251
specific hybridization probes. Since short DNA hybridization probes form stable duplexes with
their rRNA target only if there is near-perfect Watson-Crick base pair complementarity,
probes can be tailored for high specificity.
Computer assisted analyses of aligned rRNA sequences can be used to identify regions of
nucleotide sequence that are unique to a given species (or other taxon). Initially, an
approximately 20 nucleotide sequence (or longer) is identified that is unique to a particular
group or species. Oligonucleotide probes complementary to these diagnostic sequences are
then prepared, using an automated DNA synthesizer. Ideally these oligonucleotides should
form perfect double-stranded nucleic acid duplexes with the target sequence, but have several
mismatches with the homologous rRNA sequence of nontarget organisms. The specificity and
fidelity of probe binding is confirmed by hybridization of the probe with a collection of
rRNAs from a variety of different species. Typically, the probe is radiolabelled and hybridized
to rRNA immobilized on a nylon filter. Probe binding is detected by autoradiography.
Hybridization and wash temperatures are determined empirically for each new probe. A rough
estimate for the dissociation temperature (Td) of short oligonucleotide probes is:
Td "" 4°(number of G+C residues) + 2°(number of A+T residues) (Wallace and Miyada,
1987). In general, hybridization and wash temperatures about lOoC below the Td of a
perfectly matched duplex yield strong hybridization signals with good specificity. A well
designed probe should give a strong positive hybridization signal with rRNA from the targeted
organism, but demonstrate no background binding to rRNA of heterologous species.
Radiolabelled rRNA-specific probes have been used in a number of environmental studies.
Quantitative estimates of the representation of specific groups are obtained by hybridizing
radiolabelled probes to nucleic acids extracted from natural samples. Relative binding of the
specific probe is normalized to that of a universal probe, which binds to all rRNA species,
and therefore provides an estimate of total rRNA. This approach has been taken to estimate
the abundance of particular microbial species present in bovine rumen, and their response to
environmental perturbation (antibiotic challenge, Stahl et al., 1988). Giovanonni et al. (1990a)
have estimated the abundance of specific marine bacterioplankton groups with this method.
specific hybridization probes. Since short DNA hybridization probes form stable duplexes with
their rRNA target only if there is near-perfect Watson-Crick base pair complementarity,
probes can be tailored for high specificity.
Computer assisted analyses of aligned rRNA sequences can be used to identify regions of
nucleotide sequence that are unique to a given species (or other taxon). Initially, an
approximately 20 nucleotide sequence (or longer) is identified that is unique to a particular
group or species. Oligonucleotide probes complementary to these diagnostic sequences are
then prepared, using an automated DNA synthesizer. Ideally these oligonucleotides should
form perfect double-stranded nucleic acid duplexes with the target sequence, but have several
mismatches with the homologous rRNA sequence of nontarget organisms. The specificity and
fidelity of probe binding is confirmed by hybridization of the probe with a collection of
rRNAs from a variety of different species. Typically, the probe is radiolabelled and hybridized
to rRNA immobilized on a nylon filter. Probe binding is detected by autoradiography.
Hybridization and wash temperatures are determined empirically for each new probe. A rough
estimate for the dissociation temperature (Td) of short oligonucleotide probes is:
Td "" 4°(number of G+C residues) + 2°(number of A+T residues) (Wallace and Miyada,
1987). In general, hybridization and wash temperatures about lOoC below the Td of a
perfectly matched duplex yield strong hybridization signals with good specificity. A well
designed probe should give a strong positive hybridization signal with rRNA from the targeted
organism, but demonstrate no background binding to rRNA of heterologous species.
Radiolabelled rRNA-specific probes have been used in a number of environmental studies.
Quantitative estimates of the representation of specific groups are obtained by hybridizing
radiolabelled probes to nucleic acids extracted from natural samples. Relative binding of the
specific probe is normalized to that of a universal probe, which binds to all rRNA species,
and therefore provides an estimate of total rRNA. This approach has been taken to estimate
the abundance of particular microbial species present in bovine rumen, and their response to
environmental perturbation (antibiotic challenge, Stahl et al., 1988). Giovanonni et al. (1990a)
have estimated the abundance of specific marine bacterioplankton groups with this method.