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- June 2001
The genomics era has revolutionised drug discovery
and biological assay development at every stage
of the pharmaceutical process. Biochip technology
now allows the simultaneous and efficient analysis
of thousands of genes, transcripts and proteins
of potential therapeutic interest.
EuroBiochips 2000 provided delegates with a thorough
briefing of the biochip industry; what recent
advances and technologies can be applied to your
area of interest; how to overcome potential problems;
how to develop and implement effective biochip
programmes; predictions for the future; and, more
importantly, how you can use biochip technologies
to decrease your development time and ultimately
reduce costs. to harness the power of IT to provide
real competitive advantage across all disciplines.
- Data
Analysis and interpretation: case study
- Genomic
information extraction using massively parallel
sequencing approaches on self-forming DNA
microchips
- Identification
of differentially expressed genes in prostate
cancer using Affymetrix GeneChip technology
- EuroBiochips
2001 - Executive Summary
For further information
and details of the comprehensive documentation
available from this event, please visit: www.eurobiochips.com
Details of EuroBioChips 2002 will be available
soon, please visit: www.eurobiochips.com
1. Data Analysis and interpretation: case study
[top]
Teresa Garcia, Ph.D.
Biotechnology and Bioinformatics, Hoechst-Marion
Roussel/Aventis Pharma (teresa.garcia@aventis.com)
We have initiated, as part of our osteoporosis
research efforts, a program to identify differentially
expressed genes in the course of osteoblastic
differentiation and maturation. For that purpose,
60 samples defining different treatments and kinetics,
were isolated from four different mouse cell lines
which represented different stages of osteoblastic
differentiation in response to BMP2 treatment.
These samples were hybridized to > 600 high-density
Affymetrix oligonucleotide arrays representing
34,325 mouse genes. The presentation will describe
our current usage of Affymetrix mouse chips as
well as the technological and bionformatics capabilities
developed in collaboration with the Aventis Cambridge
Genomics Center to perform gene expression analysis
in a high throughput and high quality manner.
The total data set, derived from the 750 scans
generated, was analyzed using the proprietary
software tools (GECKO) developed to agglomerate,
query and annotate the data. Stringent quality
control procedures were implemented, including
the use of replicates and statistical analysis
to assess experimental and biological variability.
Candidate genes were identified by both a deductive
-based on "expert queries" and biological knowledge-
and an inductive approach - based on the usage
of known biological markers. A data reduction
process was implemented to consolidate the data
resulting in the identification of > 100 target
genes for further validation and selection. The
selection of candidate genes susceptible to play
an important role in the biology of osteoformation
will be discussed.
2. Genomic information extraction using massively
parallel sequencing approaches on self-forming
DNA microchips [top]
Pascal Mayer
Serono Pharmaceutical Research Institute, 14,
chemin des Aulx, 1228
Plan-les-Ouates, Geneva, Switzerland.
Pharmacogenomics is believed to play a major role
in the future of the pharmaceutical industry.
This will generate needs for large scale DNA sequence
analysis which can only be matched with dramatically
faster and less expensive DNA sequencing methods
than those presently available.
Our Genomic Technology Group is developing such
a new technology. It is based on a new auto-patterning
process to simultaneously micro-array onto a 1x1cm
surface up to millions of different DNA samples
which are all sequenced simultaneously using a
new sequencing by synthesis method. The result
of the sequencing reaction is visualized with
a microscope and a CCD camera and the sequence
data is obtained by real-time image analysis.
We are presently driving the technology to its
full potential. Based on our present results,
we expect to ultimately reach a throughput of
up to 50 million bases of raw sequence data per
day per setup and to reduce the cost to 1/50,000
Euro per base sequenced.
3. Identification of differentially expressed
genes in prostate cancer using Affymetrix GeneChip
technology [top]
Christian Pilarsky, Ph. D.
To identify cancer associated genes, transcript
profiling using high density arrays is the method
of choice. For this purpose metaGen has designed
a cancer GeneChip containing known tumor associated
Genes and cDNAs which were shown to be differentially
expressed in EST libraries of several tumors by
bioinformatics methods. This GeneChip was used
to identify differentially expressed genes in
prostate cancer tissue.
Since prostate tumors are heterogeneous in their
composition microdissection is needed to obtain
reliable data. Tissue from 24 prostate carcinoma
and corresponding benign tissue was harvested
by manual microdissection and RNA was isolated.
For GeneChip hybridization the RNA was amplified
by repetitive in vitro transcription.
Our results show overexpression of several genes
in prostate cancer in comparison to benign tissue
including the cyclin activating kinase. Most of
these genes have not been associated previously
with prostate tumors.
Analysis of specific gene expression by chip hybridization
represents a new approach to identify cancer associated
genes. These genes need to be further validated
to establish their role as target genes. Furthermore
a subset of those genes could be used to type
prostate cancer based on their expression profile
rather than on morphology. This will lead to new
possibilities for the diagnosis and prognosis
of prostate cancer.
4. EuroBiochips 2001 - Executive Summary
[top]
Simone Weyand and Dietmar Blohm
University of Bremen
More than two dozen international biochip conferences
have been announced for this year to be held in
United States and Europe. Most of them have a
commercial background, so as the EuroBiochips
2001, held from 6-8th of June in Munich, organized
by IBC Global Conferences Ltd., London. Obviously,
in this field enough new scientific results are
produced and new technologies being developed
to attract an auditorium, able to afford the fee
of about 3.000 DM for a three-day conference,
or the number of people who are willing to enter
this field is still high enough to fill these
auditoria. The EuroBiochips 2001 meeting at least
was one of the few fulfilling both of these expectations.
It showed, that the interest in and the demand
on this technology have not yet declined and that
there are still huge scientific and commercial
hopes in this field.
The conference has clearly bee dedicated to the
technical development and only a few lectures
included detailed biological results obtained
with microarrays or similar technical solutions.
The conference was divided in the following topics:
Applications in drug discovery, bioinfor-matics
and data analysis, business of biochips, diagnostics,
emerging technologies, micro-fluidic technologies
and protein arrays. The more than 300 participants
enjoyed about 40 lectures and 16 posters, most
of them scientifically and technically at the
top level. A few new methods, like the scanometric
approach of using gold nanoparticles enhanced
by silver reduction for hybridisation detection
and the mask less synthesis of oligonucleotides
on the chip have not been presented. But nevertheless,
the conference gave an almost comprehensive overview
of all aspects of the microarray technology including
microfluidics and bioinfor-matics. An extra bonus
was the accompanying exhibition, in which 28 companies
demon-strated their arrayers, microarray-chips
and related lab ware, scanners, software tools,
services and other new developments.
Although construction, properties and application
of these microarray components have been the general
theme of this meeting, the focus of attention
at this conference was clearly the progress in
microfluidics. One of the most convincingly demonstrated
advances in this field was the new laced concept,
which seems now to bring truly in reality was
often and too early has been announced as lab-on-the
chip technology.
In this respect, the lectures of S. O'Connor (Nanostream
Inc.) and B. Carvalho (Tecan) as well as from
O. Larsson (Amic AB) and R. Ehrnström (Gyros AB)
were of special interest. They demonstrated, that
almost all important lab techniques, like pumping,
valving, passive and active fluid distribution,
metering, mixing, solid phase binding, washing,
elution, filtration, cell growth and lyses, centrifugation,
separation, heating and different methods of signal
detection can be carried out in 96 or more parallel
units integrated into one single laced. This astonishing
versatility and accuracy is achieved by specially
designing the diameters, length, geometry, angles,
density and material surfaces of the micro channel
structures in relation to the viscosity, the surface
tension and other properties of the fluidic solutions
to be processed during the analytic run, in which
the driving forces are easily controlled by varying
acceleration and velocity of the labCD. The execution
of 48 enzyme inhibition reactions in a total volume
of 12 µl using fibber optic detection, and multiplex
PCR as well as online PCR reactions including
the lyses of E. coli cells have been realized.
"Fit the chip to the assay not vice versa!" was
a statement in this context, underlining the technical
progress recently made in material sciences and
fabrication technology to produce micro channels
and microstructures for special functions. Examples
are micro chambers for perfect mixing microfluidic
streams and pressure dependent valves, opening
e.g. only around 5 psi and closing at lower as
well as at higher pressure, simply controlled
by the speed of the labCD. More than 40 different
polymers, including Teflon, are presently in use
for producing special surface qualities and 400
more are expected to be available in the next
years to come. Using this technical opportunities,
about 100 chromatographic systems have been realized
on one single labCD prototype. Applications are
seen for high throughput drug discovery, genomics,
proteomics, clinical and molecular diagnostics
as well as the analytical routine in food and
feed industry and other fields.
This type of system obviously has the potential
to become the platform of choice for the true
lab-on-the-chip and to be introduced quickly into
the clinical routine, because most of the bio-chemistry
and lab techniques presently in use in clinical
analytics can directly be integrated into this
technology. The only technique presented at the
EuroBiochips 2001, competitive with this kind
of approach, was the so-called eSensor platform
from Clinical Micro Sensors and Motorola. Tim
Tiemann described in his lecture "DNA-diagnostics
- mass applied geno-mics", that the eSensor 4800
system contains biochip cartridges producing an
electronic readout without the need of washing
steps based on hybridisation reporter molecules.
The desktop or handheld versions of these devices
allow analysing 36 post amplification samples
of any DNA or RNA targets per chip and are simple
and flexible to use by non-skilled users, because
they work without any optical or fluidic elements.
At the moment there are panels for pathogens,
SNP's and genetic diseases available. For introducing
this technology successfully to the clinical routine
laboratory the only scientifically relevant points
are sensitivity and specificity, whereas the following
factors were designated as the most important
ones: costs for establishing the technique in
the lab and cost per test panel, the ease of using
the system, the time needed per test, regulatory
issues and intellectual property rights.
This level of applicability in "real life" situations
outside the research laboratory has not yet been
reached by any other commercial system based on
microarray technology, although D. Hall (Agilent)
reported about a highly integrated program for
preparing the first "preplot-human1-array" (on
two slides), based on inkjet-technology for producing
the chips, a special hybridisation chamber, a
"second generation" scanner including a 48 slide
carousel and a high performance software Therefore
D. Blohm (University of Bremen) might be right
to empha-size, that the microarray technology
is at present still exclusively a research method,
because it's five separate modules, the chip,
the arrayer, the hybridisation station, the scanner
and the software tools are too complex to be integrated
into a robust, automatable and easy to use stand
alone equipment. Such a gene sensor device is
being developed as a flow-through system based
on re-usable chips and with a label free readout
for nucleic acids as well as for proteins and
other biomolecules. It is expected to have the
additional advantage of enabling the measurement
of binding kinetics in each individual spot of
a microarray, even under vary-ing experimental
conditions.
Although there were no reports about the influence
of the chip-quality and other steps of the complex
experimental procedure on the final hybridisation
results, the issue of data quality played a less
important role during this conference as compared
with other recent meetings in this field. However
M. Iyer (Corning) announced at this congress an
astonishing efficient new chip producing process,
which allows spotting 10.000 different oligonucleotides
on a slide in about six seconds. May be this kind
of technological breakthrough is helping to bring
down the prices of microarrays and to expand their
application and to speed up the definition of
technical standards for this technology. An other
non-contact, high efficient microarray pro-duction
strategy has been developed and presented by HSG-IMIT
(Villingen/Schwenningen) as the so-called topspot
system, which is able to spot more than 1.000
oligonucleotids in about ten seconds.
Strong efforts are being made to transfer the
knowledge accumulated for DNA microarrays into
the field of proteomics. M. Pawlak (Zeptosens)
presented e.g. a well characterized pro-totypic
system, composed of microarrays with 3.066 spots/cm²,
a special reader with a linear dynamic range of
3,5 decades and a proprietary software. Based
on the evanescent fluores-cence method this device
has a detection limit of about 500 zeptomole and
works with 1-10 pg/ml protein in a serum sample
of 15 µl without the need of special purification
steps. The next technological step, to produce
tissue microarrays for analysing e.g. the pathology
of cells, tissues and organs, as illustrated by
O. Kallioniemi (NIH) at this conference, gave
an imagination of the potential for future developments,
because traditional methods allow to analyse a
molecular marker in one given tumour, but with
microarrays it is possible to analyse a whole
spectrum of molecular markers in tumours at the
population level.
The patent situation is in all fields of this
technology still a matter of controversy, in the
area of nucleic acid as well as in proteomics
and other types of arrays, because the final outcome
of the opposition and litigation proceedings about
the basic microarray patents, corresponding to
the EP 619 321, owned by Affymetrix, and EP 373
201, owned by Oxford Gene Technolo-gy, is not
yet foreseeable. Therefore D. Trösch (Reinhardt
Söllner Ganahl) clearly explained, that everyone
who is involved in the microarray business is
highly recommended to monitor closely the patent
situation and the corresponding proceedings.
As a concluding message, the current trends in
the field of microarray technology presented at
the EuroBiochips 2001 conference, can be summarised
as follows: Ten years after it's first steps of
development the microarray methodology has not
yet left the research laboratory but is now moving
quickly towards real-life applications. In connection
with the rapid progress in microfluidic techniques
and technical breakthroughs concerning the production
of low cost but high quality chips as well as
further miniaturisation and higher degrees of
multiplexing a huge potential of further technical
developments can be expected.
Simone Weyand and Dietmar Blohm
University of Bremen, FB2-UFT
Department of Biotechnology and Molecular Genetics
Leobener Straße, D-28359 Bremen
Tel. +49 (0) 421-218-4780
Fax. +49 (0) 421-218-7578
Email: dhb@biotec.uni-bremen.de
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