Every cell in the human body carries an
individual's genetic information in DNA, of which genes are specific parts
that encode for proteins to allow biological activity to occur. Whether
certain genes are active or not can be measured using microarrays, which
can probe tens of thousands of genes simultaneously. What is the
technology behind these arrays? How can one put that many features in such
a small space?
| Sep
1, 2005 |
| By:
Tom
A. van de Goor |
| Pharmaceutical
Discovery |
|
Tom A. van de Goor
|
Each human cell contains an estimated 30,000 genes. At birth these genes
are present and, unless mutations occur, remain stable during the person's
entire life. The genetic make-up determines what a cell can do and whether
there is an inherent susceptibility to a particular disease.
Researchers have identified several diseases that are caused by genetic
predisposition. One example is cystic fibrosis, which is caused by a
defective gene that affects cells that produce mucus, sweat, saliva and
digestive juices. The viscosity of these secretions is much more dense in
cystic fibrosis patients. Screening parents for these genes helps them
assess their risk of having children with the disease. Each year
approximately 3200 babies in the US are born with cystic fibrosis and 1 in
25 Caucasians are carriers for the disease. There are several techniques
that can be used to probe genes. Quantitative polymerase chain reaction (PCR)
is used when the number of genes is limited (around 100 or so) but arrays
excel in massively parallel investigation.
The Starting Point
Figure 1. The link between genomics,
proteomics and drug development. Research identifies genetic
predisposition to disease. With microarray experiments, RNA levels
can be determined, indicating whether the gene is active or not.
However, at the protein level disease is expressed in the cell.
Drugs targeting these proteins are developed. Genotyping can be
used during clinical trials to have the most effective patient
group, while protein identification can be used as a diagnostic
for disease.
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Depending upon a variety of circumstances, including disease, genes get
turned on or off. This causes mRNA to be produced, forming the template
for the production of proteins. mRNA is an indication of what proteins the
cell is trying to express, although it is not always a good correlation to
the protein levels, which determines the actual cell activity (Figure 1).
Also, the response of a cell to disease or an external effect such as a
drug response typically is not a single gene event. In the May 15, 2003,
issue of Cancer Research, a group from the University of Michigan
(Ann Arbor, Michigan, USA) used microarrays to identify 158 genes
associated with pancreatic cancer that were differentially expressed as
compared to people with a healthy pancreas. Authors of a paper in Cancer
Research in January 15, 2002, used microarrays with more than 23,000
features to predict the response to anti-cancer drugs in terms of efficacy
and toxicity on a group of patients. Determining individual response to
medication in studies such as these is a first step toward personalized
medicine and, perhaps, more effective treatments overall. But what exactly
are these extremely useful life science tools? How are they created? And
what is their role — current and future — in the drug discovery
process?
Types of Microarrays
The first arrays, created in the mid 80s, were called macro arrays.
They were fabricated by spotting DNA probes on a membrane-type material
with spot sizes of about 300 microns, which limited the density of the
spots to about 2000 probes. They mostly were used for DNA clones, PCR
products or oligonucleotides and typically were used with
radioactively-labeled targets.
Figure 2. An example of a
custom-made pin spotter (Image courtesy of Pat Brown Lab, Stanford
University).
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Next came microarrays, which were
created by using pin spotters. These are pin-based robotic systems that
can dispense an accurate volume of a DNA solution in a spot of about 150
microns onto a glass slide. DNA clones, PCR products or presynthesized
oligonucleotides can be bound to the glass surface to create high-density
arrays. An example of such a system is shown in Figure 2.
The Process
By the mid 90s, researchers were
using microarrays to investigate differences in expression profiles for
tissues of different types to evaluate bacterial cell lines in different
stages of their life cycle, to investigate which genes were involved in
cell division and to investigate drug dosing series. The general process
for using microarrays is as follows: templates for genes of interest are
obtained and amplified by PCR. Following purification and quality control,
aliquots (~5 nL) are printed on coated glass microscope slides. Total RNA
from both the test and reference sample is fluorescently labeled with
either Cy3– or Cy5–dUTP using a single round of reverse transcription.
The fluorescent targets are pooled and allowed to hybridize, under
stringent conditions, to the clones on the array.
Figure 3. Expression profiling using
cDNA microarrays.
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Fluorescence detection of the probe
response is done using a confocal laser microscope. Monochrome images from
the scanner are imported into software, where the images are
pseudo-colored and merged. The software attaches information about the
clones — including gene name, clone identifiers, intensity values,
intensity ratios, normalization constants and confidence intervals — to
each target. Data from a single hybridization experiment is viewed as a
normalized ratio (that is, Cy3/Cy5), in which significant deviations from
1 (no change) are indicative of increased (>1) or decreased (<1)
levels of gene expression relative to the reference sample. In addition,
data from multiple experiments can be examined using any number of data
mining tools. This process is described in Figure 3.
Figure 4. Agilent inkjet technology
for synthesis of oligonucleotide 60-mer arrays. Probes are built
up on the surface of the array by precision deposition of
reagents.
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Rather than making arrays in the
laboratory using spotters, several vendors offer DNA microarrays that have
been synthesized in situ on a surface, creating high-density
oligonucleotide arrays that have up to 500,000 probe sequences. The first
company to commercialize this type of technology was Affymetrix (Santa
Clara, California, USA), which uses a proprietary light-directed
oligonucleotide synthesis approach. A solid support is derivatized with a
covalent linker molecule terminated with a photo labile protecting group.
Light is directed through a mask to deprotect and activate selected sites,
and protected nucleotides couple to the activated sites. The process is
repeated, activating different sets of sites and coupling different bases,
allowing arbitrary DNA probes to be constructed at each site. Affymetrix
uses a different detection scheme than previously described where mRNA is
reverse transcribed into cDNA and then copied into biotinylated cRNA. The
biotin-streptavidin complex then is used to add the fluorescent tag. The
complex has a very high binding constant, allowing for very stringent
binding conditions. The reason for the very high density of the Affymetrix
array (up to 500,000) while there are only 30,000 human genes is the fact
that they use only 25-mer probes, which don't offer the required
specificity. To overcome this issue, up to 20 sequences per gene are used
to increase the overall confidence level.
Figure 5. Phosphoramidite chemistry
allows high-yield synthesis of oligonucleotide probes, allowing
long probes to be synthesized.
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Another vendor offering oligonucleotide
arrays is Agilent Technologies. Its process is shown in Figure 4. The
accuracy of its inkjet printing technology is used to build the
oligonucleotides on standard format glass slides using phosphoramidite
chemistry, as shown in Figure 5.
Figure 6. An Agilent 25,000-feature
mouse catalog array.
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The advantages of using commercially
produced microarrays are the quality and uniformity of the arrays. With
inkjet technology there is no need to create expensive masks, allowing
full flexibility and cost-effective production of microarrays. Because of
the reaction efficiency of the chemistry, 60-mer probes can be created,
allowing for increased sensitivity and specificity as compared to shorter
probes. Coverage with a 44,000-feature array, as is shown in Figure 6, is
therefore similar to the higher-density Affymetrix arrays. A lot of
attention is put into the sequence selection, where modeling tools are
used to optimize the probes that are selected for the array in order to
avoid differences between splice variants.
Alternative Approaches with Microarrays
As effective and efficient as
microarray technology is, further approaches are being investigated. One
of the most time-consuming steps in utilizing microarray technology is
hybridization. During this step, the target molecules have to find the
complementary probe on the surface, typically by molecular diffusion. To
decrease time needed for the hybridization step, Nanogen Inc. (San Diego,
California, USA) developed an electronic microarray, utilizing the natural
charge of the DNA molecules. Applying an electric current to individual
test sites on the microarray enables rapid movement and concentration of
the molecules. Molecular binding onto the microarray is accelerated up to
1000 times as compared to traditional passive methods. Nanogen's
technology involves electronically addressing biotinylated DNA samples,
hybridizing complementary DNA reporter probes and applying stringency to
remove unbound and nonspecifically-bound DNA after hybridization. Density,
however, currently is limited to 100 test sites.
Another alternative to the
fluorescence detection used in most systems is Applied Biosystems Inc.'s (ABI,
Foster City, California, USA) chemiluminescence system, which overcomes
the use of high-priced fluorecent dyes. An increase in detection
sensitivity is obtained by combining a three-dimensional (3-D) substrate
surface with the chemiluminescence chemistry on nylon substrate bound to
glass slides. The spot size is about 150 microns, allowing for up to
18,000 probes. In ABI's system, the 60-mer oligonucleotide probes are
synthesized prior to spotting on the substrate.
Past, Present and Future of Microarrays
Microarray technology today is an
integral part of drug discovery. It is used for target identification in
investigating the gene expression profile of disease. It then is used to
screen potential drug candidates by looking at which genes are affected by
the respective drugs. Next, it is used in target validation in large
patient groups and is starting to be used in ADME/Tox studies. An
increasing number of catalog arrays is available, and custom arrays are
available commercially as well, giving increased access for researchers to
different model systems. In addition, equipment is readily available to
create microarrays by spotting in the laboratory, providing researchers
with maximum flexibility. New applications are becoming available with
additional advantages. An example of this new readiness is the formation
of Agendia (Amsterdam, The Netherlands), a Dutch company with roots in the
Dutch Cancer Institute (Amsterdam, The Netherlands) and the goal of
becoming the leading provider of gene expression services. Using
microarray technology from Agilent, Agendia profiled almost 300 patients
for 70 gene prognosis profiles that were earlier discovered. The gene
expression profile studied is a more powerful predictor of the outcome of
disease in young patients with breast cancer than standard systems-based
examination of tissue samples. Patient specific treatment is a first step
towards personalized medicine.
Another new application is
comparative genomic hybridization (CGH). In this approach, researchers
look directly at the genomic DNA, rather than the expression profile of
RNA, allowing direct measurement of the copy number of a given gene. This
is important in cancer research, allowing faster chromosome mapping.
Another new technique is location
analysis — combining DNA protein complexes with antibody affinity
purification to study where proteins attached to the DNA and where the
promotor regions are.
Perhaps microarray technology will
soon be ready to make it to the big time — the doctor's office — where
patients will receive their own genetic profile, which will determine both
personalized treatment in case of disease or suggested lifestyle changes
that could prevent health problems in the future.
Tom A. van de Goor is project
manager for LC–MS solutions at the Integrated Biology Solutions unit of
the Life Science and Chemical Analysis business at Agilent Technologies in
Santa Clara, California, USA. He can be reached at tom_vandegoor@agilent.com
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