Comparison of DNA extraction protocols in Calliandra molinae Standl. for future molecular analyses

Abstract

Calliandra molinae is a shrub or small tree in the Fabaceae family, distributed in Mesoamerica and classified as "endangered" by the IUCN Red List due to habitat fragmentation. Its conservation requires molecular studies that depend on high-quality DNA, but DNA extraction in plants is challenging due to secondary metabolites that affect its purity and amplification.

This study compared three DNA extraction protocols (Collins, CTAB, and the Promega kit) to evaluate their efficiency in terms of concentration, purity, and PCR amplification, aiming to identify the most suitable method for future genetic analyses in C. molinae. Results showed that the Collins protocol, especially with samples preserved in silica gel, yielded the best DNA concentrations (86.33 ng/μL ± 50.61) and the highest purity values. The commercial kit produced the highest concentration (149.83 ng/μL ± 132.49), but with lower purity, while the CTAB method resulted in the lowest concentrations, likely due to interference from polysaccharides and secondary metabolites.

Overall, DNA purity was below optimal values for all protocols, suggesting contamination with proteins, phenols, and other compounds. In the ITS amplification test, only one of the eight processed samples yielded a positive result (12.5% success), corresponding to an extraction using the Collins protocol and silica gel preservation. None of the other methods produced successful amplifications.

The findings suggest that the presence of contaminants limits PCR efficiency and that optimization of DNA purification is necessary to improve performance in future studies. In conclusion, although the Collins protocol combined with silica gel showed better results, the low amplification rate highlights the need to further improve and standardize methods to obtain DNA with greater purity and reproducibility in C. molinae and related species.