,
Fax: +27 12 362 3947
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