Biology Reference
In-Depth Information
DNA Isolation, G + C Content
The DNA was isolated using a French pressure cell (Thermo Spectronic, Rochester,
NY) and was purified by chromatography on hydroxyapatite as described [51]. The G
+ C content (M%) of the DNA was determined by the midpoint value of the thermal
denaturation profile (Tm) with a Perkin Elmer spectrophotometer at 260 nm, pro-
grammed for temperature increases of 1.0°C/min [52]. Tm was determined by the
graphic method described by Ferragut and Leclerc [53] and the G + C content was
calculated using Owen and Hill's equation (1979).
PCR Amplification of the 16S rDNA Gene Coding Sequence and Sequencing
Purified genomic DNA was used for PCR amplification of the16S rDNA gene. The
following two sets of primers were used on the basis of the highly conserved regions
of halobacterial 16S rDNA sequences as described by Ihara [48]: f1 (5' ATTCCGGTT-
GATCCTGC 3'), r1 (5' TTTAAGTTTCATCCTTG 3'), and f2 (5' AACCGGATTAG-
ATACCC 3'), r2 (5' GTGATCCAGCCGCAGATTCC 3'). The PCR was performed
with 35 cycles of 30 s at 94°C, 30 s at 37°C and 90 s at 72°C. The PCR products were
analyzed by 1.5% agarose gel electrophoresis. The products were purified from the
gel using S.N.A.P. Gel Purification Kit (Invitrogen) and sequenced directly by the
dideoxy chain termination method [54].
Phylogenetic Analysis
The obtained sequences were compared with previously described 16S rDNA se-
quences of halophilic archaeas from the NCBI database. The sequences were aligned
by using CLUSTAL X 1.83 [55, 56] and phylogenetic trees were constructed by the
neighbor-joining method with Kimura two-parameter calculation in MEGA 3.1 soft-
ware. The confidence levels for branching orders were evaluated by the bootstrap
method [57].
DNA-DNA Hybridization
The DNA-DNA hybridization studies were performed as described by De Ley [58]
under consideration of the modifications described by Huss [59] using a Cary ® 100
Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6 × 6 multicell
changer and a temperature controller with in-situ temperature probe (Varian, Inc.).
DNA Fingerprint
The DNA from the isolates was used for AP-PCR fingerprint assay. Three small prim-
ers were used: T3GC (5' CCCAKTCGTGAWTCATGCT 3'), T3R, (5' TCCTCAYT-
TAATNAMCATGCT 3'), and Rsh (5' ATCAAAAT 3'). The PCR was performed for
35 cycles, starting with 10 s of denaturation at 92°C followed by 60 s of annealing at
30°C and 90 s of elongation at 72°C. The reaction mixture contained 10 ng of genomic
DNA, 3 mM MgCl 2 , 1 μM of each primer, 0.2 μM of dNTPs, and 2 U of Taq Poly-
merase (Invitrogen) in a final volume of 20 μl. The PCR results were visualized in a
1.5% agarose gel electrophoresis and analyzed by Image Kodak Software.
 
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