Rapid, direct extraction of DNA from soils for PCR analysis using polyvinylpolypyrrolidone spin columns

Berthelet, Marc; Whyte, Lyle G.; Greer, Charles W.

doi:10.1111/j.1574-6968.1996.tb08128.x

Cited by 159 publications

(102 citation statements)

References 20 publications

(1 reference statement)

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“…Direct extraction of DNA introduces less bias than methods which are based on cell extraction prior to lysis (von Wintzingerode et al 1997). To extract DNA, the microorganisms in the soil can be lysed mechanically by bead-beating (Moré et al 1994) (Berthelet et al 1996) (Purdy et al 1996), sonication (Degrange and Bardin 1995), freezethawing (Lee et al 1996), grinding in liquid nitrogen (Johnston and Aust 1994) (Volossiouk et al 1995) or enzymatically, using enzymes such as proteinase K (Wikstrom et al 1996;Zhou et al 1996), lysozyme (Porteous et al 1994) or pronase. A combination of these treatments may also be applied (Tsai and Olson 1991) (Tebbe and Vahjen 1993) (Picard et al 1992).…”

Section: Introductionmentioning

confidence: 99%

DNA isolation from soil samples for cloning in different hosts

Kauffmann

Schmitt

Schmid

2004

Applied Microbiology and Biotechnology

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Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas (Entcheva et al. 2001), Arthrobacter and Rhodococcus (Borneman et al. 1996) and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA from them extracted. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.3

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Section: Introductionmentioning

confidence: 99%

DNA isolation from soil samples for cloning in different hosts

Kauffmann

Schmitt

Schmid

2004

Applied Microbiology and Biotechnology

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“…After precipitating, washing and drying, the DNA was resuspended in sterilized distilled water. The crude extract of DNA was treated with polyvinylpolypyrrolidone (PVPP) and sephacryl S-400 spin columns as described by Berthelet et al (1996) to remove the PCR inhibitors such as humic acids. Untreated and treated DNA (5 ml) were loaded onto a 0.7% agarose gel with SYBR safe TM for electrophoresis at 60 V for 2 h with HindIII-digested k DNA as the molecular weight marker (Invitrogen, Carlsbad, CA).…”

Section: Description Of the Sites And Samplingmentioning

confidence: 99%

Phylogenetic analysis of bacterial communities in the shrimp and sea cucumber aquaculture environment in northern China by culturing and PCR–DGGE

Zhang

Juck

et al. 2010

Aquacult Int

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In this study, we investigated the bacterial communities in the shrimp and sea cucumber culture environment, including shrimp ponds (SP), sea cucumber ponds (SCP), mixed-culture ponds (MCP) and the effluent channel (EC) in Qingdao, China. Bacteria cultivation showed that the counts of heterotrophic, nitrate-reducing and sulfate-reducing bacteria in the sediment of SP were higher than that in the sediment of SCP and MCP, varying between 8.7 9 10 4 and 1.86 9 10 6 ,2.1 9 10 4 and 1.1 9 10 5 ,and9.3 9 10 1 and 1.1 9 10 4 CFU g -1 , respectively. In contrast, the counts of ammonium-oxidizing and nitrifying bacteria in the sediment of SP was lower than that in the sediment of SCP and MCP. Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA gene and dendrogram analyses showed that bacterial diversity in the mixed-culture environment was higher than that in the monocultures. The similarity of bacterial community between EC and SCP or MCP was higher than that between EC and SP. These results indicated that sea cucumber culture played a significant role in influencing the environmental bacterial communities that were composed mainly of Flavobacteriaceae (64.3%), Bacteriodetes (21%) and delta proteobacteria (14.7%), including the genera of Croceimarina, Lutibacter, Psychroserpens and so on. The results explained the benefit of sea cucumber culture in shrimp ponds at the level of microbial ecology.

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“…Canadian scientists have contributed to the development and implementation of both nucleic acid-based and chemical biomarker-based methods now widely used for assessing soil microbial biodiversity without the need for isolation and cultivation. Some examples include methods for soil DNA extraction (Berthelet et al 1996), achieving taxa-specific (Watson et al 1995) and quantitative (Leung et al 1997) PCR, resolving mixed PCR products by denaturant gradient gel electrophesis (Vallaeys et al 1997), detecting specific signature DNA sequences in soil DNA by reverse genome probing (Telang et al 1994), and sequencing genes cloned directly from soil DNA (Seow et al 1997). have explored the use of signature phospholipids, extracted directly from soil and identified by chemical methods, for assessing the global biodiversity of soil microbial communities.…”

Section: Canadian Research On Agriculturalmentioning

confidence: 99%

Bacteria in agricultural soils: Diversity, role and future perspectives

Topp¹

2003

Can. J. Soil. Sci.

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Bacteria in soil are very diverse, very numerous, and functionally important, and have historically been an important object of research by Canadian microbiologists. Only a small fraction of bacteria in soils are amenable to culturing in the laboratory, limiting the ability to study these organisms. Canadian scientists have contributed to the development and implementation of both nucleic acidbased and chemical biomarker-based methods now widely used for assessing soil microbial biodiversity without the need for isolation and cultivation. Pesticide degradation, and the cycling of nitrogen in soils are used here to illustrate the significance of bacterial biodiversity to soil functions relevant to human and environmental health, and crop production . There remains much to be discovered about the genetic and functional biodiversity of soil bacteria, and much to be gained from this knowledge. A number of recommendations are made for future research in soil bacteriology. Key words: Soil quality, bacteria, microbial biodiversity, pesticide biodegradation, nitrogen cycling.

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Rapid, direct extraction of DNA from soils for PCR analysis using polyvinylpolypyrrolidone spin columns

Cited by 159 publications

References 20 publications

DNA isolation from soil samples for cloning in different hosts

DNA isolation from soil samples for cloning in different hosts

Phylogenetic analysis of bacterial communities in the shrimp and sea cucumber aquaculture environment in northern China by culturing and PCR–DGGE

Bacteria in agricultural soils: Diversity, role and future perspectives

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