Comparative Study of SSCP Pattern of V3 and V4-V5 Region of 16S rRNA of Metagenomic DNA for the Identification Rumen Microbial Diversity

A solid understanding of rumen microbes is essential to strategise the manipulation of rumen for the improvement of animal production. Recent days, nucleic acid based methods are being adopted to enumerate the uncultured rumen microbiome. The present study describes the adaptability of PCR-SSCP based DNA finger printing profile in the study of rumen microbial diversity. All the hundred DNA samples isolated from the goat rumen liquor were subjected for the amplification of 16S rRNA gene using Bacterial universal primer. V3 region of 16SrRNA was amplified using first round PCR product as template. Sixty three samples which had showed positive for the amplification of V3 region were screened for SSCP and found eleven different SSCP patterns. The product of first round PCR of those fifteen samples which had produced eleven different SSCP patterns were once again subjected for the amplification of V4-V5 region and screened for SSCP. Comparison of SSCP pattern of V3 region and V4-V5 region revealed that the SSCP pattern of V3 region was distinct and variable with the samples. The sequence analysis of V3 and V4-V5 region and phylogenetic tree indicated that the DNA sequence of V3 region was found to cluster differently while two of five sequences of V4-V5 region clustered together. The difference observed in the phylogenetic tree of V3 region found to be in association with the SSCP profile of V3 region. In addition the microorganism identified based on sequence analysis of V3 region is in accordance with the reports reported earlier. It is concluded that V3 region of 16S rRNA was a better target for the DNA fingerprinting studies and PCR-SSCP of V3 region could be a cost effective DNA fingerprinting technique to study the microbial diversity of the rumen. Pattern and Region for the µ l reaction mixture l0 pmol primer, µ g of template DNA and µ l of 2X PCR master mix. The PCR amplification of bacterial 16S rRNA was performed by thermal cycler using following program: Initial denaturation of 10 min at 94 o C, followed by 30 cycles of denaturation at 94 o C for 60 s, annealing at 50 o C for 60 s and elongation at 72 o C for 60 s, with final extension at 72 o C for 5 min. The first round PCR product was used as template DNA for second round PCR targeting V3 region and V4-V5 region of 16S rRNA using respective set of primers. Amplification of V3 region was performed using touchdown method which consisted of an initial denaturation at 94 o C for 4 minutes, 15 cycles of denaturation at 94 o C for 1 minute, annealing at 65 o C for 1 minute and extension at 72 o C for 1 minute in which the annealing temperature was reduced by 0.5 o C/cycle from the preceding cycle and the 15 cycles of 94 o C for 1 minute, 55 o C for 1 minute and 72 o C for 1 minute followed by final elongation at 72 o C for 4 minutes. The PCR product size (193bp) was checked by 1% agarose gel electrophoresis. PCR amplification for V4-V5 region was started with initial denaturation at 94 ˚ C for 4 minutes. A total of 30 cycles, each including 60s at 94 o C, 60s at 50 o C, 60s at 72 o C was followed by final elongation 5minutes at 72 o C. The expected