DNA Template Preparation Research Articles

Precise large deletions by the PCR-based overlap extension method.

The authors describe an efficient method for generating large deletions (>200 nts) of precise length using the PCR-based method of gene splicing by overlap extension (1). This method is technically simple and less time consuming than conventional loop-out mutagenesis techniques requiring preparation of a single-stranded DNA template.

Comparison of Template Preparation Methods from Foods for Amplification of Escherichia coli O157 Shiga-Like Toxins Type I and II DNA by Multiplex Polymerase Chain Reaction

Escherichia coli O157:H7 has been responsible for several recent food-borne outbreaks in the United States. To protect the public health, it is essential that rapid and sensitive methods be developed for detection of this pathogen in foods. Methods were compared for preparation of template DNA for the polymerase chain reaction (PCR) from enrichments of food homogenates seeded with E. coli O157:H7. Samples were enriched for 6 h at 37°C in modified tryptic soy broth supplemented with vancomycin, cefsulodin, and cefixime. Aliquots of the enrichments (10 ml or 1 ml) were analyzed by either washing twice with physiological saline or incubating with antibodies to O157 coupled to immunomagnetic beads (Dynal®) followed by resuspending and boiling the samples. A portion of the preparation was used in a multiplex PCR to amplify a 274-bp fragment from the sltI gene and a 364-bp fragment from the sltII gene. PCR amplification of 1-ml portions of enrichment broth was successful at inoculation levels of about 10 cells per g of food. Increasing the test sample volume to 10 ml and/or using an immunomagnetic separation step improved the PCR detection sensitivity to about 1 cell per g; the entire analysis can be completed within 12 h.

Optimization of PCR-based diagnostics for human cytomegalovirus.

Various techniques of DNA template preparation for the PCR-based analysis of human CMV in biological fluids have been compared. Structural polymorphism of a CMV DNA segment (part of the major immediate early gene) in clinical isolates is described; the molecular markers (nucleotide substitutions, deletions, insertions) localized in the analyzed amplicon appear to be suitable for molecular-epidemiological studies. A scheme of spreading of the molecular markers in the population is suggested.

A High-Throughput Plasmid DNA Preparation Method

Genome projects require a high-throughput method for DNA template preparation, which traditional protocols can rarely provide. We introduce here a new protocol for plasmid and cosmid DNA preparation based on alkaline denaturation in a 96-well format. The essential improvement made in this protocol on previous 96-well preparation includes the purification by Pro-Cipitate in 96-well microfilters. The yield and quality of DNA are sufficient for restriction digestion, radioactive sequencing, and automated fluorescent sequencing. It is also significantly more cost-effective than several of the commercial mini-prep kits.

Nucleotide Incorporation Opposite Degenerate Bases byTaqDNA Polymerase

Abstract Oligodeoxyribonucleotides containing degenerate bases, 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P), 2-amino-6-methoxyaminopurine (K), and N6 -methoxyadenine (Z) were made on a DNA synthesizer. The oligonucleotides were used for the preparation of DNA templates by enzymatic ligation of DNA fragments. The templates were annealed with a primer and extended with Taq DNA polymerase and dNTPs to reveal the nucleotide(s) incorporated opposite the modified bases. Analysis of the products showed that A was inserted opposite the pyrimidine P and T opposite the purines, K and Z. Incorporation of a small amount of C opposite K was also observed.

Alkali treatment for rapid preparation of plant material for reliable PCR analysis.

For plant genetics, it would be useful to monitor easily the segregation of different alleles using the polymerase chain reaction (PCR). Preparation of DNA templates from individual plants needs to be rapid and reliable. A one tube protocol is described that involves subjecting plant tissue pieces to alkali, neutralization and heat denaturation prior to PCR analysis, and that proved to be much faster and more reliable than published protocols.

Automated magnetic preparation of DNA templates for solid phase sequencing.

An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.

High-throughput fluorescent DNA sequence analysis: Methods and automation

A strategy for high-throughput DNA sequence analysis using the fluorescent dye-primer chemistry is described. This strategy combines rapid preparation of template DNA using a modification of the polymerase chain reaction (PCR), automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Asymmetric PCR provides a reliable means of simultaneously producing sufficient and uniform quantities of several template DNAs directly from bacterial colonies or bacteriophage plaques. The automation scheme for subsequent processing and analysis of DNA samples allows a DNA sequencing facility to operate the fluorescent sequencer twice daily at its full capacity of 24 samples. Furthermore, the methods and instruments described eliminate much of the labor required by technicians to obtain a high level of data output. As described here, a DNA sequencing laboratory equipped with a single automated fluorescent sequencer can perform analysis of up to 48 fluorescent-labeled reactions every day, with an average output of over 10,000 bp per sequence run.

Advances in DNA sequencing technology

Efforts to map and sequence the genomes of the human and other species have stimulated efforts to improve the technology required for these endeavors. During the last year, these efforts have produced substantial advances in DNA template preparation, sequencing chemistry, and gel analysis.

Development of an automated procedure for fluorescent DNA sequencing

We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.

Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli

A new series of expression vectors that direct high-level overproduction of gene products in Escherichia coli is described. All contain strong bacteriophage λ promoters, pR and pL, arranged in tandem so that both promote transcription into genes inserted into or between unique restriction sites. The vectors also direct expression of the λ cl857 gene (from its natural promoter, pM), which enables their use in any E. coli host strain to effect controlled expression by shifting the temperature of cultures from 30 to 42°C. The vectors pCE30, pND201, pPT150 and pMA200U are derivatives of the high-copy-number plasmid pUC9. Vector pCE33 is an analogous derivative of the heat-inducible runaway-replication plasmid. pMOB45, and directs overproduciion of proteins by virtue of increase in both gene dosage and transcription following treatment at 42°C. The vectors pND201 and pPT150 bear a ribosome-binding site (RBS) perfectly complementary to the 3′ end of E. coli 16-S rRNA a few bp upstream from a unique HpaI site. Ways in which they may be used to improve the efficiency of translation of mRNA by substitution of a natural RBS with selection for optimal spacing from an ATG (or GTG) start codon are described. The phagemid vector pMA200U is a direct analog of pCE30 designed to facilitate preparation of single-stranded DNA templates for use in oligodeoxyribonucleotide-directed mutagenesis of overexpressed genes.

Semiautomated preparation of DNA templates for large-scale sequencing projects

The rate limiting step in a large-scale sequencing project is the generation of single-stranded DNA templates. We describe a fast, semiautomated procedure, using 96-well microtitre plates, in which 192 templates can be readily prepared in 1 day. The technique can be carried out manually or can be semiautomated using a robot pipetting device. We also provide evidence for the reliability and applicability of this method to a large-scale sequencing project.

A novel strategy for one-step cloning of full-length cDNA and its application to the genome of barley stripe mosaic virus

We have devised a novel vector-primer strategy for cloning of full-length (FL) cDNA which can be applied to non-polyadenylated RNA species. Single-stranded plasmid DNA is used to prime first-strand synthesis by reverse transcriptase, and plasmids covalently linked to FL cDNA are then circularized by the annealing of a specific oligodeoxyribonucleotide (band-aid oligo). Only limited nucleotide sequence data are required from the termini of each RNA species to be cloned to design the plasmid primer and band-aid oligo. The band-aid strategy has been applied to the cloning of barley stripe mosaic virus genomic RNAs, and found to be both rapid and efficient. A strategy for the preparation of linear double-stranded plasmid DNA templates (suitable for run-off in vitro transcription) which is independent of restriction sites present within the cloned cDNA is also described.