Rapid method for direct extraction of DNA from soil and sediments

Tsai, Yu-Li; Olson, Betty H.

doi:10.1128/aem.57.4.1070-1074.1991

Cited by 542 publications

(240 citation statements)

References 20 publications

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“…A volume of 8 ml of lysis solution I (0.15 M NaCl, 0.1 M EDTA (pH 8.0), 10 mg lysozyme ml 3I ) were added to the washed pellet and incubated at 37³C with occasional mixing for 90 min, and then 8 ml of lysis solution II (0.1 M NaCl, 0.5 M Tris-HCl (pH 8.0), 10% sodium dodecyl sulfate) were added. The sample was frozen at 380³C for 20 min and thawed in a 65³C water bath for 20 min, and this freezing and thawing cycle was repeated three times [24]. The lysate was centrifuged at 7500Ug at room temperature for 10 min.…”

Section: Genomic Dna Extraction and Puri¢cationmentioning

confidence: 99%

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Thermophilic bacterial communities in hot composts as revealed by most probable number counts and molecular (16S rDNA) methods

Blanc

1999

FEMS Microbiology Ecology

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Thermogenic composts are known to host a variety of thermophilic micro-organisms that were recently investigated by cultural means and identified as Thermus thermophilus, Bacillus spp., and Hydrogenobacter spp. In this paper, we present a classical, cultural enumeration of thermophilic populations on the one hand, and a molecular investigation of the bacterial community by restriction enzyme analyses of a clone library of bacterial 16S rRNA genes on the other hand. Bacterial diversity, revealed by the clone analyses of four samples, was shown to undergo a dramatic change between the young (13^18-day) and the old (39^41-day) samples, possibly linked to the general decrease in temperature and the physicochemical evolution of organic matter during the composting process. Among the 200 clones investigated, 69 clones could be identified as Thermus thermophilus and thermophilic Bacillus spp. These results proved both taxa to be among the dominant bacterial populations at the highest temperatures reached by thermogenic composts. z

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Section: Genomic Dna Extraction and Puri¢cationmentioning

confidence: 99%

“…To obtain ampli¢able DNA, it proved necessary to repeat the CTAB step to remove contaminating substances, such as humic acids (Andrew Ogram, personal communication; [24]). Consequently, 750…”

Section: Genomic Dna Extraction and Puri¢cationmentioning

confidence: 99%

Thermophilic bacterial communities in hot composts as revealed by most probable number counts and molecular (16S rDNA) methods

Blanc

1999

FEMS Microbiology Ecology

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“…These approaches are time-consuming and inefficient in removing contaminants, such as humic substances, from soil [16]. Different purification protocols, such as treatment with polyvinyl polypyrrolidone (PVPP) [15,16], 22], cation-exchange resins [29], Sephadex [20,27,30], or glass milk [26,27], have been used for extracting DNA from agricultural soils or sediments with varying degrees of success.…”

Section: Microorganismsmentioning

confidence: 99%

“…UG2Lr is a genetically modified strain that produces rhamnolipid biosurfactants [27]. The strain contains a hxAB / LacZY marker inserted into the chromosome by Tn7 transposition 127,331 and is also resistant to rifampicin (50 ,ug ml-') and ampicillin (50 pg ml-').…”

Section: Bacterial Strain P Aeruginosamentioning

confidence: 99%

Survival of Îº-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Leung

Cassidy

Holmes

et al. 1995

FEMS Microbiology Ecology

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A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty‐two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g−1 of dry soil based on the polymerase chain reaction (PCR). Survival of κ‐carrageenan‐encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence‐based nonselective plating and PCR‐amplification of a tnsA fragment. After freeze‐thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 109 cfu g−1 of dry soil to below the detection threshold of both selective (14 cfu g−1 of dry soil) and nonselective (1 × 103 cfu g−1 of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 106 to 2.9 × 108 cfu g−1 of dry soil after a 3‐week incubation at 22°C and declined to 6.3 × 106 cfu g−1 of dry soil after the freeze‐thaw treatment.

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“…Samples were collected and processed as reported by Peter et al (2000) and Kocherginskaya et al (2001), and stored at -20∞C until DNA extraction was performed. Extraction of total genomic DNA from rumen samples was performed using the method of Tsai and Olson (1991) with addition of 50 mg ml -1 of proteinase K to samples following the freeze-thaw cycles.…”

Section: Rumen Sampling and Total Dna Extractionmentioning

confidence: 99%

Suppressive subtractive hybridization as a tool for identifying genetic diversity in an environmental metagenome: the rumen as a model

Galbraith

Antonopoulos

White

2004

Environmental Microbiology

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Molecular techniques previously used for genome comparisons of closely related bacterial species could prove extremely valuable for comparisons of complex microbial communities, or metagenomes. Our study aimed to determine the breadth and value of suppressive subtractive hybridization (SSH) in a pilot-scale analysis of metagenomic DNA from communities of microorganisms in the rumen. Suppressive subtractive hybridization was performed using total genomic DNA isolated from rumen fluid samples of two hay-fed steers, arbitrarily designated as tester or driver. Ninety-six subtraction DNA fragments from the tester metagenome were amplified, cloned and the DNA sequences were determined. Verification of the isolation of DNA fragments unique to the tester metagenome was accomplished through dot blot and Southern blot hybridizations. Tester-specific SSH fragments were found in 95 of 96 randomly selected clones. DNA sequences of subtraction fragments were analysed by computer assisted DNA and amino acid comparisons. Putative translations of 26 (32.1%) subtractive hybridization fragments exhibited significant similarity to Bacterial proteins, whereas 15 (18.5%) distinctive subtracted fragments had significant similarity to proteins from Archaea. The remainder of the subtractive hybridization fragments displayed no similarity to GenBank sequences. This metagenomic approach has exposed an unexpectedly large difference in Archaeal community structure between the rumen microbial populations of two steers fed identical diets and housed together. 16S rRNA dot blot hybridizations revealed similar proportions of Bacteria and Archaea in both rumen samples and suggest that the differences uncovered by SSH are the result of varying community structural composition. Our study demonstrates a novel approach to comparative analyses of environmental microbial communities through the use of SSH.

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Rapid method for direct extraction of DNA from soil and sediments

Cited by 542 publications

References 20 publications

Thermophilic bacterial communities in hot composts as revealed by most probable number counts and molecular (16S rDNA) methods

Thermophilic bacterial communities in hot composts as revealed by most probable number counts and molecular (16S rDNA) methods

Survival of Îº-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Suppressive subtractive hybridization as a tool for identifying genetic diversity in an environmental metagenome: the rumen as a model

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