Sea cucumbers play important ecological and economic roles, making accurate species identification and genetic studies essential for their conservation and management. However, genomic DNA isolation in sea cucumbers is often challenged by the presence of ossicles and complex dermal structures. This study aimed to develop a modified method for high purity genomic DNA isolation by evaluating different tissue types and preparation techniques. A single specimen of Holothuria fuscocinerea from Karimunjawa was analyzed using anatomical and molecular approaches. Six extraction treatments were applied, including anterior tissue (P0), smooth muscle tissue (P1), dermal tissue treated with bleach (P2), homogenized muscle tissue (P3), gonad (P4), and body secretion (P5). DNA quality and concentration were assessed using Nanodrop and Quantus, followed by PCR amplification targeting the mitochondrial COI gene. Anatomical observations revealed the presence of buttons, rod, and tables ossicles, with button type being dominant. The results showed that dermal tissue treated with bleach as chemical methods for ossicle removal produced the highest DNA yield (4.76 ng μL-1 Nanodrop and 3.53 ng μL-1 Quantus) and relatively better purity compared to other tratments. PCR amplification of anterior tissue using the COIceF/ceR primer successfully generated clear DNA bands in all samples (100%), with a high identification success rate for H. fuscocinerea, indicating the suitability of the extracted DNA for molecular analysis. This study demonstrated that ossicle removal and appropriate tissue selection are critical factors in improving DNA extraction efficiency in sea cucumbers.
