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2.6.3. PCR amplification
Two different sets of primers were tested in this study to select those that yielded
the best results to compare DGGE patterns based on their resolution and observed
diversity (Table 1). Primer set Bact-0968-GC-F / Bact-1401-R was used to amplify the
V6 to V8 regions of the 16S rRNA gene [47, 48] and PRBA-338-GC-F and PRUN-518-
R amplified the V3 hypervariable region [44]. PCR was performed using the Taq DNA
polymerase kit from Life Technologies (Gaithersburg, Md). PCR mixtures (50 μl)
contained 0.5 μl Taq polymerase (1.25 U), 20 mM Tris-HCl (pH 8.5), 50 mM KCl, 3
mM MgCl 2 , 200 μM of each deoxynucleoside triphosphate, 5 pmol of the primers, 1 μl
of DNA template, and UV-sterilized water. The samples were amplified in a T1
thermocycler (Whatman Biometra, Göttingen, Germany), and the cycling conditions for
each pair of primers are listed in Table 1. Aliquots (5 μl) were analyzed by
electrophoresis on 1.5% (wt/vol) agarose gels containing ethidium bromide to check for
product size and quantity.
2.6.4. DGGE analysis
The amplicons obtained from the intestinal lumen-extracted DNA and the
probiotic strains were separated by DGGE according to the specifications of Muyzer et
al. [39] using a Dcode TM system (Bio-Rad Laboratories, Hercules, CA).
Electrophoresis was performed in an 8% polyacrylamide gel (37.5:1 acrylamidebisacrylamide;
dimensions, 200 by 200 by 1 mm) using a 30 to 55% denaturing gradient
for separation of PCR products. The gels contained a 30 to 55% gradient of urea and
formamide increasing in the direction of the electrophoresis. A 100% denaturing
solution contained 7 M urea and 40% (vol/vol) deionized formamide. PCR samples
were applied to gels in aliquots of 13 μl per lane. The gels were electrophoresed for 16
h at 85 V in 0.5 X TAE (20 mM Tris acetate [pH 7.4], 10 mM sodium acetate, 0.5 mM
Na 2 -EDTA) [50] buffer at a constant temperature of 60 ºC and subsequently stained
with AgNO 3 [51].
Gel image processing and comparative analysis of banding patterns was done
with Bionumerics version 4.0 (Applied Maths BVBA, Sint-Martens-Latem, Belgium).
Similarity between DGGE profiles was determined by calculating similarity indices of
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