An efficient and rapid protocol for plant nuclear DNA preparation suitable for next generation sequencing methods

Abstract

Premise of the study: In this study, we developed a nuclear DNA extraction protocol for Next Generation Sequencers (NGS). Methods and Results: We applied this extraction method to grapevines and coffee trees, which are known to contain many secondary metabolites. The nuclear DNA obtained was sequenced by the 454/GS-FLX method. We obtained excellent results, with less than 4% cytoplasmic DNA, in a similar way to a BAC (Bacterial Artificial Chromosome)-building protocol. We also compared our protocol with a classic DNA extraction using specific cytoplasmic DNA amplification. Results showed a lower cytoplasmic DNA contamination with the new protocol. Conclusions: The method presented here is fast and economical. The DNA obtained is of high quality, with a low level of cytoplasmic DNA contamination, and very efficient for the construction of sequencing libraries. © 2011 Botanical Society of America.