Microarrays

Microarrays represent an exciting new development in microbial community analysis. Nucleic acid hybridization is the principle on which the technique is based. The main difference between past protocols and microarrays is that the oligonu-cleotide probes, rather than the extracted DNA or RNA targets, are immobilized on a solid surface in a miniaturized matrix. Thus, thousands of probes can be tested for hybridization with sample DNA or RNA simultaneously. In contrast to other hybridization techniques, the sample nucleic acids to be probed are fluorescently labeled, rather than the probes themselves. After the labeled sample nucleic acids are hybridized to the probes contained on the microarray, positive signals are detected by use of CSLM or other laser microarray scanning device (Fig. 4.7). A fully developed DNA microarray could include a set of probes encompassing virtually all known natural microbial groupings and thereby serve to monitor the population structure simultaneously at multiple levels of resolution (see Table 4.1; Guschin et at., 1997; Ekins and Chu, 1999; Wu et at., 2001). Such an array would potentially allow for an enormous increase in sample throughput. A major drawback of microarrays for use in soil ecology studies currently is their need for a high copy number of target DNA/RNA to obtain a signal that is detectable with current technologies. Targets in concentrations of less than 103-104 are difficult to detect

Relative Abundance detection

Sample 1 Sample 2

hybridize hybridize

Soil sample or bacterial isolate

^ Isolate mRNA.

Make cDNA by reverse transcription, using fluor-escently labeled nucleotides.

) Apply the cDNA mixture to a microarray, a microscope slide on which copies of single-stranded DNA fragments from the organism's genes are fixed, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray.

Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot (yellow) represents a gene expressed.

mRNA molecules

Labeled cDNA molecules (singlestrands)

DNA microarray

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