Amplified ribosomal DNA restriction analysis (ARDRA)

The ARDRA technique is basically based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. The genomic DNA extraction, 16S rDNA PCR amplification and purification of the PCR product was carried out as previously described [9]. For ARDRA analysis, 20 μl (50 ng) of 16S rDNA purified PCR products were digested by 1.5 U of three different restriction enzymes HaeIII, MspI, HinfI (New England Biolabs, UK) according to manufacturer’s instruction and incubated for 3 h at 37 °C. The resulting digested fragments together with 100 bp ladder (Merck Genei, India) were resolved by gel electrophoresis at 60 V on 2% agarose gels in 1 × TAE buffer containing 10 μg/ml of EtBr. The gel profile obtained were analyzed by considering, the character state “1” for clearly detected bands in the gel track and assigned “0” if it was absent or impossible to determine. The data matrix thus generated was calculated by Dice similarity coefficient. Each pairwise comparison was constructed from the similarity matrix by the unweighted pair group method with arithmetic mean (UPGMA) using DICE similarity coefficient and the TreePlot program in NTSYSpc 2.02e analysis package (Applied Biostatistics Inc., New York).