245
transcriptase. Individual rDNA genes can then be clonally isolated and sequenced as described
above. Successful application of this method was used to study the species diversity of hot
spring microbial mats (Ward et al., 1990). An advantage of this method is that in theory all
clones obtained contain rRNA genes. Very vigorous lytic protocols can be used, which may
significantly enhance the extraction of nucleic acids. There is, however, a drawback to the
method. Production of full-length cDNAs is problematic, because reverse transcriptase tends
to stop at modified bases or complex secondary structural elements. Thus full length rRNA
clones may be difficult to obtain (Ward et al., 1990).
Another promising technique involves the specific amplification of small subunit rRNA genes
from mixed population DNA. This is achieved via the polymerase chain reaction (peR),
which exploits the thermostable properties of Thermus aquaticus DNA polymerase (Taq
polymerase, Sakai et al., 1988). Oligonucleotide primers, which hybridize to the distal ends
of a specific, targeted gene, initiate the synthesis of new DNA strands. Taq polymerase
catalyzes the synthesis of nascent strands of the targeted gene, which is flanked by the
oligonucleotide primers. Repeated cycles of DNA denaturation (@ 94°C), reannealing of
oligonucleotide primers (@ 40°C), and primer extension (@ n°C), are possible, since
thermostable Taq polymerase can withstand repeated heating cycles at 94°e. The
denaturationireannealingiprimer-extension cycle can be repeated up to 40 times, resulting in
an exponential increase of the DNA from the targeted gene. Very small amounts of starting
total DNA (typically nanograms) can be used to amplify microgram quantities of the specific,
targeted gene(s) of interest.
For subsequent phylogenetic analysis, amplified small subunit rRNA genes can be cloned, and
the rRNA genes of individual clones sequenced (Medlin et al., 1988; Giovanonni et al.,
1990a). This technique has been exploited to amplify small subunit RNA genes from both pure
cultures and mixed populations. The peR approach has the advantage of being extremely
rapid, since all the clones obtained will contain rRNA genes. One disadvantage of the
approach is that, depending on the reaction conditions, Taq DNA polymerase can have a
relatively high error rate (Ennis et al., 1990; Eckert and Kunkel, 1990), which may be
detected when amplification products are cloned, and the individual clones are sequenced.
transcriptase. Individual rDNA genes can then be clonally isolated and sequenced as described
above. Successful application of this method was used to study the species diversity of hot
spring microbial mats (Ward et al., 1990). An advantage of this method is that in theory all
clones obtained contain rRNA genes. Very vigorous lytic protocols can be used, which may
significantly enhance the extraction of nucleic acids. There is, however, a drawback to the
method. Production of full-length cDNAs is problematic, because reverse transcriptase tends
to stop at modified bases or complex secondary structural elements. Thus full length rRNA
clones may be difficult to obtain (Ward et al., 1990).
Another promising technique involves the specific amplification of small subunit rRNA genes
from mixed population DNA. This is achieved via the polymerase chain reaction (peR),
which exploits the thermostable properties of Thermus aquaticus DNA polymerase (Taq
polymerase, Sakai et al., 1988). Oligonucleotide primers, which hybridize to the distal ends
of a specific, targeted gene, initiate the synthesis of new DNA strands. Taq polymerase
catalyzes the synthesis of nascent strands of the targeted gene, which is flanked by the
oligonucleotide primers. Repeated cycles of DNA denaturation (@ 94°C), reannealing of
oligonucleotide primers (@ 40°C), and primer extension (@ n°C), are possible, since
thermostable Taq polymerase can withstand repeated heating cycles at 94°e. The
denaturationireannealingiprimer-extension cycle can be repeated up to 40 times, resulting in
an exponential increase of the DNA from the targeted gene. Very small amounts of starting
total DNA (typically nanograms) can be used to amplify microgram quantities of the specific,
targeted gene(s) of interest.
For subsequent phylogenetic analysis, amplified small subunit rRNA genes can be cloned, and
the rRNA genes of individual clones sequenced (Medlin et al., 1988; Giovanonni et al.,
1990a). This technique has been exploited to amplify small subunit RNA genes from both pure
cultures and mixed populations. The peR approach has the advantage of being extremely
rapid, since all the clones obtained will contain rRNA genes. One disadvantage of the
approach is that, depending on the reaction conditions, Taq DNA polymerase can have a
relatively high error rate (Ennis et al., 1990; Eckert and Kunkel, 1990), which may be
detected when amplification products are cloned, and the individual clones are sequenced.