SAND-MEDIATED DNA EXTRACTION FROM ORCHIDS FOR GENOMIC APPLICATIONS

Abstract

DNA extraction is an essential routine procedure for downstream applications in genetics and molecular biology. Since DNA was extracted for the first time, countless protocols have been developed yielding excellent results; however, the diversity of plant families and the distribution in remote areas represent a challenge for these protocols. Orchidaceae, one of the most species-rich plant families in the world, is a group with abundant polyphenols and polysaccharides in its tissues. In addition, sample collection in remote areas represents a challenge, consequently, conventional protocols can be inappropriate for obtaining DNA. In this way, CTAB-based gel preservation has emerged as an excellent alternative to easily collect, store and transport samples, moreover, alternatives such as sterile sand and DNA stabilization solutions during grinding allow a suitable DNA extraction. Here, we combined saturated NaCI-CTAB gels, sterile sand, sucrose-based DNA stabilization buffer, and conventional CTAB solution for obtaining DNA from fresh and stored samples for up to 32 days at room temperature, and described, from sample collection to spectrophotometer-obtained quantification, a non-organic method for obtaining DNA from orchids species for genomic applications. This protocol allows not only the extraction of suitable quantities of high molecular weight DNA of adequate purity and integrity from different orchid species without the use of expensive equipment and cumbersome reagents such as liquid nitrogen and toxic phenols, and/or time-consuming procedures, but also, the use of this DNA in downstream applications such as PCR-based genotyping, restriction enzyme analysis, and most genomic applications.