The Use of DNA Chip Technology to Better
Understand Neuromuscular Disorders
This section is designed to give a brief, but technical overview
on microarray, or DNA chip technology and how it relates to
the neuromuscular disease research we are performing.
The understanding of inherited muscle disorders has progressed
rapidly within the last decade. It is believed that mutations
in a diverse group of muscle genes causes a smaller number
of functional pathways and structural networks to become disrupted.
As a result of these common pathogenetic mechanisms, muscle
pathology and clinical diseases share many characteristics.
To elucidate the similarities and differences between various
neuromuscular disorders, we are using DNA microarray technology
to identify the changes that occur in gene expression in a
group of muscular dystrophies and myopathies. New hypotheses
about muscular pathogenesis can be created by the descriptive
information obtained from these microarray studies.
Why Microarray?
DNA microarrays examine the levels of messenger RNA (mRNA) expressed.
Although it would be most useful to examine levels of protein
activity, localization, or concentration, this is not yet feasible.
However, as pointed out by Brown (Schena et al., 1995), just
as natural selection has optimized the biochemical properties
of gene products, it has also optimized gene transcription.
| In general, levels of protein
and mRNA activities are regulated in parallel. By knowing
when and where a gene is expressed, insight into its biological
role can be inferred. Since microarrays have the ability
to screen thousands of genes simultaneously, gene expression
patterns can be analyzed to provide information about
the state of the cell or tissue. |
 |
How does DNA Microarray work?
The method of analysis consists of sample preparation, hybridization,
staining and scanning of arrays, and data analysis.
Sample Preparation
The first step is to extract the total RNA from the tissue sample.
This RNA extraction contains messenger RNA (mRNA), ribosomal
RNA (rRNA) and transfer RNA (tRNA). Depending on the type and
source of the tissue, the mRNA can vary from 1-5% of the total
RNA extraction. A double stranded copy DNA ( ds cDNA) is created
next. Since the mRNA contains a poly-A tail, it is specifically
used in the reaction to create the ds cDNA. The ds cDNA is then
used to create a biotin-labeled copy RNA (cRNA) [Figure1]. The
biotin-labeled cRNA will act as the “target” and will
be hybridized to the probe array.
Hybridization of the “target”
to the probe array
Our probe arrays are the GeneChip‚ purchased from Affymetrix‚.
Tens to hundreds of thousands of different oligonucleotide probes
are synthesized onto each array using photolithography and combinatorial
chemistry. The placement of each oligonucleotide on the array
and its specific sequence is known. Different arrays can be
purchased depending on the organism and tissue used [Figure2].
Fifteen micrograms of out biotin-labeled cRNA
is added to the array and incubated overnight at 45C. Since
our “target” is cRNA, it will bind to the specific
complementary oligonucleotide sequence on the probe during
this incubation. Unbound “targets” are then washed
off [Figure 3].
Staining of the bound “target”
The bound biotin-labeled cRNA “target” is then stained.
A solution of Streptavidin-Phycoerythrin (SAPE) is added. SAPE
has high affinity for biotin and several SAPE molecules will
bind to each biotin molecule. A wash step is performed to remove
unbound SAPE. The chip is then ready to be scanned at 488nm.
The SAPE bound to the biotin-labled cRNA emits light at 570nm
and is proportional to the amount of bound “target”
at each location on the probe array [Figure 4].
Data Analysis
The array can then be analyzed for absolute expression or as
a comparison between different arrays. The absolute expression
examines the hybridization intensity to determine if the “target”
bound to which individual oligonucleotide on the probe. Two
different chips can be compared. Each individual oligonucleotide
on the probe is compared between the two chips. The intensity
of the bound “target” is used to determine if an increase,
decrease or no change has occurred.
To learn more about microarrays, you can visit the below
web sites.
•National
Human Genome Research Institute
•DNA
Microarray Methodology (flash animation)
•Affymetrix
