可用于微生物群落分子生态学研究的活性污泥总DNA提取方法研究

活性污泥样品经液氮速冻、沸水浴融化、溶菌酶处理和 SDS裂解后 ,99%以上细胞裂解。所提取的 DNA经琼脂糖凝胶电泳检测和荧光法浓度测定 ,其片断大小在 2 0 kb左右 ,产量可达 1 .75 6± 0 .1 mg/g MLSS。样品 ABS2 6 0 nm/ABS2 80 nm的比值为 1 .96± 0 .2。以提取的总 DNA为模板 ,进行细菌核糖体小亚基 1 6Sr DNA基因 V3区和多组分苯酚羟化酶大亚基基因 (Lm PHs)的 PCR扩增 ,均获得成功 ,为活性污泥中微生物群落的分子生态学研究提供了一种简便、可靠的 DNA提取方法。     

Rapid extraction of plasmids from Clostridium perfringens.

Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens. The first method involved lysis of 1 to 2 ml of C. perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl. DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel. The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS). Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel. Of 57 strains of C. perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method. These new plasmids were of higher molecular mass and ranged up to 68 megadaltons. Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA. An additional 159 isolates of C. perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.     

Rapid extraction of plasmids from Clostridium perfringens

Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens. The first method involved lysis of 1 to 2 ml of C. perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl. DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel. The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS). Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel. Of 57 strains of C. perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method. These new plasmids were of higher molecular mass and ranged up to 68 megadaltons. Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA. An additional 159 isolates of C. perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.     

Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations

Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained. Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 +/- 0.03 M (pH 12.9 +/- 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs. The extent of denaturation is shown to increase with time of exposure and NaOH concentration. Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield. While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form. In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA. The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10(1)-10(5) s(-1)) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10(4)-10(5) s(-1)). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.     

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.     

An easy and rapid method for isolation of entire mycobacterial genome for application in pulsed-field gel electrophoresis

A DNA extraction suitable for mycobacterial lysis in gentle conditions compatible with genome analysis by pulsed-field gel electrophoresis is presented. Effects of preliminary treatments with SDS, Triton X-100, and hexane on mycobacterial outer layer were observed by electron microscopy. The most efficient procedure, performed on cells from liquid or solid medium, consisted of treatment by Triton X-100, agarose embedding of the cells, and further treatment with -amylase followed by lysozyme and SDS-proteinase K.     

临床标本细菌基因组DNA提取方法探讨

目的优化细菌基因组DNA提取方法,使其适合临床细菌分子生物学检测需要。方法分别采用专用DNA提取液法、热裂解法、溶菌酶法、热裂解法与碱性裂解法组合改良法,对纯培养细菌和临床标本中细菌基因组DNA进行提取。结果专用DNA提取液法、溶菌酶法提取成功率为100%,热裂解法革兰阳性菌提取成功率为0%,革兰阴性菌成功率为100%,碱性裂解液法在NaOH浓度大于4 mmol时提取成功,临床标本在NaOH溶液超过20 mmol/L并含2%SDS时细菌基因组DNA的提取成功率为100%。结论热裂解法与碱性裂解法组合改良法提取细菌基因组DNA方便快速、简单实用,适用临床标本检测。     

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.     

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.     

Comparison of different methods for the isolation and purification of total community DNA from soil

The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine-HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40-90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments.     

A rapid method for the screening of plasmids from Clostridium acetobutylicum and other Clostridium sp.

Summary A rapid method was developed for plasmid DNA screening from micro volume cultures of Clostridium acetobutylicum. It involves protoplastization by lysozyme, nuclease inhibition by incorporating EDTA or DEP, followed by lysis with high concentration of SDS. The whole lysate is applied directly to electrophoretic analysis.     

Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment.

This study reports improvements in two of the key steps, lysis of indigenous cells and DNA purification, required for achieving a rapid nonselective protocol for extracting nucleic acids directly from sodium dodecyl sulfate (SDS)-treated sediment rich in organic matter. Incorporation of bead-mill homogenization into the DNA extraction procedure doubled the densitometrically determined DNA yield (11.8 micrograms of DNA.g [dry weight] of sediment-1) relative to incorporation of three cycles of freezing and thawing (5.2 micrograms of DNA.g [dry weight] of sediment-1). The improved DNA extraction efficiency was attributed to increased cell lysis, measured by viable counts of sediment microorganisms which showed that 2 and 8%, respectively, survived the bead-mill homogenization and freeze-thaw procedures. Corresponding measurements of suspensions of viable Bacillus endospores demonstrated that 2 and 94% of the initial number survived. Conventional, laser scanning epifluorescence phase-contrast, and differential interference-contrast microscopy revealed that small coccoid bacterial cells (1.2 to 0.3 micron long) were left intact after combined SDS and bead-mill homogenization of sediment samples. Estimates of the residual fraction of the fluorescently stained cell numbers indicated that 6% (2.2 x 10(8) cells.g [dry weight] of sediment-1) of the original population (3.8 x 10(9) cells.g [dry weight] of sediment-1) remained after treatment with SDS and bead-mill homogenization. Thus, lysis of total cells was less efficient than that of cells which could be cultured. The extracted DNA was used to successfully amplify nahR, the regulatory gene for naphthalene catabolism in Pseudomonas putida G7, by PCR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies to the most tightly bound proteins in eukaryotic DNA

DNA released from eukaryotic cells by proteases/SDS or by alkali/SDS still contains distinct proteins which are not removed by these cell lysis procedures nor by subsequent phenol treatment. The proteins most tightly bound to DNA can only be isolated by degradation of DNA. In contrast to the protein-DNA complexes, the protein material isolated after degradation of DNA is sensitive to protease treatment. Moreover, the isolated protein material tends to form aggregates which are insoluble in buffers not containing detergents. They are only poorly soluble in buffers containing SDS. The partially solubilized material can be separated by SDS-polyacrylamide gel electrophoresis into two main bands. Antibodies were raised in rabbits against the polypeptides contained in these main bands. Immunofluorescence micrographs are presented of cells treated with the antibodies. The results indicate that the proteins characterized by their involvement in extremely stable protein-DNA complexes also occur independently of DNA in eukaryotic cells.     

Identification of a biochemically unique DNA-membrane interaction involving the Escherichia coli origin of replication.

DNA-membrane complexes have been obtained from Escherichia coli by using a freeze-thaw lysis procedure that avoids lysozyme and detergents. Complexes made in this manner and containing DNA near the origin of replication are uniquely sensitive to ionic strength, Pronase, and trypsin. There is approximately one such complex per chromosomal origin. The sensitivities suggest that origin-specific binding is mediated by a protein. By using these unique characteristics to distinguish origin-specific complexes from the majority of DNA-membrane binding sites, it was found that the origin-specific binding persists after termination of chromosomal replication.     

Direct extraction of microbial community DNA from humified upland soils

This paper describes a protocol effective at extracting high yields of high-purity microbial community DNA from humified soils. DNA was extracted from soil by lysozyme, SDS and freeze–thaw lysis, precipitated and then subjected to a double caesium chloride density gradient centrifugation stage before concentrating and washing. Evaluation using three soils yielded up to 30 μg DNA g⁻¹ dry soil, with absorbance ratios at 260 : 230 nm and 260 : 280 nm of 1·6–2·0. The DNA extracted from the three soils was digested by four restriction enzymes and a 16S rDNA eubacterial product was amplified by PCR. These tests indicated that the DNA obtained by the protocol was sufficiently pure for molecular biological analysis.     

Isolation OP DNA Prom Yeast by Chromatography on Hydroxyapatite

A simple procedure is described for isolation of purified non degraded total DNA from yeast cells. The procedure involves conversion of the cells into sphero-plasts by enzymatic treatment, lysis of the sphero-plasts in 8 M urea - 0.24 M sodium phosphate buffer -0.01 M EDTA (ethylendiamintetraacetic acid, sodium salt) - 1% SDS (sodium dodecyl sulphate), deproteiniza-tion of the lysate with chloroform-phenol and separation of the DNA from proteins, RNA and other contaminants by hydroxyapatite chromatography. The yield is about 90% of the DNA in the starting material (sphero-plasts).     

Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.     

Control of Lysis of T4-infected Escherichia coli

The lysis of Escherichia coli B/5 infected with T4Dr48 could be delayed by addition of 9-aminoacridine (9AA). Infected cells showed an early period of maximal response followed by a decline in sensitivity. The ultimate rate of lysis was also affected by the dye. Deoxyribonucleic acid (DNA), protein, and lysozyme synthesis began at the normal time in complexes inhibited by 9AA addition. The rates of synthesis of these macromolecules were lower in the presence of the dye, with DNA and lysozyme synthesis being more strongly affected than total protein synthesis. Penicillin-sensitive cell wall synthesis stopped at about 10 min after infection. Inhibition of oxidative metabolism by early potassium cyanide addition prevented lysis in the presence of intracellular lysozyme. The cyanide-sensitive event occurred at about 20 min in normal infections, and between 30 and 40 min in 9AA-inhibited infections. 9AA could alter both the time at which the cyanide-sensitive event occurred and the time of lysis. Addition of chloramphenicol did not prevent lysis once intracellular lysozyme was present. Lysis from without of infected cells consisted of three phases: an initial sensitivity, followed by a short period of resistance, and then a return to sensitivity in normal infections. The demonstration of the late return to sensitivity depended on the presence of intracellular lysozyme, and could be delayed by 9AA addition.     

DNA Extraction from Activated Sludges

To optimize the cell lysis step for DNA extraction from activated sludge samples, two floc dispersion methods (sonication versus stirring with a cation exchange resin), and three cell lysis treatments (lysozyme + SDS, sonication in a water bath, and thermal shock) were tested. For dispersion, stirring with cation exchange resin was more efficient than sonication. The cell lysis procedures were applied in two sequences, and DNA was quantified after each cell lysis treatment. Lysozyme + SDS was the most effective step in the cell lysis procedures. The cell lysis treatment sequences giving the highest DNA yields were not the same for all the sludges. The differences in sludge microbial compositions and floc structures required specifically adapted cell lysis protocols. The proposed protocols were highly efficient for DNA extraction, yielding about 50 mg DNA g⁻¹ volatile suspended solids, and allowed PCR amplification of 16S rDNA. Received: 26 September 1998 / Accepted: 13 February 1999