DNA Barcoding for Vertebrate Identification

For the identification of birds, mammals, reptiles and fish, a 303 bp fragment of mtDNA cox1 gene was PCR-amplified with primers AVS2F and AVS3R as described in Tull et al. (2022 (link)). PCR reactions were carried out in a total volume of 20 μL with 1× Phusion HF Buffer (Thermo Fisher Scientific), 0.2 mm dNTP, 0.25 μm of each primer and 0.4 U Phusion Hot Start II DNA Polymerase and 2 μL of purified DNA. The PCR mixture was initially denatured at 98°C for 30 s, followed by 10 touchdown cycles for 10 s at 98°C, 20 s at 60°C (reducing the temperature 1°C per cycle) and 30 s at 72°C, followed by 30 cycles of 10 s at 98°C, 20 s at 50°C and 30 s at 72°C. In case the PCR was negative due to highly degraded DNA, we performed a second analysis by PCR-amplifying a shorter, 183 bp fragment of mtDNA 12S rRNA gene, using primers Ave12F and Ave12R, described in Oja et al. (2017 (link)). PCR products were checked using 2% 1×TAE gel-electrophoresis and visualized under UV radiation using ethidium bromide.
PCR products were purified, sequenced and nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to identify various taxa, such as reptiles, fish and birds.