A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.     

A DNA extraction method for RAPD analysis from plants rich in soluble polysaccharides

A simple procedure for the isolation of DNA from mature leaf tissue was developed. This procedure purified DNA using Sephacryl S-1000 column and PEG 8000 precipitation. Polysaccharide-like components were successfully removed from DNA samples from species in which polysaccharides were found to be difficult to remove by phenol/chloroform extraction. The DNA obtained by this method was suitable for PCR, RAPD, enzyme digestion, and Southern-blot analyses.     

Direct capture and cloning of receptor kinase and peroxidase genes from genomic DNA.

A direct DNA capture and cloning procedure with magnetic bead separation was used to isolate receptor kinase like and peroxidase genes from oat (Avena sativa) and wheat (Triticum aestivum L.) genomic DNA, respectively. In this procedure, the digoxigenin-labeled probe DNA and target genomic DNA fragments were mixed, denatured, and hybridized. The double-helix complexes formed were captured with anti-digoxigenin immunoglobulin-coated magnetic beads and then cloned into either the lambdaBlueSTAR or pUC18 vector. The effectiveness of this procedure was demonstrated by using two specific DNA probes to capture receptor-like kinase genes and surrounding sequences from oat genomic DNA and a peroxidase gene from wheat genomic DNA.     

Simple preparation of parapoxvirus genome DNA for endonuclease analysis

We compared five methods for improved extraction of very-large parapoxvirus DNA from infected cells: (i) alkaline-lysis procedure followed by phenol extraction; (ii) modified Hirt procedure, which was a neutral lysis procedure followed by phenol extraction; (iii) Hirt procedure; (iv) method used for extraction of vaccinia virus DNA; and (v) standard procedure using virus purification with an ultracentrifuge and protease-sodium dodecyl sulfate-phenol treatment. The alkaline-lysis procedure was more rapid, inexpensive and simpler than the other methods. Moreover, with this method it is not necessary to prepare any special facilities, reagents and kits. Although the extracted DNA was still crude, we could reproducibly prepare viral DNA from 2 X 10(6) infected cells in less than 2 hr and it could be readily digested by restriction endonuclease. This method will aid rapid genetic classification of parapoxvirus.     

Purification of DNA for bacterial productivity estimates

[methyl-3H]thymidine-labeled DNA from natural populations of aquatic bacteria was completely separated from RNA and protein by hydroxylapatite chromatography. The procedure was validated by monitoring increases in Escherichia coli cell count, A550, DNA concentration, and thymidine incorporation into DNA isolated by the proposed technique. The procedure can be used in the field and does not rely on the use of acid-base hydrolysis or volatile organic solvents.     

Integration of progeny simian virus 40 DNA into the host cell genome

A procedure was developed for the separation of cellular DNA of productively infected monkey kidney cells from free simian virus 40 DNA. The application of this procedure allowed the investigation of progeny viral DNA integration into the host cell DNA by nucleic acid hybridization techniques. The purification consisted of precipitation of the cellular DNA by Hirt's (1967) method, velocity centrifugation in alkaline sucrose gradients, equilibrium centrifugation in ethidium bromide/CsCl solution, and an additional velocity centrifugation in an alkaline sucrose gradient. The efficiency of each step of the procedure was determined by monitoring the amount of contaminating free viral DNA. Purified cellular DNA, isolated from cells late after infection, contained approximately 0/sd006% free viral DNA, but as much as 2% integrated simian virus 40 DNA. This corresponds to more than 20,000 integrated virus genome equivalents per cell, as determined by DNA-DNA reassociation kinetics. Integration of simian virus 40 DNA into the cellular DNA became detectable at 24 hours after infection, and increased with the increase in the rate of viral DNA synthesis.     

Comparison of three procedures for isolating DNA from bacteria.

Three methods employing chloroform-isoamyl alcohol (CI), phenol, or enzymes, were evaluated for isolating DNA from Escherichia coli, Bacillus subtilis, and Arthrobacter globiformis. For the amounts of reagents employed at optimum conditions in the CI and phenol procedures, 0.4-0.9 mg of DNA/g wet weight of cells was isolated. Using the enzymatic procedure, approximately twice as much DNA was isolated. DNA isolated by the CI procedure contained 0.03-0.09% protein and 0.08-0.12% RNA. DNA isolated by the phenol procedure contained 0.02-0.05% protein and 2.2-2.6% RNA. DNA isolated by an enzymatic procedure, which is described in detail, contained 32.2-45.7% protein and 0.3-0.6% RNA. DNA isolated by all three procedures are double-stranded and at least 10(6) in molecular weight, as suggested by data from thermal transition analyses and transformations. These data emphasize that the desired characteristics of DNA for experimental purposes must be considered in selecting an isolation procedure.     

Isolation of genomic DNA from the earthworm speciesEisenia fetida

Our interest in detecting genotoxic exposure in earthworms led us to isolate high quality DNA from theEisenia fetida species. For that, we compared a modification of the conventional phenol-chloroform extraction procedure, usually refered to as the Maniatis procedure, to two commercially available kits reportedly eliminating multiple partitions in phenol and chloroform, namely the Qiagen and Nucleon protocols. From the 260 nm optical density values, the commercial kits extracts hinted toward higher DNA recovery with those procedures. However, the 260/280 nm ratios indicated that the quality of the DNA isolated with the modified Maniatis procedure was purer than that isolated with the commercial kits, the latter being most probably contaminated by proteins and/or RNA. The Maniatis procedure was slightly modified by the introduction of a potassium acetate step for protein precipitation and by shortening the proteinase K treatment from 12–18 h to only 2 h. The higher quality of the DNA isolated by phenol-chloroform extraction was confirmed by quantification with the fluorescent 3,5-diaminobenzoic acid assay. Preliminary results suggest that the modified Maniatis procedure herein described is not only applicable for DNA adducts studies using³²P-postlabelling techniques but is also suitable for DNA extraction from other earthworm species such asLumbricus terrestris.     

Technical note: Bone DNA extraction and purification using silica-coated paramagnetic beads

The goal of this study was to develop a simple method to improve DNA recovery from challenging bone samples. To this end, an optimized procedure was developed that combined the demineralization and DNA extraction into a single step, followed by DNA purification using an automated silica-coated paramagnetic bead procedure. This method replaced a previous silica-membrane-based procedure, which was able to recover sufficient DNA to obtain full autosomal and Y chromosome STR profiles from greater than 90% of the samples, including samples greater than 20 years old. The development process began with the evaluation of buffer and demineralization systems to determine the best reagent combination. During the developmental process, we observed that the addition of EDTA and DTT affected silica-based DNA purification methods by raising the pH of the digest buffer. The protocols with buffer ATL, PK, EDTA, and DTT followed by lowering the pH with sodium acetate just before purification resulted in the best yields. The method reduced the extraction volume from 10 to 1.5 ml and used commercially available reagents already being utilized in forensic DNA casework. Because of the simplicity and small volume needed for the procedure, many steps where contamination could be introduced have been eliminated or minimized. This study demonstrated a new method of recovering DNA from bone samples capable of extracting trace quantities of DNA, removing potential inhibitors, and minimizing the potential for exogenous DNA contamination.     

A preparation of short DNA chains synthesized in vivo after introduction into DNA from live cells of trioxsalen crosslinks is not enriched by replication initiation regions

The experiments undertaken in order to verify the trioxsalen-crosslinking method suggested by Russev and Vassilev for isolation of eukaryotic replication origins are described. It was found that the preparation of viral DNA isolated by the above mentioned method from CV-1 cells lytically infected with SV40 was not enriched in sequences including SV40 replication origin. The hybridization pattern of DNA preparation isolated by the trioxsalen-crosslinking procedure from chicken erythroblastosis cells with the cloned fragments of globin gene domain was found to be identical to those of the total DNA probe. The DNA fraction enriched in replication origins was isolated from the same cells with the aid of nascent DNA strand extrusion method by Zannis-Hadjopoulos et al. The hybridization pattern of this DNA fraction with the cloned fragments of chicken alpha-globin gene domain was different from those of total DNA. Taking together, the results of our experiments demonstrate that trioxsalen-crosslinking procedure does not lead to the isolation of replication origins from the objects studied in the present investigation.     

Methods for detection of Anticarsia gemmatalis nucleopolyhedrovirus DNA in soil.

Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0. 25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the AgMNPV DNA preparations were diluted 10- or 100-fold. AgMNPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.     

Isolation of a single-stranded plasmid from Clostridium acetobutylicum NCIB 6444.

The cryptic plasmid pDM6 was isolated from late exponential-phase cells of Clostridium acetobutylicum NCIB 6444 by either alkaline lysis or electroporation. The application of high voltage during electroporation resulted in higher DNA yield than did the alkaline lysis procedure. However, electroporation-induced plasmid release generated high amounts of single-stranded DNA compared with the alkaline lysis procedure, which generated both double-stranded DNA (monomer and dimer forms) and single-stranded DNA.     

Methods for Detection of Anticarsia gemmatalis Nucleopolyhedrovirus DNA in Soil

Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0.25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the AgMNPV DNA preparations were diluted 10- or 100-fold. AgMNPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.     

Isolation of a single-stranded plasmid from Clostridium acetobutylicum NCIB 6444

The cryptic plasmid pDM6 was isolated from late exponential-phase cells of Clostridium acetobutylicum NCIB 6444 by either alkaline lysis or electroporation. The application of high voltage during electroporation resulted in higher DNA yield than did the alkaline lysis procedure. However, electroporation-induced plasmid release generated high amounts of single-stranded DNA compared with the alkaline lysis procedure, which generated both double-stranded DNA (monomer and dimer forms) and single-stranded DNA.     

太湖新银鱼线粒体DNA的制备

建立了适合太湖新银鱼等小型鱼类ｍｔＤＮＡ分离纯化的有效方法。改进匀浆条件,采用ＤＮａｓｅＩ和ＲＮａｓｅ纯化方法,可用常规方法由小量太湖新银鱼中制备出ｍｔＤＮＡ。采用改进的碱裂解法,能够在２小时内由单尾太湖新银鱼中快速分离纯化出适于限制性分析用的ｍｔＤＮＡ。     

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.     

A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA

A simple new procedure was described for producing a sequential series of overlapping clones for use in DNA sequencing. The technique used single-stranded M13 DNA and complementary DNA oligomers to form specific cleavage and ligation substrates. It was, therefore, independent of the sequence of the DNA cloned into the vector. Deletions of varying sizes were generated from one end of the insert through the 3' to 5' exonuclease activity of T4 DNA polymerase. The approximate size of the deletion and therefore the starting point for DNA sequencing could be estimated by electrophoresis of the subcloned phage DNA on a agarose gel. This greatly reduced the number of templates that must be sequenced to obtain a complete sequence. The entire procedure could be carried out in one tube in less than a day. The procedure was used to subclone and sequence the maize mitochondrial 18 S rDNA and 5' flanking region (2622 bases) in less than a week. Other applications of oligomers and single-stranded DNA in the construction of insertions, deletions, and cDNAs are discussed.     

猕猴桃叶DNA的AFLP分析

以猕猴桃幼叶为材料提取基因组DNA并进行AFLP扩增。对主要实验步聚包括DNA纯化、DNA的酶切、酶切片段与接头的连接、PCR扩增、电泳、以及银染等方面的反应参数进行比较和优选,初步摸索出适合于猕猴桃叶为材料的AFLP程序,并得到较为清晰可辩的AFLP指纹图谱,为开展猕猴桃遗传多态性研究和在分子水平上开展物种生物学分析提供一个实用的手段。     

Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification

The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye-ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N-nitroso-N-methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks.     

Sequence complexity of circular Epstein-Bar virus DNA in transformed cells

A simplified procedure, based on several methods previously used to isolate circular DNA molecules from bacteria, was derived for the preparation of covalently closed circular viral DNA molecules from large quantities of lymphocytes transformed by Epstein-Barr virus. The protocol can be applied both to virus nonproducer lines and to lines containing cells activated to virus production. Sufficient amounts o highly purified viral DNA of intracellular origin were obtained from B95-8 and Raji cells to allow direct visual analysis of their sequence complexities after cleavage with EcoRI and separation of fragments by gel electrophoresis. No major differences in complexity were observed between circular DNA and linear virion DNA from B95-8 cells. The fragment patterns observed in this fashion agree well with those detected by conventional blotting and hybridization methods. The procedure can also be used as an analytical method to assay for small amounts of circular Epstein-Barr virus DNA molecules in other transformed cells. In this connection, no circular Epstein-Barr virus DNA was detected in Namalva cells.