PCR amplification of 16s rDNA
Ribosomal DNA was amplified directly from a single colony grown on agar LB plates
using Taq polymerase as recommended by the manufacturer (Sigma, Aldrich).
Reaction mixtures contained 2mM MgCl2, 10mM TrisHCl, pH 8.3, 50mM KCl 200*M
deoxynucleoside triphosphates, 2.5 units of Thermus aquaticus DNA polymerase,
0.2 *M of each oligonucleotide primer. Thermal cycling was as follows: denaturation
at 95°C for 1.5 min., annealing at 55°C or 60 °C for 1.5 min. and
extension at 72°C for 1.5 min. for a total of 30 cycles.
The oligonucleotide primer sequences to identify and amplify rDNA genes were
EubacF: agagtttgatcctggctcag; EubactR: ggttaccttgttacgactt, for eubacteria;
Ribosomal DNA Cloning and Sequencing
ArcheaF: ttccggttgatccygccgga; ArcheaR: yccggcgttgamtccaatt, for archea;
EukF: aacctggttgatcctgccagt; EukR: tgatccttctgcaggttcacctac, for eukaryotes.
PCR amplified DNA, after the purification using the Nucleospin® Extract,
a ready to use system for direct and fast purification of PCR products from
agarose gels (Macherey-Nagel), was directly sequenced using Thermo Sequenase
Cy 5.5 Dye Terminator Cycle Sequencing kit following the protocol suggested
by the manufacturer (Amersham Pharmacia Biotech). The products were separated
in a gel matrix using the automated sequencing instrument SeQ 4x4 personal sequencer
system. The purified, amplified eubacterial rDNAs were cloned by using the SureClone
TM Ligation kit (Amersham Pharmacia Biotech). Insert-containing clones were
identified by agarose gel electrophoresis of small-scale plasmid preparation
(Maniatis et a., 1982). Plasmid templates were sequenced as reported above.
Determined sequences were aligned to previously determined rDNA sequences using
the program Clustal W at http://www.ddbj.nig.ac.jp
A total of 6 RAPD primers were tested for their ability to provide informative
and reproducible RAPD profiles. RAPD1 (5'ggtttccgccc3'), RAPD2 (5'ggatcctgac3'),
RAPD3 (5'cggcccctgt3'), RAPD4 (5'tcccgctgcg3'), RAPD2T (5'ggttcctgtc 3'), RAPD4C(5'agggcgacgc
The results of the experiments with primer 1 and 2 are reported here, because
they show clearly that the different clones have different genetic profiles.
The PCR reaction mixture (25ml) contained 1X PCR buffer supplemented wih 3mM
MgCl2 solution (Sigma Aldrich) 200mM each of the four deoxynucleoside triphosphates
(dNTPs, Amersham Pharmacia Biotech), 500ng of one or two 10 nucleotide primers
(MWG-Biotech AG) 0.5 U of Taq DNA Polymerase (Sigma-Aldrich) and a small part
of a single colony from agarose plate. PCR amplification was performed on a
Perkin Elmer 2400 Thermal Cycler using the following cycling program: after
denaturation at 94°C for 3 min., the reaction mixtures undergo 45 cicles
of denaturation at 94°C for 0.5min., annealing at 32°C for 1min. and
extension at 72°C for 2 min., with an additional final 10min. extension
at 72°C. 15ml of each amplification products were separed on 1% agarose
gel stained with 0.5mg/ml ethidium bromide, examined and photographed under
a UV light. A 1.0 kb ladder DNA sample (MBI Fermentas) was used as a molecular
weight marker. The RAPD reproducibility was established running all reactions
in three independent experiments.
Scanning Electron Microscopy
These analyses were performed in the "Centro Interdipartimentale di Microscopia
Elettronica" of the University of Naples Federico II.