Plant and metagenomic DNA extraction of mucilaginous seeds

• Published in: MethodsX 2014-01, Vol.1 (C), p.225-228
• Main Authors: Ramos, Simone N.M., Salazar, Marcela M., Pereira, Gonçalo A.G., Efraim, Priscilla
• Format: Article
• Language: English
• Subjects: Cupuassu; DNA extraction; Metagenomic; PCR; Plant; Seeds
• ISSN: 2215-0161; 2215-0161
• DOI: 10.1016/j.mex.2014.09.005

Stages of sample treatment: (a) seeds in fermentation boxes; (b) frozen cupuassu beans; (c) maceration of beans with liquid nitrogen; (d) fat extraction under stirring with petroleum ether; and (e) initial step of DNA extraction process. The pulp surrounding the seeds of some fruits is rich in mucilage, carbohydrates, etc. Some seeds are rich in proteins and polyphenols. Fruit seeds, like cacao (Theobroma cacao) and cupuassu (Theobroma grandiflorum), are subjected to fermentation to develop flavor. During fermentation, ethanol is produced [2–6]. All of these compounds are considered as interfering substances that hinder the DNA extraction [4–8]. Protocols commonly used in the DNA extraction in samples of plant origin were used, but without success. Thus, a protocol for DNA samples under different conditions that can be used for similar samples was developed and applied with success. The protocol initially described for RNA samples by Zeng et al. [9] and with changes proposed by Provost et al. [5] was adapted for extracting DNA samples from those described. However, several modifications have been proposed:•Samples were initially washed with petroleum ether for fat phase removal.•RNAse was added to the extraction buffer, while spermidin was removed.•Additional steps of extraction with 5M NaCl, saturated NaCl and CTAB (10%) were included and precipitation was carried out with isopropanol, followed by washing with ethanol.