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.
Nick-translation was used to incorporate Alexa 647-12-OBEA-dCTP (Molecular Probes) into 2 micrograms of purified P. roseola hsp82-1 amplification product following standard protocols (Sambrook et al. 1989) and using Alexa 647-12-OBEA-dCTP and unlabeled dCTP in a ratio of 1:1. The average length of labeled probe fragment was 200-400 nucleotides. Labeled DNA was separated from unincorporated dNTPs by passage through a G50-150 Sephadex (Sigma) spin column, precipitated with 10 x 10-6 g of blocking DNA (sheared salmon sperm DNA, Eppendorf) and resuspended in 0.2 ml hybridization solution (4x SSC pH 7.0, 0.1% SDS, 50% deionized formamide, 0.14 g/L sheared salmon sperm DNA and 0.2 g/L tRNA).
Hybridization and Visualization
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.
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