"MINES" method for genomic DNA extraction from deep biosphere biofilms

Successful and efficient extraction of high quality, high molecular weight genomic DNA from the environmental samples is an essential primary step to understand the genetic, metabolic and evolutionary characteristics of the microbial communities. Deep mine biofilm samples that contain high amounts of mucoid exopolysaccharide often pose difficulties to obtaining refined community DNA. To circumvent this hindrance, we report our "MINES" method which we developed for optimal biofilm DNA recovery suitable for all types of high-resolution downstream applications. The method is also suitable for samples collected from landfill compost, kitchen digest (KD), and for Gram-positive Geobacillus sp. strain WSUCF1 and Gram-negative E. coli DH5α strains. In one form of the method, use of a gentle preprocessing technique to loosen the mucoid layer, combined with a multi-lytic polyzyme treatment to maximize yields from all cell types in the biofilm sample, yielded >1 μg of high molecular weight DNA (16-20 kb) per gram of the biofilm sample, with an A260/280 and A260/230 ratio of about 2. Furthermore, amplification of 16S rRNA genes as well as restriction digestion with BamHI and HindIII suggest that the newly developed method can minimize any inhibitory effects of contaminants. Results indicate that it is an appropriate methodology for the extraction of total genomic DNA for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging. IMPORTANCE: Our present knowledge of microorganisms and their enzymes from deep mine subsurfaces is based largely on laboratory studies of pure microbial cultures. These methods tend only to hit nearly 1% of the entire microbial community. In this regard, metagenomics, has emerged as a strategic approach to explore unculturable microbes through the sequencing and analysis of DNA extracted from the environmental samples. This research paper discusses our "MINES" method for genomic DNA extraction from deep biosphere biofilm samples.