Feasibility of transferring fluorescent in situ hybridization probes to an 18S rRNA gene phylochip and mapping of signal intensities

Abstract

DNA microarray technology offers the possibility to analyze microbial communities without cultivation, thus benefiting biodiversity studies. We developed a DNA phylochip to assess phytoplankton diversity and transferred 18S rRNA probes from dot blot or fluorescent in situ hybridization (FISH) analyses to a microarray format. Similar studies with 16S rRNA probes have been done determined that in order to achieve a signal on the microarray, the 16S rRNA molecule had to be fragmented, or PCR amplicons had to be <150 bp in length to minimize the formation of a secondary structure in the molecule so that the probe could bind to the target site. We found different results with the 18S rRNA molecule. Four out of 12 FISH probes exhibited false-negative signals on the microarray; eight exhibited strong but variable signals using full-length 18S RNA molecules. A systematic investigation of the probe's accessibility to the 18S rRNA gene was made using Prymenisum parvum as the target. Fourteen additional probes identical to this target covered the regions not tested with existing FISH probes. Probes with a binding site in the first 900 bp of the gene generated positive signals. Six out of nine probes binding in the last 900 bp of the gene produced no signal. Our results suggest that although secondary structure affected probe binding, the effect is not the same for the 18S rRNA gene and the 16S rRNA gene. For the 16S rRNA gene, the secondary structure is stronger in the first half of the molecule, whereas in the 18S rRNA gene, the last half of the molecule is critical. Probe-binding sites within 18S rRNA gene molecules are important for the probe design for DNA phylochips because signal intensity appears to be correlated with the secondary structure at the binding site in this molecule. If probes are designed from the first half of the 18S rRNA molecule, then full-length 18S rRNA molecules can be used in the hybridization on the chip, avoiding the fragmentation and the necessity for the short PCR amplicons that are associated with using the 16S rRNA molecule. Thus, the 18S rRNA molecule is a more attractive molecule for use in environmental studies where some level of quantification is desired. Target size was a minor problem, whereas for 16S rRNA molecules target size rather than probe site was important.

FIG. 1.

Consensus secondary structure of the 18S rRNA gene according to previous studies (3, 8, 9). The binding sites of the probes are displayed as red lines for the probes that resulted in no signals and in green lines for the probes that resulted in signal-to-noise ratios above 2.0. Blue lines indicate the primers that were used to amplify the target DNA.

FIG. 2.

The secondary structure formed from three different fragments showing the accessibility of the Chlo01 probe site and the hybridization of three different fragments of the 18S DNA amplified from clone HE001005-53, a chlorophyte alga, isolated in PICODIV (23).

FIG. 3.

Comparison of the signal intensity of the full-length amplicon hybridized with published probes from 18S and 16S rDNA. 16S rDNA data are taken from the study of Lane et al. (13). 18S rDNA data are from Table 3 and represent normalized signals.