An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine

This report describes an improved immunochemical procedure to stain cells in suspension for incorporated bromodeoxyuridine (BrdUrd) and total DNA content. The procedure consists of five steps: chromatin proteins are extracted by treating with 0.1 M HCl and 0.7% Triton X-100 to facilitate DNA denaturation and to minimize nonspecific staining; cellular DNA is denatured by heating to 100 degrees C in distilled water; BrdUrd in single-stranded DNA (ssDNA) is stained using an immunochemical procedure; autofluorescence is reduced using sodium borohydride (NaBH4); and DNA is stained with the fluorescent dye propidium iodide. With this procedure, the BrdUrd incorporated by CHO cells during periods as short as a few seconds can be detected using flow cytometry. In addition, the stoichiometry of the immunofluorescent staining procedure is high.     

Cytochemistry for bromodeoxyuridine/DNA analysis: stoichiometry and sensitivity

This report describes an improved immunochemical procedure for staining cells in suspension for amount of incorporated bromodeoxyuridine (BrdUrd) and total DNA. In this procedure, cellular DNA is partially denatured by extracting the cells with 0.1 M HCl and then heating them to 80 degrees C in a 50% formamide solution. The cells are then immunofluorescently stained using a monoclonal antibody against BrdUrd in single-strand DNA (ssDNA) and counterstained for DNA content with propidium iodide (PI), a dye that fluoresces preferentially when bound to double-strand DNA (dsDNA). We show that the relative amounts of immunofluorescently stained BrdUrd in ssDNA and PI in dsDNA can be altered reciprocally by changing the formamide concentration, denaturation time, and denaturation temperature. We show that this new immunochemical staining procedure allows more complete DNA denaturation so that fivefold lower levels of BrdUrd incorporation can be quantified. In addition, we show that the BrdUrd-linked immunofluorescence achieved using the new denaturation procedure is more linearly related to cellular BrdUrd content than that achieved after acid DNA denaturation. However, cell loss is sufficiently severe with the thermal denaturation procedure that it may not be applicable to all cell types.     

Specific interaction between mouse liver non-histone chromosomal proteins and mouse DNA demonstrated by a sequential DNA-protein binding procedure.

The binding of mouse liver chromosomal proteins to DNA has been investigated using the nitrocellulose filter binding technique. Careful purification of the DNA involving nuclease S1 digestion and prefiltration through a nitrocellulose filter is used to reduce background binding in the absence of protein to less than 1%. Procedures involving direct binding of protein to labeled DNA, competition of binding of labeled DNA by unlabeled DNA, and dissociation of DNA . protein complexes with time do not indicate significant preference for binding to mouse DNA relative to Escherichia coli DNA. This specificity is demonstrated much more clearly by a novel type of procedure, which we call a sequential binding procedure. In this procedure non-specific binding proteins are sequestered by incubation with an excess of unlabeled E. coli DNA prior to addition of labeled DNA. Under these conditions, labeled mouse DNA is bound to filters to a 3- to 4-fold greater extent than labeled E. coli DNA.     

Isolation of replication forks from growing Ehrlich ascites cells

A procedure is described which permits the large-scale isolation of essentially complete replications forks from the DNA of Ehrlich ascites cells. The whole nuclear DNA is first isolated by a method which involves minimal hydrodynamic shear. The DNA is then degraded by cryolysis, a freeze-thawing procedure, to a size providing the otherwise very labile forked structures with a sufficient resistance against shear forces. Finally, the Y-shaped structures of replicating DNA are separated by nitrocellulose column chromatography. When the newly formed strands of replicating DNA were density-labeled with 5-bromodeoxyuridine the DNA fraction isolated by this procedure banded in isopycnic CsCl gradients at a density expected for Y-shaped molecules with two light-heavy branches and one light-light branch and sedimented significantly faster than the corresponding bulk DNA fraction through neutral sucrose gradients. The forked molecules could be visualized by electron microscopy. The essential step of the procedure is the cryolysis which produces fragments from larger DNA structure essentially at random. When the cryolysis is omitted the forked structures are disrupted within the highly susceptible regions around the branching point.     

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.     

Screening recombinant clones containing sequences homologous to Escherichia coli genes using single-stranded bacteriophage vector

Detection and isolation of Escherichia coli clones carrying vectors with foreign DNA sequences partially homologous to specific E. coli genes is difficult because denatured DNA in the host genome can hybridize with the probe. In this paper we present a procedure which simplifies this task by using bacteriophage M13 as the cloning vector. The procedure takes advantage of the secretory properties of the phage, as well as the property of nitrocellulose membrane to bind protein and single-stranded DNA but not double-stranded DNA. This procedure is shown to be effective in identifying E. coli clones containing sequences of Chlamydomonas reinhardtii chloroplast DNA that are homologous to the rpoC gene of E. coli. We suggest that this procedure can be used generally for rapid isolation of DNA sequences that are homologous to E. coli genes.     

An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: In situ purification and stable binding of DNA on paper filters

An improved procedure is presented for the binding to filter paper and subsequent purification of DNA from plasmid-containing bacterial colonies. The procedure includes treatments with NaOH, enzymatic digestion, and organic solvent extraction of the filter-bound DNA. This method allows isolation of DNA in a reusable form from thousands of colonies in several hours. Double-labeling experiments with [³H]thymidine and [¹⁴C]proline indicated that (i) during purification the DNA:protein ratio is increased several hundredfold; (ii) little or no DNA is lost during the procedure; (iii) the resultant purified DNA is tenaciously bound to the paper. Thus, the final filter-bound DNA allows multiple sequential hybridizations of different probes to one filter.     

A rapid chemical procedure for isolation and purification of chromosomal DNA from gram-negative bacilli

A rapid and simple method for preparing chromosomal DNA from gram-negative bacilli is presented. It is based on the alkaline (NaOH 0.03 m) lysis of cell walls. The resulting emulsion is purified by proteinase K (0.625 mg/g of wet wt), SDS, and the deproteinizing agent (chloroform isoamyl alcohol). The purity, molecular nature, and yield of DNA obtained by the present method are compared with those of DNA extracted by Marmur's procedure and a Marmur's modified procedure. We have developed and standardized this original method to isolate double-stranded DNA, free of proteins and RNA contamination and with a significantly higher yield of DNA than the two other methods. This procedure is particularly useful for strains with low growth and can be applied in every field concerned with DNA analysis.     

The production of single-stranded DNA suitable for sequencing using the polymerase chain reaction

A simple and reliable procedure for the amplification of single-stranded DNA suitable for sequencing is described. This procedure employs the polymerase chain reaction and implements modifications pertaining to the purification of the double-stranded DNA product prior to single-stranded DNA amplification. The most consistent sequencing reactions are obtained when the double-stranded DNA product is purified by centrifugation with a microconcentrator prior to single-stranded DNA amplification and the overall amount of specific primers and number of cycles used, in both single-stranded and double-stranded DNA polymerase chain reactions, are reduced.     

Large scale preparation of DNA for vast DNA excess hybridization

A method for the preparation of relatively large quantities of sonicated DNA for use in the vast DNA excess hybridization technique is reported. The procedure, applied here to rat liver DNA, involves centrifugation of DNA from sonicated nuclei to equilibrium in a CsCl gradient, RNase and pronase digestion, and phenol extraction. Compared with DNA purified by the Marmur procedure, the product has a lower contamination with protein and RNA, takes about one third the time to prepare, and has better hybridization characteristics.     

Fluorometric quantification of DNA in cells and tissue

The validation of a simple and rapid DNA solubilization procedure is described. Quantitative extraction of intact, polymerized DNA was achieved by cell lysis or tissue homogenization in an ammonium hydroxide-Triton X-100 solution. The solubilization procedure inactivates endogenous DNAase and increases the fluorescence-enhancement activity of the extracted DNA, thereby eliminating the need for enzyme treatment or exposure to high salt solutions. The extracts can be utilized directly in a sensitive fluorescence-enhancement assay with bisbenzimidazole (Hoechst 33258) reagent. Estimates of DNA cell content were unaffected by the number of cells lysed or the volume of lysate employed in the assay. In all cases, the solubilized DNA estimates were linear and parallel to the bovine DNA standard. The optimum range for estimation of DNA in this assay is 5-150 ng. In addition, estimates of DNA obtained with this method and the standard diphenylamine assay were in excellent agreement. This simple, one-step DNA extraction procedure can be utilized in conjunction with Hoechst reagent to obtain quantitative estimates of DNA levels in cell or tissue extracts.     

Model studies on the acriflavine-Feulgen reaction

The specificity and quantitative reliability of the Feulgen-acriflavine-SO2 procedure was tested on polyacrylamide model films containing DNA. Noncovalent binding of acriflavine to DNA was observed when the washing procedure, as used in the classical way, was applied. The noncovalently bound acriflavine could be removed with an extra wash in acid-ethanol. The presence of SO2 in the staining solution has been found to enhance covalent binding significantly. The absorbance of films stained by our Feulgen-acriflavine-SO2 procedure is directly proportional to that obtained by the classical Feulgen-pararosanilline-SO2 procedure. The acriflavine-Feulgen procedure has also been tested using a commercial and a purified dye. The use of purified acriflavine, compared to a commercial sample did not result in a significant difference in the maximum absorbance value of stained DNA nor in the absorption or the fluorescence emission spectra of acriflavin covalently bound to DNA.     

DNA-mediated gene transfer without carrier DNA

DNA-mediated gene transfer is a procedure which uses purified DNA to introduce new genetic elements into cells in culture. The standard DNA-mediated gene transfer procedure involves the use of whole cell DNA as carrier DNA for the transfer. We have modified the standard DNA-mediated gene transfer procedure to transfer the Herpes simplex virus type 1 thymidine kinase gene (TK) into TK- murine recipient cells in the absence of whole cell carrier DNA. The majority (8/10) of carrier-free transformant lines expressed the TK+ phenotype stably, in sharp contrast to our results with carrier-containing DNA-mediated gene transfer. There was a wide range in donor DNA content among independent transformants. Further analysis on one transformant line using DNA restriction digests and in situ hybridization provided evidence that, in the absence of whole cell carrier DNA, multiple donor DNA sequences became integrated at a single chromosomal site.     

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.     

A procedure for the isolation and purification of plasmid DNA from Rhizobium meliloti

A procedure is described for the isolation and purification of the DNA of plasmids that are indigenous to the agriculturally important nitrogen-fixing bacterium Rhizobium meliloti. The procedure involves the lysis of bacteria with an ionic detergent or a mixture of ionic and nonionic detergents, the extraction of total DNA from precipitated membrane-DNA complexes, the enrichment of supercoiled plasmid DNA by the selective alkaline denaturation of chromosomal DNA, and a further purification of plasmid DNA using cesium chloridepropidium diiodide gradients. This procedure yields pure plasmid DNA in amounts of 30 to 50 μg per liter of a culture of cell density of approximately one A550 unit. The DNA thus obtained has been found to be of sufficient purity to serve as substrate for the most commonly used restriction endonucleases.     

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.     

Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand

DNA with a 5′-adenylpyrophosphoryl cap (5′-adenylated DNA; AppDNA) is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5′-Adenylated DNA is also useful for in vitro selection experiments. Efficient preparation of 5′-adenylated DNA is therefore desirable for several biochemical applications. Here we have developed a DNA adenylation procedure that uses T4 DNA ligase and is more reliable than a previously reported approach that used the 5′-phosphorylated donor DNA substrate to be adenylated, a DNA template, and ATP but no acceptor strand. Our improved DNA adenylation procedure uses the above components as well as an acceptor strand that has a strategically chosen C-T acceptor-template mismatch directly adjacent to the adenylation site. This mismatch permits adenylation of the donor DNA substrate but largely suppresses subsequent ligation of the donor with the acceptor, as assayed on nine different DNA substrates that collectively have all four DNA nucleotides represented at each of the first two positions. The new DNA adenylation procedure is successful using either laboratory-prepared or commercial T4 DNA ligase and works well on the preparative (2 nmol) scale for all nine of the test DNA substrates.     

Rapid generation of subclones for DNA sequencing using the reverse cloning procedure

A fast and reliable procedure for generating subclones necessary for sequencing long stretches of DNA has been developed. The reverse cloning procedure involves cloning a fragment of DNA into a single-stranded plasmid or phage vector containing a polycloning region; synthesizing variable lengths of double-stranded DNA using a "Universal Primer"; isolating the double-stranded DNA; and force cloning the double-stranded DNA fragments into a complementary vector with the polycloning region in the reverse orientation. The resulting clones can be sequenced, using the same Universal Primer and T7 DNA polymerase, to provide overlapping DNA sequences. The reverse cloning procedure can be used to construct deletion mutations.     

DNA isolation from dry and fresh samples of polysaccharide-rich plants

DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yield with a single protocol; thus, even closely related species may require different isolating protocols. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to isolate DNA from tissues containing high levels of polysaccharides. The procedure is applicable to both dry and fresh tissues and was tested on chickpea seeds, soybean, and wheat leaves. This method solved the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides. The isolated DNA proved amenable to PCR amplification and restriction digestion.     

A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNase

A preparative procedure for the large-scale isolation of plasmid DNA without the use of RNAse is described. Crude plasmid DNA is prepared using a standard boiling method. High-molecular-weight RNA is removed by precipitation with LiCl, and low-molecular-weight RNA is removed by sedimentation through high-salt solution. The procedure is inexpensive, rapid, simple, and particularly suitable for processing several large-scale preparations simultaneously. A similar procedure has been developed for preparation of lambda-phage DNA.