Optimized Protocol for DNA Extraction in Three Theobroma Species.

Abstract

DNA extraction is a crucial step in molecular biology research, particularly for genetic and genomic analyses. These studies require a high concentration of high-quality DNA, which is often a challenge for underexplored species or when the available plant material consists of aged tissue. To address these challenges, the cetyltrimethylammonium bromide (CTAB)-based DNA extraction method has been optimized to improve efficiency and yield. The process begins with an overnight incubation of plant tissue macerated with liquid nitrogen in a solution containing a high concentration of CTAB (4%). Subsequently, the mixture undergoes two washes with chloroform: isoamyl alcohol. The nucleic acids are then precipitated using isopropanol, followed by a wash with 70% ethanol to ensure purity. Finally, the purified DNA is resuspended in ultrapure water. This optimized procedure produces high-quality DNA suitable for various downstream applications, including PCR and sequencing, even from older leaves of the three Theobroma species: T. cacao, T. bicolor, and T. grandiflorum. Additionally, this protocol significantly enhances throughput and allows for the parallel processing of a substantially larger number of samples compared to conventional techniques. Key features • An efficient CTAB-based DNA extraction protocol provides high-quality nucleic acids from older leaves by increasing CTAB concentration and incubation time in lysis buffer. • Provides reliable yields in the three Theobroma species: T. cacao, T. bicolor, and T. grandiflorum. • A high-throughput workflow reduces processing time and increases daily sample capacity, supporting large-scale genomic investigations.