Optimization and Comparative Analysis of Phenol-Chloroform vs Salting-Out DNA Extraction Methods from Human Blood and Saliva Tissues: Assessing Yield, Purity, and Suitability for Downstream Molecular Applications

Abstract

High quality DNA extraction is fundamental for molecular diagnostics, biomedical research, and forensic sciences. Among biological sources, blood provides high yields, whereas saliva offers a non-invasive alternative. Classical methods such as phenol-chloroform and salting-out remain widely used, but their comparative performance requires further evaluation. This comparative laboratory study analyzed 60 human samples (blood and saliva) using phenol-chloroform and salting-out extraction protocols. DNA yield and purity were assessed with Nanodrop spectrophotometry, and results were compared across sample types and methods. Phenol-chloroform yielded significantly higher DNA concentrations and superior purity than salting-out. From blood, the mean yield was ~308 ng/µL with A260/A280 ~1.89 using phenol-chloroform, compared with ~18 ng/µL (A260/A280 ~1.82) using salting-out. From saliva, phenol-chloroform produced ~64 ng/µL (A260/A280 ~1.87) versus ~38 ng/µL (A260/A280 ~1.75) with salting-out. Blood was the most reliable DNA source, but saliva provided adequate DNA quality for PCR-based and genotyping applications. Phenol-chloroform extraction remains the superior method for obtaining high-yield, high-purity DNA, particularly from blood. However, due to biosafety risks associated with organic solvents, the salting-out method offers a safer and low-cost alternative suitable for routine diagnostics and use in resource-limited settings.