Detection of coxsackie B virus infection using a rapid screening method

A Western blot method (WB) was adapted for rapid screening of antibodies against coxsackie virus B1-B6 in sera from patients with newly diagnosed insulin-dependent diabetes mellitus, myocarditis or febrile syndrome of suspected coxsackie viral aetiology. The use of a mixture of all 6 coxsackie virus B serotypes as the common antigen permitted a very rapid and inexpensive detection of antibody-positive sera for preliminary diagnosis and further detailed assay. Comparison of the results with those obtained in parallel run virus-neutralization tests showed a higher sensitivity and comparable specificity of WB.     

A rapid method for the purification of extrachromosomal DNA from eukaryotic cells.

A simple and efficient procedure to purify the low molecular weight extrachromosomal DNA from eukaryotic cells is described. Gentle lysis of cells with urea and sodium dodecyl sulfate in 0.24 M phosphate buffer (pH 6.8) is followed by the removal of high molecular weight bulk DNA by centrifugation. Protein and RNA are removed from the supernatant by hydroxyapatite chromatography in urea/phosphate buffer. Urea is then removed with 0.15 M phosphate buffer and the extrachromosomal DNA, virtually free from protein and RNA, is finally eluted in 0.5 M phosphate buffer. The procedure allows the recovery of about 99% simian virus 40 (SV40) DNA from infected monkey kidney cells in the extrachromosomal fraction. In normal mouse, monkey, andhuman cells, approximately 1% of total cell DNA appears to be extrachromosomal.     

A rapid method for extraction and purification of DNA from dental plaque.

A rapid method based on previously described DNA extraction procedures was developed for the isolation of DNA from dental plaque samples. The isolated DNA is suitable for use in the PCR. Freeze-thawing, cell wall-degrading enzymes, and guanidine isothiocyanate were used to lyse cells and release DNA. The released DNA was adsorbed onto diatomaceous earth and purified by washing with guanidine isothiocyanate, ethanol, and acetone. The purified DNA was released from the diatomaceous earth into an aqueous buffer and analyzed by PCR with 16S rDNA primers (rDNA is DNA coding for rRNA). As judged from studies with pure cultures of a number of bacterial species, gram-negative and gram-positive organisms were lysed equally well by this procedure. The amount of PCR product was proportional to the number of cells analyzed over the range tested, 500 to 50,000 cells. On the basis of studies with plaque samples that were spiked with known quantities of the oral bacterium Treponema denticola, the DNA prepared from plaque was free of substances inhibitory to PCR. This method should have utility in molecular genetic studies of bacterial populations not only in uncultured plaque samples but also in other complex bacterial assemblages.     

A rapid, cost-effective method of assembly and purification of synthetic DNA probes >100 bp

Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.     

A simple and rapid purification method for Escherichia coli DNA polymerase I.

We report a simple, three-step method for the purification of Escherichia coli DNA polymerase I. Its advantages over other procedures are ease and rapidity, the absence of an autolysis or any high speed centrifugation step, and applicability to large quantities of material. In addition, RNA polymerase can be isolated as a by-product. We have applied this method to purify DNA polymerase both from wild type E. coli cells and from cells bearing a lambda prophage carrying the polA gene (Kelley, W.S., Chalmers, K., and Murray, N.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5632-5636). This latter source amplifies the amount of DNA polymerase in the cells by at least 10-fold.     

An improved method for rapid purification of covalently closed circular plasmid DNA over a wide size range

An improved method has been developed for the large-scale purification of covalently closed circular (CCC) plasmid DNA molecules of sizes ranging from 4·3 to 73 kb. This protocol uses an alkaline-lysis procedure followed by acid-phenol extraction but with several modifications to previously reported methods. The principal modification is the replacement of NaCl by MgCl₂ in the extraction buffer to improve yield and to remove chromosomal and other non-CCC plasmid DNA. Plasmid DNA can be purified in less than 1 h and used successfully in restriction enzyme analysis and cloning experiments.     

A rapid method for purification of organelles for DNA isolation: self-generated percoll gradients

Self-generated Percoll gradients have been used for rapid purification of crude chloroplasts and mitochondria, obtained by common differential centrifugation techniques. Such purified organelles were used for isolating DNA from safflower (Carthamus tinctorius L.), carrot (Daucus carota L.), various Solanaceae, and numerous somatic hybrids. The method is simple, has the advantage of not requiring DNase, and is particularly well suited when only limited amounts of aseptically grown shoots are available. As judged by restriction enzyme analyses and chloroplast DNA cloning experiments, the DNAs are of sufficient purity for many molecular biological applications without CsCl gradient purification.     

A rapid method for detection of plant genomic instability using unanchored-microsatellite primers

In order to assess the feasibility of microsatellite primers as markers for genomic instability, we conducted a study of DNA stability in cauliflower callus. A protocol is described for the rapid screening of a large number of putative variant calli and plants. Genomic DNA is isolated and screened by microsatellite primers. We believe that inter-simple sequence repeat PCRs can conveniently detect and measure common genetic events underlying plant genomic instability. These include deletions, amplifications, translocations, insertions, recombination or chemical alterations. Our results indicate that instability occurred in an early step in the process of callogenesis. The technique is fast, reproducible, and is a new application for ISSR markers.     

一种快速微量提取植物叶片DNA的方法

介绍了一种快速提取微量DNA的方法。该方法简单易行,无需任何特殊设备,所需样品量少。提取的DNA纯度高,D260nm/D280nm在1．9-2．1之间,可满足RAPD、SSR、转基因植株的PCR检测等以PCR扩增为基础的实验需要。     

Investigation on the interaction of DNA and electroactive ligands using a rapid electrochemical method

A rapid method for investigation of the interaction of DNA and electroactive ligands based on an electrochemical equation for irreversible processes is presented. The binding constant (K) and the size of binding site (s) are simultaneously obtained from the dependence of the current on the amount of added DNA in voltammetry. A non-intercalative binder (Hoechst 33258) and two DNA-intercalators (mitoxantrone (MXT) and actinomycin D (AMD)) were examined in experiments. It was found that the binding constant of Hoechst 33258, mitoxantrone and actinomycin D, were 2.1 x 10(8), 8.9 x 10(9) and 9.1 x 10(9) cm(3) mol(-1); and the size of their binding sites were 4, 3 and 8, respectively. The study provides a convenient and sensitive approach for estimating affinity parameters and outlining the interaction between DNA and electroactive targeting compounds.     

A rapid method of gene detection using DNA bound to Sephacryl

A rapid method of gene detection has been developed utilising DNA fragments immobilized on resins and a sandwich hybridization assay. This method permits the detection of restriction fragment length polymorphisms (RFLPs) without the need to immobilize sample DNA. The method is based on the use of two non-overlapping DNA restriction fragments, one of which is attached to a resin (fragment A) and the other 32P-labelled (fragment B). Fragments A and B will not hybridize to each other unless there is a DNA or RNA fragment capable of hybridizing to both A and B present in the same reaction. Hybridization in this instance will result in the resin being radioactively labelled. The RFLP associated with the mutation causing sickle-cell anaemia was used as a model to develop the method. The resin Sephacryl S-500 appeared most suited to our method for two reasons: (i) DNA immobilization experiments using two coupling procedures and four resins indicated that Sephacryl S-500 bound the most DNA with very little non-covalent coupling. (ii) Hybridization experiments with DNA bound to a number of resins showed that DNA bound to Sephacryl S-500 hybridized most efficiently with a low level of nonspecific hybridization. Using optimum hybridization conditions 5 X 10(-18) mol of beta-globin DNA could be detected. The method has been used to distinguish between DNA from sickle, heterozygote and normal patients.     

A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide

We describe a modification of the DNA extraction method, in which cetyltrimethylammonium bromide (CTAB) is used to extract nucleic acids from plant tissues. In contrast to the original method, the modified CTAB procedure is faster, omits the selective precipitation and CsCl gradient steps, uses less expensive and toxic reagents, requires only inexpensive laboratory equipment and is more readily adapted to high-throughput DNA extraction. This protocol yields approximately 5-30 microg of total DNA from 200 mg of tissue fresh weight, depending on plant species and tissue source. It can be completed in as little as 5-6 h.