PCR amplification and sequencing

For amplification of 16S rRNA region and control region of mitochondrial DNA, primers were designed as shown in Table 2. Same fragments from each sample was amplified in three DNA extracts to validate results. Annealing temperature was changed and the annealing time was extended. Primers were designed using Primer 3 plus and using mtDNA reference sequence of Struthio camelus. As DNA quantity was extremely low, Titanium Taq DNA Polymerase (Roche) was used, which is mostly used for High-Throughput genotyping using MassArray System (Agena Biosciences).

PCR reaction was set up in 5 μl total volume per reaction. The reaction mixture contained 2 μl of eluted DNA, reaction buffer supplied by the manufacturer (Agena Biosciences), dNTPs at 200 μM each, Taq polymerase (Roche) at 0.1 unit (0.02 μl at 5 U/μl, primers at 200 nM and MgCl₂ at the final concentration as mentioned in the protocol. PCR cycling conditions for Titanium Taq DNA polymerase (Invitrogen) included: initial heating to 95°C, followed by 45 cycles of 94°C for 20 sec, 56°C for 30 sec, and 72°C for 1 min, and a final step of 72°C for 3 min. After PCR, the products were checked on 3% agarose gel (Sigma Aldrich) using 50bp DNA ladders (NEB) and the expected product sizes were confirmed. PCR products were purified using Shrimp Alkaline Phosphatase (SAP) from Agena Biosciences as per the manufacturer protocol.