Optimizing Cetyltrimethylammonium Bromide (CTAB) DNA Extraction Protocol of Dipterocarpaceae Wood Tissues to Support Population Origin Verification in Global Timber Trade

A DNA database is crucial for timber’s authentication in transparent and sustainable global trade. DNA extraction and applications are critical components of the database development. Wood tissues often contain high levels of metabolites that pose significant challenges for DNA extraction and downstream polymerase chain reaction (PCR) applications. This study used wood and leaf tissues from four populations of Rubroshorea curtisii (Dipterocarpaceae) for modified cetyltrimethylammonium bromide (CTAB) DNA extraction and PCR amplification using trnL–trnF chloroplast DNA primer. The protocol was developed and optimized by incorporating bead-beating methods and enhanced buffer composition (polyvinylpyrrolidone, β-mercaptoethanol) to improve cell lysis and remove inhibitors. The protocol efficiency was evaluated by visualizing the DNA band by electrophoresis, assessing the quality of the DNA sequence chromatogram, and comparing the produced result with the sequence in GenBank (NCBI). This study found visible and consistent DNA bands in leaf tissues, but inconsistent in wood. PCR amplification of the chloroplast trnL–trnF region produced clear and single amplicons from both tissues, showing that the extracted DNA was of sufficient quality for molecular analyses. Sequences produced a clear and readable electropherogram, which subsequent NCBI BLASTn validation confirmed high quality of the result, with 100% query coverage and strong similarity to Dipterocarpaceae reference sequences. In addition to amplification success, sequence alignment showed polymorphic regions, including both single-nucleotide polymorphisms and indels, which differentiated two chloroplast haplotypes corresponding to the island of origin. These results suggest the modified CTAB protocol enables successful PCR amplification and further genetic analyses specifically to verify timber origin in global trade.