DNA extraction and PCR optimization for DNA Barcode analysis of commercially grown coffee varieties of Nepal

Abstract

The isolation of high quality Genomic DNA is an essential criterion for further Molecular analysis. Coffea genus is well known for its high amount of polyphenols, polysaccharides and other secondary metabolites that degrades the quality of the DNA isolation needed for further downstreaming processes. The present work was carried out with the goal of obtaining simple and efficient DNA isolation protocol generating high quality amplification for Barcoding. The protocol involves modifying the CTAB extraction, incorporating use of polyvinylpyrrolidone and β-mercaptoethanol yielding quality DNA with ratio (A260/280) between 1.8-2.0 indicating low contamination. The PCR conditions were optimized for high amplification on the basis of optimal concentration of MgCl₂ (3mM), primer (0.5 µM), Taqpolymerase (0.2 U), 50-60 ng of DNA template and cycle conditions as: initial denaturation of 94°C for 4min followed by 35 cycles of denaturation at 94°C for 50 sec, annealing at 54°C for 50 sec and extension at 72°C for 80 sec, followed by final extension at 72°C for 7 min. The optimal conditions produced high amplified reproducible data. Thus, the optimized method proposed enabled a simple and fastDNA extraction and PCR amplification for Coffeegenus, may serve as efficient tool for further molecular analysis.