Bacterial Communities in Various Conditions of the Composting Reactor Revealed by 16S rDNA Clone Analysis and DGGE - Sustainable Biotechnology: Sources of Renewable Energy - page 171

or 99% similarity) or evolutionary distance (e.g. < 0.01). These values can then be
used to plot a coverage curve (C vs D) that describes how well the library represents
the total community given varying criteria of uniqueness. The homologous coverage
(Cx) is calculated by the following formula Cx 1-(Nx/n), where Nx is the number

of unique sequences in the sample and n is the total number of sequences [20].

=

Construction of a Phylogenetic Tree

Sequences are aligned using the Clustal W program version 1.7 [21, 22], and all
sites with gaps in any sequences and the regions of PCR primers are removed
from the alignment. The phylogenetic trees are constructed by the neighbor-joining
method [23] or the maximum-likelihood method [24] using the PHYLIP (Phylogeny
Inference Package) program version 3.5c ( http://evolution.genetics.washington.
edu/phylip.html ) [25]. The stability of relationships was assessed by performing
bootstrap analyze of the neighbor-joining data based on 1,000 resamplings.

3.2.2 Denaturing Gradient Gel Electrophoresis (DGGE)

PCR Amplification of 16S rDNA

Community DNA was used as the template DNA for PCR. The bacterial vari-
able V3 region of 16S rDNA was amplified by PCR with a forward primer, with
a GC-clamp on the 5 terminals, B341F-GC (5 -CGCCCGCCGCGCCCCGCGC
CCGTCCCGCCGCCCCCACCCGCCTACGGGAGGCAGCA-3 , E . coli position
341-356) and reverse primer B515R (5 -TTACCGCGGCTGCTGGCAC-3 E .
coli position 533-515), which are modified Ellis et al. primer sequences [26].
Amplification of 16S rDNA by touch-down PCR using DNA polymerase comprised
3 min at 94 ◦ C, 10 cycles of 30 s at 94 ◦ C, 30 s at 65 ◦ C, 30 s at 72 ◦ C, and the anneal-
ing temperature was lowered for 1 ◦ C every 2 cycles from 65 to 60 ◦ C. Then, 30
cycles of 30 s at 94 ◦ C, 30 s at 60 ◦ C, 30 s at 72 ◦ C and a final extension of 3 min
at 72 ◦ C was carried out. The PCR products were purified by using commercially
available purification kit (i.e. GFX PCR DNA and Gel Band Purification Kit, GE
Healthcare, UK).

Denaturing Gradient Gel Electrophoresis

Procedures for gel casting and electrophoresis were according to manufacturer's
instructions. DGGE was performed on an 8% polyacrylamide gel with a 20-60%
denaturant gradient, where 100% denaturant corresponds to 7 M urea and 40%
formamide. Electrophoresis was run at a constant temperature of 58 ◦ C for 4 h at
200 V, using a DCode universal mutation detection system (Bio-Rad Laboratories,
UK). PCR products separated on the gel were stained with 20 L of ethidium

bromide (10 mg/mL) in 200 mL of the buffer, which was used for electrophore-
sis, for 30 min and washed with the buffer for 30 min (3X 10 min washes) and
then photographed with ultraviolet irradiation. A substitute for ethidium bromide,

μ