Recovery of DNA from soils and sediments

Experiments were performed to evaluate the effectiveness of two different methodological approaches for recovering DNA from soil and sediment bacterial communities: cell extraction followed by lysis and DNA recovery (cell extraction method) versus direct cell lysis and alkaline extraction to recover DNA (direct lysis method). Efficiency of DNA recovery by each method was determined by spectrophotometric absorbance and using a tritiated thymidine tracer. With both procedures, the use of polyvinylpolypyrrolidone was important for the removal of humic compounds to improve the purity of the recovered DNA; without extensive purification, various restriction enzymes failed to cut added target DNA. Milligram quantities of high-purity DNA were recovered from 100-g samples of both soils and sediments by the direct lysis method, which was a greater than 1-order-of-magnitude-higher yield than by the cell extraction method. The ratio of labeled thymidine to total DNA, however, was higher in the DNA recovered by the cell extraction method. than by the direct lysis method, suggesting that the DNA recovered by the cell extraction method came primarily from active bacterial cells, whereas that recovered by the direct lysis method may have contained DNA from other sources.     

Recovery of crenarchaeotal ribosomal DNA sequences from freshwater-lake sediments.

We report several novel environmental sequences of archaea from the kingdom Crenarchaeota, recovered from anaerobic freshwater-lake sediments in Michigan. A nested PCR approach with Archaea- and Crenar-chaeota-specific primers was used to amplify partial Small-subunit ribosomal DNAs. Phylogenetic analysis of seven sequences shows that these DNAs represent a monophyletic lineage diverging prior to all recently identified crenarchaeotal phylotypes isolated from temperate environments. Including our lineage, all uncultured crenarchaeotal sequences recovered from moderate or cold environments form a distinct, monophyletic group separate from the "genuine" thermophilic crenarchaeota. Our finding extends the emerging picture that crenarchaeota, thought until recently to be solely extreme thermophiles, have radiated into an unexpectedly large variety of ecologically important, temperate environments.     

DNA recovery from soils of diverse composition.

A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.     

Comparison of methods of DNA extraction from stream sediments.

In Upper Three Runs Creek (Aiken, S.C.) and many other environments, less than 1% of bacteria visible microscopically can be cultured. Exploitation of molecular biology techniques has led to development of new methods, such as extraction of nucleic acids from soils or sediments, to study the dominant, nonculturable bacteria. The purpose of this study was to compare three published methods of DNA extraction that fall into two general categories: those in which cells are lysed in sediments (the Ogram and Tsai and methods [A. Ogram, G. S. Sayler, and T. Barkay, J. Microbiol. Methods 7:57-66, 1987; Y. L. Tsai and B. H. Olson, Appl. Environ. Microbiol. 57:1070-1074, 1991]) and those in which cells are removed from sediments prior to lysis (the Jacobsen method [C. S. Jacobsen and O. S. Rasmussen; Appl. Environ. Microbiol. 58:2458-2462, 1992]). DNA yield varied with extraction method; the Ogram method had a significantly higher yield than the other methods. However, DNA extracted via the Ogram method was badly sheared and contained a smaller proportion of eubacterial DNA. The Tsai method was less time consuming than the other methods, but DNA samples were of lower purity. If DNA purity is of paramount concern (as would be the case if PCR was to be performed) and quantity is not important, the Jacobsen method is recommended because of the low concentration of contaminants. If DNA is to be used directly in DNA-DNA hybridizations, the Ogram method is recommended since it gives maximal yields.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of diverse halobenzoate-degrading denitrifying bacteria from soils and sediments

Denitrifying bacteria capable of degrading halobenzoates were isolated from various geographical and ecological sites. The strains were isolated after initial enrichment on one of the monofluoro-, monochloro-, or monobromo-benzoate isomers with nitrate as an electron acceptor, yielding a total of 33 strains isolated from the different halobenzoate-utilizing enrichment cultures. Each isolate could grow on the selected halobenzoate with nitrate as the terminal electron acceptor. The isolates obtained on 2-fluorobenzoate could use 2-fluorobenzoate under both aerobic and denitrifying conditions, but did not degrade other halobenzoates. In contrast, the 4-fluorobenzoate isolates degraded 4-fluorobenzoate under denitrifying conditions only, but utilized 2-fluorobenzoate under both aerobic and denitrifying conditions. The strains isolated on either 3-chlorobenzoate or 3-bromobenzoate could use 3-chlorobenzoate, 3-bromobenzoate, and 2- and 4-fluorobenzoates under denitrifying conditions. The isolates were identified and classified on the basis of 16S rRNA gene sequence analysis and their cellular fatty acid profiles. They were placed in nine genera belonging to either the alpha-, beta-, or gamma-branch of the Proteobacteria, namely, Acidovorax, Azoarcus, Bradyrhizobium, Ochrobactrum, Paracoccus, Pseudomonas, Mesorhizobium, Ensifer, and Thauera. These results indicate that the ability to utilize different halobenzoates under denitrifying conditions is ubiquitously distributed in the Proteobacteria and that these bacteria are widely distributed in soils and sediments.     

Characterization of fluoranthene- and pyrene-degrading bacteria isolated from PAH-contaminated soils and sediments

Sixteen environmental samples, from the United States, Germany and Norway, with histories of previous exposure to either creosote, diesel fuel or coal tar materials, were screened for bacteria which could degrade high molecular weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A modified version of the spray plate technique was used for the isolations. Using fluoranthene (FLA) and pyrene (PYR) as model HMW PAHs, we isolated 28 strains on FLA and 21 strains on PYR. FLA degraders were defined as able to grow on FLA but not PYR. PYR degraders grew on both PAHs. All PYR degraders were found to be Gram-positive and all FLA degraders were Gram-negative. GC-FAME analysis showed that many of the PYR degraders were Mycobacterium spp and many of the FLA degraders were Sphingomonas spp. Comparison of the metabolic characteristics of the strains using the spray plate technique and direct growth studies revealed that more than half of the FLA degraders (59%) were able to cometabolize PYR (ie, they produced clearing zones or colored metabolites on spray plates but did not grow on the PAH) and the ability of many of these strains to cometabolize fluorene, anthracene, benzo[b]fluorene, benzo[a]anthracene and benzo[a]pyrene was significantly affected by pre-exposure to phenanthrene. Studies on the metabolic products produced from PYR cometabolism by strain EPA 505 suggested the possibility of attack at two different sites on the PYR molecule. However, the inability to derive degradable carbon from initial opening of one of the PYR rings probably accounted for the lack of growth on this PAH by the FLA-degrading strains. The PYR degraders on the other hand, were less able to cometabolize HMW PAHs, even following pre-exposure to PHE. Characterization of the FLA degradation pathway for several of the Sphingomonas isolates indicated oxidation and ring opening through to acenaphthenone as the principle metabolite. Strain CO6, however, also oxidized FLA through fluorenone, suggesting a dual attack on the FLA molecule, similar to that observed by others in Mycobacterium spp. Journal of Industrial Microbiology & Biotechnology (2000) 24, 100–112. Received 01 May 1999/ Accepted in revised form 01 November 1999     

Rapid method for direct extraction of DNA from soil and sediments.

A rapid method for the direct extraction of DNA from soil and sediments was developed. The indigenous microorganisms in the soil and sediments were lysed by using lysozyme and a freeze-thaw procedure. The lysate was extracted with sodium dodecyl sulfate and phenol-chloroform. In addition to a high recovery efficiency (greater than 90%), the yields of DNA were high (38 and 12 micrograms/g [wet weight] from sediments and soil, respectively). This method generated minimal shearing of the extracted DNA. The crude DNA could be further purified with an Elutip-d column if necessary. An additional advantage of this method is that only 1 g of sample is required, which allows for the analysis of small samples and the processing of many samples in a relatively short (7 h) period.     

Rapid method for direct extraction of DNA from soil and sediments.

A rapid method for the direct extraction of DNA from soil and sediments was developed. The indigenous microorganisms in the soil and sediments were lysed by using lysozyme and a freeze-thaw procedure. The lysate was extracted with sodium dodecyl sulfate and phenol-chloroform. In addition to a high recovery efficiency (greater than 90%), the yields of DNA were high (38 and 12 micrograms/g [wet weight] from sediments and soil, respectively). This method generated minimal shearing of the extracted DNA. The crude DNA could be further purified with an Elutip-d column if necessary. An additional advantage of this method is that only 1 g of sample is required, which allows for the analysis of small samples and the processing of many samples in a relatively short (7 h) period.     

直接从土壤中提取DNA的方法

研究微生物的多样性 ,即微生物的种类和数量的多少是评价土壤质量的重要指标。由于土壤微生物种类繁多 ,数量巨大 ,加上土壤中 99%的种类难以通过传统的平板分离技术来进行培养[1],人们必须借助其他技术来解决。近年发展的非培养技术 ,如ＢＩＯＬＯＧ微量板分析技术[2 ]、细胞壁磷脂酸分析技术[3]和分子生物学方法[4 - 6 ],克服了培养的环节 ,对微生物生态学研究产生极大的推动作用。其中分子生物学是应用最广、最有发展潜力的技术。它的主要步骤是通过直接提取土壤中的ＤＮＡ ,经纯化处理后 ,利用合适的引物扩增 1 6ＳｒＲＮＡ基因 ,通过分…     

Microbial degradation of ethylenediaminetetraacetate in soils and sediments

[¹⁴C]ethylenediaminetetraacetate (EDTA) was shown to be slowly biodegraded to ¹⁴CO₂ in soils and sediments under aerobic conditions and by microorganisms in mixed liquid culture. EDTA chelates of Cu, Cd, Zn, Mn, Ca, and Fe added to soil were equally degraded, while Ni-EDTA was degraded more slowly.     

Review of methodologies for extracting plant-available and amorphous Si from soils and aquatic sediments

There is a variety of methodologies used in the aquatic sciences and soil sciences for extracting different forms of Si from sediments and soils. However, a comparison of the published extraction techniques is lacking. Here we review the methodologies used to extract different Si fractions from soils and sediments. Methods were classified in those to assess plant-available Si and those to extract Si from amorphous silica and allophane. Plant-available Si is supposed to comprise silicic acid in soil solution and adsorbed to soil particles. Extraction techniques for plant-available Si include extractions with water, CaCl₂, acetate, acetic acid, phosphate, H₂SO₃, H₂SO₄, and citrate. The extractants show different capabilites to desorb silicic acid, with H₂SO₃, H₂SO₄ and citrate having the greater extraction potential. The most common extractants to dissolve amorphous silica from soils and aquatic sediments are NaOH and Na₂CO₃, but both also dissolve crystalline silicates to varying degrees. In soils moreover Tiron is used to dissolve amorphous silica, while oxalate is used to dissolve allophanes and imogolite-type materials. Most techniques analyzing for biogenic silica in aquatic environments use a correction method to identify mineral derived Si. By contrast, in the soil sciences no correction methods are used although pedologists are well aware of the overestimation of amorphous silica by the NaOH extraction, which is most commonly used to extract silica from soils. It is recommended that soil scientists begin to use the techniques developed in the aquatic sciences, since it seems impossible to extract amorphous Si from soils completely without dissolving some of the crystalline silicates.     

Recovery of DNA segments from agarose gels

After electrophoresis, DNA can be efficiently recovered by solubilization of agarose gels with NaClO₄, followed by retention of DNA on glass fiber filters. After removal of the NaClO₄ by ethanol, the DNA can be extracted with a low salt buffer.     

Recovery of DNA from agarose gels using a modified Elutrap.

We have made a significant improvement in the electroelution device, Elutrap (Schleicher and Schuell) by substituting an agarose gel barrier, which is made from 0.6% agarose (SeaKem GTG; FMC Corporation), into the elution chamber in place of the manufacturer specified BT2 membrane. This modification substantially increases the DNA recovery from agarose gels, even in samples containing less than 1 microgram of DNA, and shortens elution times particularly for large sizes of DNA (greater than 4.4 kbp). Additionally, the gel barrier provides a reproducible quantity and quality of DNA recovery. The high quality of the eluted DNA using the modified Elutrap makes this system suitable for further DNA manipulations.     

Efficient molecular cloning of environmental DNA from geothermal sediments

An efficient and simple method for constructing an environmental library using mechanically sheared DNA obtained directly from geothermal sediments is presented. The method is based on blunt-end modification of DNA fragments followed by 3-adenylation using Vent DNA polymerase and Taq DNA polymerase, respectively. The prepared DNA fragments are then ligated into a TA cloning vector and used in the transformation of Escherichia coli. This method has been successfully applied to the cloning of ORFs derived from uncultivated prokaryotes present in geothermal sediment.