Optimized Phenol-Chloroform-Isoamyl DNA Extraction Protocol for Single Fish Eggs

Reliable DNA extraction is essential for genetic research on marine species; however, obtaining sufficient DNA from single fish eggs remains challenging. Existing protocols often require optimization to achieve high PCR efficiency. The optimized phenol-chloroform-isoamyl extraction protocol presented in this paper improves DNA yield and quality from individual eggs of Atlantic bluefin tuna (Thunnus thynnus), bogue (Boops bops), saddled seabream (Oblada melanura), and painted comber (Serranus scriba) by modifying buffer volumes, incubation times, and washing steps, following prior micropuncturing of eggs on a glass slide. DNA quality is confirmed by spectrophotometry, PCR amplification of the mitochondrial COI gene, electrophoresis, and Sanger sequencing. This method provides a low-cost and effective approach for species identification from individual fish eggs. Key features • Effective and low-cost DNA extraction method from fish eggs. • Uses only standard reagents and equipment, allowing wide application in all laboratories. • High PCR efficiency.