Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples.

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.     

Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.     

Biotransformation of polycyclic aromatic hydrocarbons by yeasts isolated from coastal sediments.

Yeast abundance in the sediments of 13 coastal sites in Massachusetts was quantified, and the potential of yeast isolates to biotransform polycyclic aromatic hydrocarbons (PAHs) was determined. Plate counts of yeasts varied between 10(2) to 10(7) CFU g (dry weight) of sediment-1. The most abundant genera isolated and identified included Candida, Cryptococcus, Rhodotorula, Torulopsis, and Trichosporon. More than 50% of the isolates from heavily contaminated sites transformed phenanthrene, as determined by spray-plate screening. The plate counts of phenanthrene-transforming yeasts correlated significantly to the sediment concentrations of phenanthrene. Transformation of [9-14C]phenanthrene and [12-14C]benz[a]anthracene by individual isolates varied greatly, ranging from 0.15 to 8.15 mumol of PAH g-1 in 120-h incubations. Of the isolated yeasts, Trichosporon penicillatum exhibited the greatest capacity for phenanthrene transformation. The ability to transform PAHs appears to be widespread among yeasts in coastal sediments.     

Estimation of bacterial cell numbers in humic acid-rich salt marsh sediments with probes directed to 16S ribosomal DNA

The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 x 10(7) to 2.5 x 10(9) (average, 1.1 x 10(9) +/- 5.2 x 10(8)) cells g of sediment-1. In September, numbers of SRB detected ranged from 5.4 x 10(8) to 7.3 x 10(9) (average, 2.5 x 10(9) +/- 1.5 x 10(9)) cells g of sediment-1. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments.     

A molecular approach to the characterization of the eukaryotic communities of an extreme acidic environment: methods for DNA extraction and denaturing gradient gel electrophoresis analysis

The diversity of the phytobenthonic community present in six acidophilic microbial mats from Río Tinto (Iberian Pyritic Belt, SW Spain) was analysed by optical microscopy and two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 18S rDNA cloned gene fragments. Sixteen DNA isolation protocols as well as two commercial DNA extraction kits were tested and their efficiency compared. Purified DNA extracts were amplified by PCR using universal eukaryotic primers and the PCR products analysed by DGGE. Bead-mill homogenization was found to be superior to the other cell lysis methodologies assayed (sonication or freeze-thawing cycles) as it allowed efficiencies of cell disruption of over 95%. The methods combining bead-mill homogenization in the presence of SDS, treatment with chemical extractants (hexadecylmethylammonium bromide or guanidine isothiocyanate) and phenol extraction resulted in DNA preparations that amplified the same number of bands when analysed by DGGE as the two commercial kits assayed. The phylogenetic affiliations of the DGGE bands were determined by a BLAST search, and nine different species related to the Chlorophyta, Ciliophora, Kinetoplastida, Ascomycota, Streptophyta and Colcochaetales taxonomical groups were identified. Similar levels of diversity were found using cloning procedures. Although not all the species observed under the microscope were detected using molecular techniques, e.g. euglenas, heliozoan, or amoebae, DGGE fingerprints showed rather well the level of diversity present in the samples analysed, with limitations similar to cloning techniques.     

革兰氏阳性细菌基因组DNA提取方法的比较及优化

【目的】基因组DNA提取效率和质量对分子生物学相关研究起着关键的作用,革兰氏阳性细菌由于细胞壁较厚、难破裂使其基因组DNA提取的难度增大,本文旨在寻找一种高效稳定的DNA提取方法。【方法】以Clostridium thermocellum和Thermoanaerobacterium thermosaccharolyticum为实验菌株,使用6种DNA提取方法对C. thermocellum基因组DNA进行提取,对比其提取效果和产率。【结果】改良的SDS-碱裂解法提取得到的DNA浓度较高(400 mg/l左右),且平行样间浓度和纯度稳定。【结论】为革兰氏阳性细菌基因组DNA提取提供参考。     

Diethyldithiocarbamate inhibits scheduled and unscheduled DNA synthesis of rat thymocytes in vitro and in vivo--dose-effect relationships and mechanisms of action

In vitro as well as in animal models, diethyldithiocarbamate (DDC) modifies the tumoricidal activity of some antineoplastic agents. To gain further information about the mechanism of action of DDC, we measured (i) in vitro and (ii) in vivo changes in DNA synthesis of rat thymocytes. (i) In vitro, the scheduled (SDS) and unscheduled (UDS) incorporation of [3H]thymidine ([3H]dT) into DNA of rat thymic cells were biphasically inhibited in a dose range of 1-1000 micrograms DDC/ml. The UV-induced UDS was totally suppressed by 10 and 100 micrograms DDC/ml. (ii) In vivo, 1-4 h following intraperitoneal administration of 250-1000 mg DDC per kg body wt., SDS and UDS were inhibited up to about 80% in a dose-dependent manner. Nucleoid sedimentation, uptake of [3H]dT into the cells, and the pattern of phosphorylation of the intracellular [3H]dT following DDC treatment did not reveal any differences to the controls. A possible effect of DDC treatment on the ribonucleotide reductase and the DNA polymerase alpha is suggested.     

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control.     

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.     

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was the only method which resulted in amplifiable RNA of good quality (ie intact 16S/23S rRNA). Sample homogenization and hot chemical (SDS) cell lysis combined with mechanical (bead-beating) lysis in presence of a DNA competitor (skim milk) contributed to improving substantially (around 23 times) the DNA yield of the PowerSoil DNA Isolation Kit. Apart from presenting NA extraction strategies for optimizing extraction parameters with corrosion samples from carbon steel, this study proposes DNA and RNA extraction procedures suited for comparative molecular analysis of total and active fractions of bacterial communities associated with carbon steel corrosion events, thereby contributing to improved MIC diagnosis and control.     

Purification of plasmid DNA using a multicompartment electrolyser separated by ultrafilter membranes

A simple, scalable method for purification of plasmid DNA is described. Plasmid DNA was released from Escherichia coli JM109 by lysis (1% SDS, 0.2 M NaOH). Then a neutralization solution (3 M sodium acetate buffer, pH 4.8) was added to precipitate genomic DNA and protein. After the clarification of the lysate, the supernatant was placed in a multicompartment electrolyser separated by ultrafilter membranes to remove the remaining contamination (RNA, genomic DNA and protein). A recovery of 75%±2% of total plasmid DNA was obtained after 60 min electrophoresis with a field strength of 8 V cm^–1 using cells at 30 g l^–1 (quantified by dry cell weight). Genomic DNA, RNA and protein were undetectable in the purified plasmid DNA solution.     

Enumeration and phylogenetic analysis of polycyclic aromatic hydrocarbon-degrading marine bacteria from Puget sound sediments.

Naphthalene- and phenanthrene-degrading bacteria in Puget Sound sediments were enumerated by most-probable-number enumeration procedures. Sediments from a creosote-contaminated Environmental Protection Agency Superfund Site (Eagle Harbor) contained from 10(4) to 10(7) polycyclic aromatic hydrocarbon (PAH)-degrading bacteria g (dry weight) of sediment-1, whereas the concentration at an uncontaminated site ranged from 10(3) to 10(4) g of sediment(-1). Isolates of PAH-degrading bacteria were obtained from these most-probable-number tubes as well as from sediment samples from noncontaminated sites and from bioreactors enriched with PAHs. The 18 resulting strains were grouped by whole-cell fatty acid analysis into two subgroups. The larger group of strains belonged to the newly described genus Cycloclasticus, whereas the other group contained members of the genus Vibrio. The Cycloclasticus group seems to be widespread in noncontaminated sediments. PAH degradation was confirmed in selected strains on the basis of removal of phenanthrene from growing cultures.     

Methylation of DNA in NaCl-adapted cells of potato

Salt-adapted and control cells of the cultivated potato, Solanum tuberosum cultivar Russet Burbank, untreated or treated with 5-azacytidine (an inhibitor of DNA methylation), were compared with respect to: a) % of cytosine methylation in total nuclear DNA, as determined by HPLC; b) fresh and dry weight. Adapted and control cells were compared also with respect to % of cytosine methylation in DNA, which was purified from DNaseI-partially-digested chromatin and size fractionated by electrophoresis in agarose gels. The growth (represented by dry weight) of the NaCl-adapted cells in saline medium lacking 5-azacytidine was similar to that of control cells in standard medium. The adaptation of the cells was correlated with some increase (+16%) of methylation in total DNA and with a much greater increase in the lower molecular weight DNA fractions which were obtained from the presumably more active chromatin. As expected, the treatment of the cells with the methylation inhibitor induced a decrease in the level of methylation. The decrease of methylation, however, was much greater in the adapted cells, whose dry weight, unlike in the control, was not affected by this treatment.Abbreviations 5-azaCyt 5-azacytidine - C cytidine - 2,4-D 2,4 dichlorophenoxyacetic acid - DW dry weight - EDTA ethylenediaminetetraacetic acid - FW fresh weight - HPLC high performance liquid chromatography - m⁵Cyt 5 methyl cytidine - RB Russet Burbank - SDS sodium dodecyl sulfate - TE 10 mM Tris and 1 mM EDTA - Tris Tris [hydroxymethyl] aminomethane     

Extraction of chromosomal DNA from Staphylococcus and Listeria by a rapid method using achromopeptidase

A rapid and simple method for preparation of chromosomal DNA from Gram-positive bacteria is reported. Susceptibility to lysis with Sodium Dodecyl Sulfate (SDS) increases when undergoing treatment with acetone before being digested by bacteriolytic enzymes. Rapid lysis of Staphylococcus and Listeria cells is obtained through a respective treatment by lysozyme with lysostaphine and by lysozyme with achromopeptidase, adding to that the effect of SDS in Tris-Hcl buffer. This procedure of preparing chromosomal DNA provides 1 to 4 mg of DNA out of 1 g of bacterial cells in a day.     

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 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.     

Microbial Ecosystem in Sunderban Mangrove Forest Sediment,North-East Coast of Bay of Bengal,India

This is the first documentation of seasonal and spatial fluctuation of the culturable microbial population collected from different zones in the sediment of the Sunderban mangrove forest. The population of cellulose degrading bacteria, [mean value of CFU 6.189 ± 1.025 × 10⁶ (g dry weight of sediment)^?1] was found to be maximum during post monsoon in the deep forest region, whereas, the fungal population [mean value of CFU 3.424 ± 0.886 × 10⁶ (g dry weight of sediment)^?1] was found to be maximum during pre-monsoon in the rooted region. The abundances of microbes, in decreasing order, studied from different zones are nitrifying bacteria [mean value of CFU 1.125 ± 0.359 × 10⁶ (g dry weight of sediment)^?1], phosphorous solubilizing bacteria (PSB) [mean value of CFU 0.805 ± 0.322 × 10⁶ (g dry weight of sediment)^?1], free living nitrogen fixing bacteria [mean value of CFU 0.417 ± 0.120 × 10⁶ (g dry weight of sediment)^?1] and sulfur reducing bacteria (SRB) [mean value of CFU 0.356 ± 0.125 × 10⁶ (g dry weight of sediment)^?1]. The content of organic carbon in the soil decreased from the deep forest region to the rooted and unrooted region but a reverse profile was found for soil salinity and soil silicate concentration. The results from the present study indicate that the monsoon cycle has a pronounced effect on the microbially dominated biogeochemistry in the sediment and consequently on the ecology of the Sundarban mangrove forest.     

Stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the gastrointestinal tract of the suckling mouse

The effects of epidermal growth factor (EGF), cortisone and thyroxine on deoxyribonucleic acid (DNA) synthesis in the esophagus, stomach, small intestine and colon have been studied in suckling mouse. Daily administration of EGF [4 micrograms/g body weight (bw)/day] during 3 days to 8-day-old mice induced a significant increase of the incorporation of [3H]thymidine into DNA in the stomach, the small intestine, and the two halves of the colon. The DNA synthesis in the esophagus remained unaffected by the EGF treatment. The maximal increase of [3H]thymidine incorporation into DNA was observed in the colon, and represented 112%. Daily administration of cortisone acetate (25 micrograms/g bw/day) or thyroxine (1 microgram/g bw/day) during 3 days to 8-day-old mice had no significant influence of the DNA synthesis of any part of the gastrointestinal tract. These results show that EGF is able to affect the DNA synthesis in the stomach, small intestine and colon of suckling mice.     

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.     

Abundance of dioxygenase genes similar to Ralstonia sp. strain U2 nagAc is correlated with naphthalene concentrations in coal tar-contaminated freshwater sediments

We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-microl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 +/- 0.7) x 10(3) to (2.9 +/- 0.3) x 10(5) copies of nagAc-like dioxygenase genes per microg of DNA extracted from sediment samples. These values corresponded to (1.2 +/- 0.6) x 10(5) to (5.4 +/- 0.4) x 10(7) copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene.