Most POAs contain short oligonucleotide (oligo) probes representing genes with diagnostic value, usually the small-subunit ribosomal RNA (16S rRNA) gene.
These arrays are commonly used to detect the presence of specific bacteria in complex samples based on unique sequence regions. Several factors make 16S rRNA ideal for microbial identification and differentiation including (1) 16S rRNAs genes are found in all bacteria; (2) there is no evidence of horizontal transfer of these genes between organisms; and (3) the genes contain both conserved and variable regions, either of which can be used for probe selection depending on the objective of the study (Olsen et al., 1986). Additionally, there is a vast amount (>100,000 sequences) of rRNA sequence data available via the Ribosomal Database Project (RDP) (Cole et al., 2005).
Because some regions of rRNA genes are highly conserved, it is often necessary to use short oligos (~20-mers) for POAs in order for the probes to be specific to individual organisms. Using shorter probes, it is possible to discriminate a single mismatch in a probe-target hybridization (Zhou et al., 2004). A commonly used POA design strategy consists of arraying several probes that perfectly match a given target along with corresponding probes containing a single mismatch (usually at the central position) relative to the target (Wilson et al., 2002a; El Fantroussi et al., 2003; Peplies et al., 2003; Urakawa et al., 2003). Detection of the target sequence is indicated by greater signal intensity for the perfectly matched probes compared with the mismatched probes. Although this strategy enables very specific detection of target sequences, it does have some potential disadvantages, which we discuss in a later section on specificity.
One of the challenges for 16S rRNA-based analysis is the innate propensity of these molecules to form stable secondary structures that may interfere with hybridization and lead to false-negative results. For example, one study (Peplies et al., 2003) reported that 17 out of 41 expected hybridization events were not detected. This possibility can be reduced by incorporating one of numerous available software programs, such as Mfold (Zuker, 2003), which can identify self-complementarity in oligo probes into the probe design process. This difficulty can also be addressed by either fragmenting the target prior to hybridization or by the inclusion of helper-probes in the hybridization mixture. The helper-probes are designed to disrupt the local secondary structure by binding to the target molecule adjacent to the actual probe binding site. However, there is a risk that the helper-probe could cause nonspecific binding if it binds too closely to the actual probe binding site (Chandler et al., 2003). Furthermore, disruption of secondary structure in one region may result in the formation of secondary structures in other regions that could possibly affect the binding sites of other probes.
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