The use of microarrays allows the identification of organisms in an ecological
sample by hybridizing DNA extracted from the sample to a set of known DNA
specimens arrayed on a solid support, usually a microscope slide (reviewed
in Call et al. 2003). The sample DNA, or more usually PCR product
derived from it, is labeled with a fluorophore so that the specific sites
of hybridization on the microarray can be visualized and the intensity of
the hybridization signal quantified. The method is conceptually identical
to Southern hybridization of a dot blot, but technological innovations allow
thousands of known DNA samples to be arrayed on a single microscope slide
and many such slides to be produced in a single batch at low cost. Microarray
hybridization has been used to assess microbial diversity and to assay for
specific pathogenic bacteria in numerous studies (reviewed in Ye et al.
2001; Call et al. 2003).
Results of simple hybridizations to a prototype Rotifer chip demonstrate that hybridization of an exact match between labeled DNA and DNA on the microarray is measurably more intense than hybridization of two sequences that differ by as little as 0.6%. This is sufficient for sub-family, sub-genus,or even sub-species identification, depending on the specific gene used in the experiment. The cross-hybridization of related sequences, particularly under lower stringency conditions, may also be useful, as it can be used to detect the presence of sequences in the ecological sample that are not present on the microarray but are related to those that are.
Ecological samples will likely contain DNA from a mixture of species,
at different concentrations depending on relative species abundance.
A protocol for quantitative assessment of rotifer species diversity using
a microarray has not yet been developed. However, a standard application
of microarray hybridization in studies of genomic transcription levels involves
competitive hybridization of two differentially labeled samples of cDNA to
the same microarray slide (Schena et al. 1996; Bowtell 1999).
This allows quantitative differentiation between transcription levels in the
two samples when the relative difference in abundance is greater than about
two-fold (Quackenbush 2002). Using the same technique, it should be
possible to quantitate >2-fold differences in species abundance between
two ecological samples. This would allow rapid and sensitive examination
of differences in species abundance in diurnal cycling, for example.
As yet a microarray is unavailable for rotifers; however, this situation may change in the near future. Below is a protocol for hybridization to a microarray that has been tested on a preliminary Rotifer chip (submitted manuscript). A note on terminology: unlike traditional (Southern, northern) hybridizations, the labeled DNA, generally more complex than that immobilized on the chip, is refered to as the target DNA and the DNA immobilized on the chip is refered to as the probe DNA.
Microarrays were denatured in deionized water at 95°C for 2 minutes,
dehydrated in 95% ethanol for 2 minutes at room temperature, and dried using
compressed air. Microarrays were rehydrated by soaking in prehybridization
solution (5x SSC pH 7, 0.1% SDS, 1% fraction V BSA) for 45-90 minutes at 42°C,
then were washed in deionized water twice for 4-5 seconds each, in isopropanol
once for 3 to 4 seconds, and dried by briefly spinning in a centrifuge (Beckman-Coulter
TS-5.1-500 rotor at 45 rcf for 2 minutes). Labeled DNA was denatured
at 95°C for 3 minutes, cooled on ice, and 20 microliters was applied
to each microarray slide. Microarrays and labeled DNA were then covered
with Hybri-Slip cover slips (Molecular Probes),
placed in hybridization chambers (Corning)
and hybridized for 18-23 hours at 42°C.
Microarray slides were washed in 2x SSC, 0.1% SDS at 42°C
for 5 minutes, then in 0.1x SSC, 0.1% SDS once for 10 minutes at room temperature
(low stringency), or four times for 5 minutes each at 60°C (high stringency).
Slides were then washed three times for 1 minute each at room temperature
in 0.1x SSC, immediately dried by briefly spinning in a centrifuge, and stored
in a dark box. Hybridization of labeled DNA to the micoarray was visualized
using a Gene-Pix 4000 B scanner (Axon Instruments) and GenePix
Pro 4.0 software.
Bowtell, D. D., 1999. Options available--from start to finish--for obtaining expression data by microarray. Nature Genet. 21: 25-32.
Call, D. R., M. K. Borucki & F. J. Loge, 2003. Detection of bacterial pathogens in environmental samples using DNA microarrays. J. Microbiol. Methods 53:235-243.
Chandler, D. P., G. J. Newton, J. A. Small & D. S. Daly, 2003. Sequence versus structure for the direct detection of 16S rRNA on planar oligonucleotide microarrays. Appl. Envir. Microbiol. 69: 2950-2958.
Quackenbush, J., 2002. Microarray data normalization and transformation. Nat. Genet. 32 sup 496-501.
Sambrook, J., T. Fritsch & T. Maniatis, 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Press.
Schena, M., D. Shalon, R. Heller, A. Chai, P. O. Brown & R. W. Davis, 1996. Parallel human genome analysis: Microarray-based expression monitoring of 1000 genes. Proc. Nat. Acad. Sci USA 93: 10614-10619.
Ye, R.W., T. Wang, L. Bedzyk & K. M. Croker, 2001. Applications of DNA microarrays in microbial systems. J. Microbiol. Methods 47:257-272.