Multicolor analysis extends potential of DNA microarrays
White light source and conventional microscope open use of entire visible spectrum
Microarray platforms are used everywhere
— from academic research centers to public health laboratories to pharmaceutical
companies. By enabling analysis of thousands of DNA sequences at a time, they have
dramatically increased throughput and helped reduce costs in bioanalytical investigations.
Typically, with “spotted” microarrays,
DNA from two samples (a control and the sample of interest) is labeled with different
color reporters and hybridized to the microarray. By comparing the reporter intensities,
investigators can determine, for instance, whether the sample is healthy or
But this is often not enough, said
Jason R.E. Shepard, a researcher in the Biodefense Laboratory of the Wadsworth
Center at the New York state Department of Health in Albany. With respect to cancer
screening and diagnosis, for example, there are various types and stages of the
disease, and it would be more useful to evaluate the sample of interest against
a number of parameters. Multiplexed arrays can provide much information by probing
many gene targets in parallel, but these analyses are still based on the relative
responses of two samples.
The multicolor microarray
platform could permit higher throughput as well as increased experimental significance.
The ability to measure multiple parameters against a control, rather than the single
parameter afforded by two-color assays, could allow investigators to test a number
of hypotheses. Shown here are four images colored to reflect the wavelengths
used and indicating where hybridization has occurred (the red spot in the bottom
left image, for example).
“It has always struck me as odd
that you’re doing an assay that has thousands of genes you can interrogate,”
he added, “and, typically, you look at only two samples.” To address
this, he adapted a microarray platform developed in the lab of David R. Walt at
Tufts University, where Shepard did his graduate studies, so that the array could
probe eight samples at a time. As described in an Analytical Chemistry paper
published online March 7, this simultaneous multicolor array hybridization affords
an analytical flexibility not available with standard two-color assays.
Researchers have developed a multicolor microarray platform that
offers an analytical flexibility not available with conventional two-color assays.
Using various color reporters, a single bead in the fiber optic-based microarray
can hybridize eight samples at a time.
The limitations of conventional microarray
platforms lie largely in the choice of excitation sources and reporters. Typically,
these platforms use laser excitation. Probing more than the usual two colors would
require additional laser wavelengths, which could lead to significantly higher costs
and larger sizes. Furthermore, the platforms generally use standard organic fluorophores
as reporters, which have limitations of their own. Emission signals can be weak
when there are relatively low concentrations of the gene target. At the same
time, the use of such fluorophores can result in photobleaching, especially with
long exposure times.
Shepard addressed these limitations
using a white light source, an epifluorescence microscope and a combination of organic
fluorophores and quantum dot reporters. The white light source and microscope allow
excitation and detection across the visible spectrum (the source is also substantially
less expensive than laser sources). The quantum dot reporters are often considerably
brighter than organic fluorophores and are more resistant to photobleaching. Also,
they offer narrow emission bands, which helps reduce spectral overlap of the reporters.
Indeed, this represents one of the
main questions addressed in the paper: How many reporters can you fit into a defined
“When I first thought of this
concept, I took every type of reporter in my lab and bought a couple more,”
Shepard said. He started with about a dozen, “but it was easy to see right
away that I was going to run into spectral overlap.” Ten reporters still
led to some bleedthrough. Finally, he determined that he could use as many as eight
reporters without spectral overlap.
It might be possible to increase the
number of reporters, he added, by using specific filters, prisms or filterless grating-based
optics. The filters discussed in the Analytical Chemistry paper were not
specifically optimized for the reporters used.
Shepard demonstrated the technique
by analyzing eight Bacillus anthracis samples simultaneously. The system
he used was based on an epifluorescence microscope made by Olympus and modified
by Optical Analysis of Nashua, N.H. A white-light mercury bulb provided excitation,
and a CCD camera made by the Cooke Corp. of Romulus, Mich., acquired images of the
fluorescence. The excitation and emission filter wheels were made by Prior Scientific
of Cambridge, UK. IPLab software from Scanalytics of Fairfax, Va., controlled the
filter wheels and shutters and processed the images.
The array platform was a 1-mm fiber
optic bundle etched and embedded with microbead sensors. The fiber optic bundle
contained approximately 50,000 3-μm array “spots,” to which labeled
target nucleic acids were hybridized. One advantage of a fiber optic-based platform
is that it offers uniform spot features, such as size and boundaries. These
can vary considerably in other array formats, complicating analysis.
The roughly 1-mm fiber optic bundle used for the microarray contains approximately
50,000 array “spots,” each of which is 3 μm in diameter. One advantage
of the fiber optic-based platform is that the features of these spots, such
as size and boundaries, are uniform, which is not always the case with other
The experiments showed that multiplexed
analysis with the new platform offered a fourfold increase in throughput with respect
to conventional two-color assays. The experiments included a polymerase chain reaction
amplification step, without which analysis of eight samples might not be as efficient.
Nonetheless, having more colors would still allow higher throughput as well as improved
experimental significance. Investigators could use the platform to explore a number
of hypotheses, measuring multiple parameters against the control instead of the
single parameter offered by conventional two-color assays. Or they could assign
multiple parameters as controls to circumscribe the unknown response.
The platform could serve a variety
of applications including pathogen diagnostics. “Particularly in the area
of biodefense,” Shepard said, “I view arrays as necessary for rapid
typing of unknown or emerging (mutating) pathogen detection. The natural propensity
for horizontal gene transfer and the ability to engineer organisms could very well
preclude detection by an assay directed at identifying a single gene target. Or
even something like the possibility of multiple infectious agents turning up together
could be problematic for conventional assays.”
In the meantime, Shepard plans to extend
the technique. “We’re going to start dabbling with different assays
in which we can do multicolor analysis,” he said. “Not just DNA microarrays,
but some live-cell imaging as well.” The technique is especially well suited
to this because the photostability of the quantum dots will allow the researchers
to track cells for hours at a time.
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