An improved method for purifying DNA from soil for polymerase chain reaction amplification and molecular ecology applications

Porteous, Lorna; Seidler, Ramon J.; Watrud, Lidia S.

doi:10.1046/j.1365-294x.1997.00241.x

Cited by 126 publications

(69 citation statements)

References 16 publications

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“…Other methods, which use mortar mill grinding [64], grinding under liquid nitrogen [73,77], ultrasonication [48,51] and microwave thermal shock [47] have also been reported. Frostegard et al [17] observed that drying soil before grinding greatly improved the lysis efficiency.…”

Section: Cell Lysismentioning

confidence: 99%

Extraction of DNA from soil

Robe¹,

Nalin²,

Capellano³

et al. 2003

European Journal of Soil Biology

254

164

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Section: Cell Lysismentioning

confidence: 99%

Extraction of DNA from soil

Robe¹,

Nalin²,

Capellano³

et al. 2003

European Journal of Soil Biology

254

164

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“…Other authors homogenized the soil samples in an extraction buffer containing PVPP and Chelex 100 resin, extracted the DNA with the same buffer supplemented with SDS, proteinase K and 10% Sarkosyl, and purified it with a mixture of phenol/clorophorm/isoamyl alcohol (25:24:1) [54]. Cell lysis was also achieved using a long treatment at 68°C in the presence of SDS and guanidine isothiocyanate followed by precipitation with polyethylene glycol (PEG-8000) and purification with CTAB, chloroform, and ammonium acetate [30,87].…”

Section: Initial Attempts For Dna Isolationmentioning

confidence: 99%

Determination of Denitrification Genes Abundance in Environmental Samples

Correa‐Galeote¹,

Tortosa²,

Bedmar³

2013

Metagenomics

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Diversity of microorganisms involved in the biogeochemical N cycle is of fundamental interest in microbial ecology. Denitrification is a key step in the cycle by which nitrate is reduced to dinitrogen gas via the soluble nitrite and the gaseous compounds nitric oxide and nitrous oxide. The process is carried out by the sequential activity of the nitrate, nitrite, nitric oxide, and nitrous oxide reductase enzyme, respectively. The fluorescence-based quantitative real-time polymerase chain reaction (qPCR) is widely used for quantification of nucleic acids in samples obtained from numerous, diverse sources. Here, we provide a well-proven methodology for isolation of DNA from environmental samples and describe relevant experimental conditions for utilization of qPCR to assay the 16S rRNA and nar/nap, nirK/nirS, c-nor/qnor, and nos denitrification genes that encode synthesis of denitrifying enzymes. The ISO 11063 standard method and MIQUE guidelines are considered with the aim to increase experimental transparency.

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“…The most popular detergent treatment includes SDS at 1% and salt concentrations of 1 M or more, often coupled with heating and shaking (Steffan et al, 1988;Bruce et al, 1992;Herrick et al, 1993;Smalla et al, 1993;Holben, 1994;Lovell and Piceno, 1994;Moré et al, 1994;Volossiouk et al, 1995;Porteous et al, 1997;Cullen and Hirsch, 1998;Edgcomb et al, 1999). A hot-SDS lysis method was first presented by Selenska and Klingmüller (1991).…”

Section: Cell Breakagementioning

confidence: 99%

“…Adding detergents and salts can help to alleviate this problem, although SDS can inhibit PCR if not removed in subsequent steps (Weyant et al, 1990). Some protocols, particularly commercially available kits, tend to use strong chaotropic agents like guanidinium salts (Tsai and Olson, 1990;Porteous et al, 1997). Degradatory enzyme digestion steps are often included in breakage regimes.…”

Section: Cell Breakagementioning

confidence: 99%

“…The soil homogenates can be centrifuged to remove soil debris, but often the extractions proceed directly with the homogenate. Many protocols facilitate breakage and the subsequent deproteination step by adding CTAB and increasing the salt concentration (Zhou et al, 1996;Porteous et al, 1997;Ranjard et al 1998;Edgcomb et al, 1999). This may also help remove humic materials.…”

Section: Nucleic Acid Extractionmentioning

confidence: 99%

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Direct Extraction of DNA from Soils for Studies in Microbial Ecology

Schneegurt

Dore²,

Kulpa³

2003

Current Issues in Molecular Biology

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Molecular analyses for the study of soil microbial communities often depend on the extraction of DNA directly from soils. These extractions are by no means trivial, being complicated by humic substances that are inhibitory to PCR and restriction enzymes or being too highly colored for blot hybridization protocols. Many different published protocols exist, but none have been found to be suitable enough to be generally accepted as a standard. Most direct extraction protocols start with relatively harsh cell breakage steps such as bead-beating and freeze-thaw cycles, followed by the addition of detergents and high salt buffers and/ or enzymic digestion with lysozyme and proteases. After typical organic extraction and alcohol precipitation, further purification is usually needed to remove inhibitory substances from the extract. The purification steps include size-exclusion chromatography, ion-exchange chromatography, silica gel spin columns, and cesium chloride gradients, among others. A direct DNA extraction protocol is described that has been shown to be effective in a wide variety of soil types. This protocol is experimentally compared to several published protocols.

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An improved method for purifying DNA from soil for polymerase chain reaction amplification and molecular ecology applications

Cited by 126 publications

References 16 publications

Extraction of DNA from soil

Extraction of DNA from soil

Determination of Denitrification Genes Abundance in Environmental Samples

Direct Extraction of DNA from Soils for Studies in Microbial Ecology

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