DNA extraction, amplification, purification and sequencing
Total genomic DNA was extracted from the intestinal contents using Gsure
Bacterial Genomic DNA isolation kit. The quality of the extracted DNA
was assessed by electrophoresis in 1% (w/v) agarose gel. The
concentration of the extracted DNAs was determined using a Nanodrop
(Thermo fisher Scientific) and then normalized to 200 ng/μl. Universal
16S rRNA genes were amplified by PCR in a volume of 25μl containing 200
ng DNA, 5X Phusion HF buffer, 10mM each dNTP, 2.5 U of Phusion DNA
Polymerase, 0.5μM of forward and reverse primers as listed before
(Weisburg, Barns, Pelletier, & Lane, 1991).
The PCR amplification was carried out with the following process:
940C for 5 min; 35 cycles at 94 0C
for 1 min, 50 0C for 30 sec, 72 0C
for 2 min and a final extension at 72 0C for 10 min.
The amplified PCR products were analysed by electrophoresis on a 1%
agarose gel and visualized under Gel Documentation System (Bio-Rad). The
PCR product was further purified using Gene JET purification column and
sequenced at sequencing was done using BigDye® terminator kit following
manufacturer’s instructions (Applied Biosystems Inc. ABI, Foster City,
CA) and products analysed on an ABI 3500xL Genetic Analyser platform (at
Eurofins Pvt Ltd, Bangalore, India). Amplicons were sequenced from both
directions using forward and reverse primers.
The purified PCR products were sequenced and aligned with the
corresponding sequences in the GenBank database by using Blast search
algorithm (Altschul et al., 1997).These sequences were deposited in the
GenBank.