Dna extraction

Dubeux, Marcos J. M., Araujo-Vieira, Katyuscia, Mott, Tamí, Neves, Jessika Maria, Faivovich, Julián & do Nascimento, Filipe A. C., 2025, Tadpole morphology, advertisement call and natural history notes of Ololygon muriciensis (Anura: Hylidae: Scinaxini), Journal of Natural History 59 (25 - 28), pp. 1949-1977 : 1950-1951

DNA extraction and molecular identification of tadpoles

Whole cellular DNA was extracted from two adults (MHNUFAL 16171–72) and one tadpole (collected with lot MHNUFAL 16168) using the phenol/chloroform extraction method ( Sambrook and Russel 2001). A fragment of the ribosomal 16S rRNA mitochondrial gene was amplified using two sets of primers: 16SarL and 16SbrH ( Palumbi et al. 2002) and An16SF and An16SR (Lyra et al. 2016). Polymerase Chain Reaction (PCR) amplification was carried out in 25 µL reactions using 12.5 µL of Master Mix PCR Buffer with 0.4 mM of each dNTP and 3 mM of MgCl2, 8.4 µL of nuclease-free water, 0.5 µL of Taq DNA polymerase (5 U/µL), 0.8 µL of each primer (10pmol) plus 2 µL of DNA template (20–100 ng /µL). The PCR protocol consisted of an initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 94°C for 45s, annealing at 55°C (for the set of primers 1) and 50°C (for the set of primers 2) for 60s, and extending at 72°C (for the set of primers 1) and 60°C (for the set of primers 2) for 60s. The PCR-amplified products were purified using isopropanol and sent to be bidirectionally sequenced using the Sanger method in ACT Gene. Forward and reverse sequences were edited using Bioedit 7.2 ( Hall 1999), and consensus sequences were generated.

The identity of the tadpole was corroborated by comparing the fragment AR/ BR (~344 bp) of the gene 16S rRNA among the two adult topotypical specimens (GenBank accession numbers PP920100 and PP920101) and one tadpole (GenBank accession number PP920099) . Our comparison revealed that these specimens share 100% similarity (overlap = 344 bp, mismatch = 0).