Comparison of Microarray Technologies
October 9, 2001
Lecturer: Larry Ruzzo
Three microarray technologies are available:
- Affymetrix GeneChip (Affymetrix)
- Spotted Glass Slides
Affymetrix GeneChip technology provides an array of 250,000 probes,
each probe containing a set of oligonucleotides of approximately 25
base pairs each representing a region of interest within a gene. As
described in the previous lecture, the array is exposed to cDNA
developed from a cell in which it is hypothesized these genes are
expressed. The cDNA then hybridizes (attaches) to complementary
sequences on the array. As with other microarray technologies, the
strategy is to identify which genes in the cell are expressed, and to
what degree, based on the extent of hybridization observed at each
probe. Fluorescent molecules attached to the cDNA create an
intensity of light that corresponds to the degree of hybridization.
Typically for each oligo that is intended to exactly match a portion
of the gene, Affymetrix ensures there is another oligo that has a
mismatch in one location. The purpose of this is to corroborate
evidence provided by a match: if one of the probes is heavily
hybridized, then the absence of hybridization at the site where the
oligo has been deliberately modified supports the hypothesis that the
hybridization at the match site was more likely due to a sequence
match than to some other source of attraction (eg, that the molecular
shape of that oligo and any similar oligo just happens to be that of
velcro). The added value of this corroboration was questioned in
GeneChips can be used to compare genes expressed in two cells, one of
which is known to be afflicted, by comparing the effect of exposing
two microarrays to the genetic contents of the cells and comparing the
hybridization patterns on those two arrays.
GeneChips are created using a photolithography technology similar to
that used in making integrated circuits, though at a 24 micron feature
size much larger than the submicron features sizes of ICs. A series
of steps proportional in length to the number of bases is required,
beginning with a chemically coated glass substrate. A
photo-protective mask is used to expose the substrate to light
selectively. Where light strikes the subtrate, it becomes active such
that with nucleotide incubation, chemical coupling occurs. This
must be repeated with different masks for each type of nucleotide,
and then repeated again a number of times equal to the length of
the oligotide. The whole process is easy to repeat in a mass
production of identical GeneChips, but, like IC fabrication, expensive
to set up. Creating the masks and carrying through a run costs
something like $100,000; each Chip is then sold for about $1000.
Inkjet technology is similar in function. The inkjet controls
application of chemicals according to software without the use
of physical masks. This makes the setup process much cheaper:
there is no need to amortize the cost of a run over a large
number of identical cells. However, the probes are less dense
than is possible using photolithography.
A third technology is the mechanical deposition of entire
cDNA or a PCR product onto an array using carefully designed
metal pen nibs controlled by a robotic arm.
The array is exposed to equal amounts of green and red fluorescent
dyed samples, corresponding to normal and affected cells,
respectively. The color at each site indicates the relative
amount of hybridization corresponding to the relative expression
in the two cells of the cDNA at that site. That is, yellow
indicates expression in both cells; black in neither; red
in only the affected cell; green only in the normal cell.
Comparison of the Three Technologies:
- High density
- Increasing number of species available
- Lower cost
- Better control than spotted glass
- Low cost
- Customizable and Kits are available for specific genes (eg, fruit fly)
- Substitution of radioactive dyes for fluorescent
increases the strength of signal per match,
allowing the use of less sample.
- Even if a gene is weakly expressed, relative intensity is
preserved and so discrepancy in expression is evident.
- Alignment of imaging equipment relies on sophisticated
(ie, imperfect) software.
- Spots range in brightness, shape, some have holes; requires
image analysis software, which again is imperfect.
- Dyes are not same brightness over all excitement levels;
may have nonlinear response curves; this can be mitigated
by swapping dyes, but that entails repeating the experiment.
- Based on ratio means method is sensitive to noise.
In 1997, no one was ready to look at more than 25 to 50 gene levels.
Now people want to look at the whole genome.
One can imagine using an array per human individual, laced with
carefully selected genes representing susceptibility to or contraction
of specific diseases, to provide useful diagnostic and preventative
medical data. And to establish insurability...
There has been a 45% annual growth in microarray revenues
during the past two years.
The expected market size is one-billion dollars by 2005.
The Transcriptional Program of Sporulation in Budding Yeast:
(an article in Science, Vol 282, October 23, 1998, by S. Chu et al.)
The process by which DNA is involved in the creation of mRNA is
called Transcription. Which traits are transcribed changes
over the life cycles of a cell; the map of this as a function
of cycle state is called a Transcriptional Program.
Budding yeast is much like that used in cooking.
Sporulation is the life cycle in which spores are produced.
Yeast is normally Diploid, but when producing spores it goes through
meiosis producing four haploid daughter cells, which are the spores.
It does this when put in a Nitrogen-rich environment as a form of
hibernation or self-preservation.
The meiosis is typically described as being divided into four phases:
early, middle, mid-late, and late corresponding to
replication/recombination, meiosis I, meiosis II, and spore
Transcription in the early and middle phases has been studied
in experiments predating this paper. 150 genes had been implicated
in the sporulation process.
In this work, the authors compare the levels of mRNA present
over time using a series of microarrays. They found 500 genes
that are induced (ie, whose population increases during the process)
and 500 that are repressed.
Additionally, using knockout experiments, they found three specific
genes that prevent sporulation but leave other development unchanged.
Graphs show that the number of mononuclear cells drop with time;
the number of spores increases; thus demonstrating that the cells
they are observing are indeed sporulating. The graphs also demonstrate
that it is not a sudden process: the cells are mixed in state over
Microarray data shows which genes are expressed at the corresponding
times. Activity is as expected, given the previous experiments,
during the various phases of activity; but clustering
the microarray intensities as functions of time into those of
similar shape (ie, those probes that experienced a quantity of
hybridization that varied similarly over the course of time)
suggests division into seven rather than four phases: a refinement
of the earlier sporulation process description.
The authors were able, in each of these seven phases, to identify from
the microarray data which genes were expressed during that phase; the
total being about 500 as compared to 150 known earlier. This gives
those who want to better understand the mechanics of the sporulation
process a great deal more direction as to where to look for clues.