Extraction of High Molecular Weight DNA from Microbial Mats

Summary

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Abstract

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA^2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes^4,5,6.

The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A_260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes⁷ suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

Protocol

1. Microbial Cell Extraction:

1. Homogenize microbial mats with a sterile grinding pestle by mixing thoroughly. Place approximately all 30 g (wet weight) of homogenized mat material into sterile container of Waring blender, add about 100 mL of 1 M NaCl (or a concentration specific to sample in use), and blend three times at medium speed for 1 min with intermittent cooling in a -20°C freezer for 1 min. Transfer the slurry into a 250 mL centrifuge bottle and fill the remaining empty volume with 1 M NaCl. Prepare NaCl solution in autoclaved DI water and filter sterilize it.
2. Further dislodge the microbial cells from the matrix by shaking (150 rpm) using ....

Discussion

Given that total cell removal from complex and highly diverse microbial mat samples is not practical, the primary concern is how well the extracted cells represent the overall microbial mat community. In a previous study, PCR-DGGE analysis of microbial 16S rRNA genes showed that the five cell removal steps used in this protocol extracts cells that are representative of the overall microbial mat community⁷. The actual number of cell extraction steps required to provide a cell pellet that is representative of th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
β-mercapt–thanol	Sigma-Aldrich	M3148
Polyethylene glycol 8000	Promega Corp.	V3011	20% in 1.2 M NaCl
Potassium acetate	Fisher Scientific		Fisher Scientific
Quant-iT dsDNA Assay kit	Invitrogen	Q33130
RNase	Epicentre Biotechnologies	MRNA092
Sodium Chloride	VWR	BDH8014	Appropriate conc.
Sodium Dodecyl Sulfate	Fisher Scientific	03-500-509	10% in water
sodium hexametaphosphate	EMD Millipore	SX0583-3	2% in water
TBE	Fisher Scientific	BP1333-1
CHEF Mapper XA System	Bio-Rad	170-3670
NanoDrop 1000 spectrophotometer	Thermo Fisher Scientific, Inc.	ND-1000
Vortexer	Scientific Industries Inc.
Ultraviolet Crosslinker	UVP Inc.
Waring blender	Waring Laboratory	LB10S