Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support.

Approaches to direct solid phase sequencing of genomic and plasmid DNA have been developed using magnetic beads, coated with streptavidin, as solid support. The DNA is immobilized through selective incorporation of biotin into one of the strands. A single stranded template, suitable for sequencing, is obtained through strand-specific elution. Using this concept, in vitro amplified plasmid DNA and chromosomal DNA were sequenced directly from single colonies. The solid phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The system was found to be suitable for sequencing using both isotope- and fluorescent-labelled primers.     

Bidirectional solid-phase sequencing of in vitro-amplified plasmid DNA

A solid-phase approach is described for manual and automated sequencing of plasmid DNA obtained directly from bacterial colonies through the polymerase chain reaction. The DNA fragment is selectively immobilized to magnetic beads and after strand-specific elution, the eluted strand, as well as the remaining immobilized strand, is used for bidirectional dideoxy sequencing. The solid-phase approach ensures that the amplification and the sequencing reactions can be performed under optimal conditions. The approach is exemplified by fluorescent sequencing of a cloned Streptomyces curacoi gene having a G + C content of more than 70%.     

The use of exonuclease III for preparing single stranded DNA for use as a template in the chain terminator sequencing method.

Exonuclease III, which degrades DNA 3' leads to 5' and is specific for duplex DNA, can be used to prepare single stranded DNA from linear duplexes. This is shown to be suitable as a template for use with the chain terminator DNA sequencing method of Sanger et al. [1]. Strategies are discussed for preparing single stranded DNA templates by this method and, in particular, its application to the sequence analysis of DNA cloned in plasmid vectors.     

Semi-automated solid-phase DNA sequencing.

Increasing the efficiency of DNA sequencing necessitates the development of systems which reduce the need for manual operations by integrating template preparation, sequencing reactions, product separation and detection. A semi-automated system, whereby PCR-amplified biotinylated genomic or plasmid DNA is immobilized on streptavidin-coated magnetic beads, has been developed.     

Efficient site-directed mutagenesis by simultaneous use of two primers.

A rapid and efficient procedure for site specific mutagenesis is described. A double primed synthesis with a 17-mer mismatch primer and a "universal" 15-mer M13 sequencing primer was used to introduce a T to A transversion into an ompF signal peptide gene cloned in the M13mp8 vector. The two primers were annealed to the circular single stranded M13 template. After a short extension and ligation reaction, a double stranded restriction fragment containing the mismatch (ompF*/EcoR1-SalI) was cut out of the partly single stranded circular DNA and inserted into pBR322. 42% of the E.coli transformants harboured plasmid with the desired mutation, which could be detected by the appearance of a new restriction site (MboII) and by dot blot hybridization of plasmid DNA with the 32P-labeled 17-mer.     

Approaches to Solid Phase DNA Sequencing

Abstract

A novel approach to enable two-directional solid-phase DNA sequencing, involving immobilization of double stranded plasmid to avidin agarose, is described. A plasmid vector, pRIT28, has been designed to allow enzymatic incorporation of biotinylated dUTP into two alternative sites.     

Dideoxy sequencing method using denatured plasmid templates

The dideoxy sequencing method in which denatured plasmid DNA is used as a template was improved. The method is simple and rapid: the recombinant plasmid DNA is extracted and purified by rapid alkaline lysis followed by ribonuclease treatment. The plasmid DNA is then immediately denatured with alkali and subjected to a sequencing reaction utilizing synthetic oligonucleotide primers. It takes only several hours from the start of the plasmid extraction to the end of the sequencing reaction. We examined each step of the procedure, and several points were found to be crucial for making the method reproducible and powerful: (i) the plasmid DNA should be free from RNA and open circular (or linear) DNA; (ii) a heptadecamer rather than a pentadecamer is recommended as a primer; and (iii) the sequencing reaction should be done at 37 degrees C or higher rather than at room temperature. The method enabled us to determine the sequence of more than a thousand nucleotides from a single template DNA.     

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.     

Rapid colorimetric detection of in vitro amplified DNA sequences

A colorimetric assay to detect immobilized amplified nucleic acids has been designed. This approach provides a rapid assay, suitable for clinical diagnosis, to analyze DNA sequences amplified by the polymerase chain reaction. The specific DNA sequences are captured on a solid support by the use of a recombinant fusion protein consisting of the Escherichia coli lac repressor and staphylococcal protein A. The biotin streptavidin system is used to detect the immobilized material. Positive samples can be analyzed by direct solid-phase sequencing. Here, we show that this nonradioactive concept can be used for analysis of Staphylococci and Streptococci and for specific detection of the protozoa Plasmodium falciparum in clinical samples.     

一种快速有效纯化DNA序列分析模板的方法

介绍一种ＤＮＡ序列分析模板的快速、有效的纯化方法。该法对ＤＮＡ模板的回收率可达９５％以上。多次测序结果表明,此法与其他常规纯化方法相比,具有简便、快速、有效、可靠等优点,其测序结果电泳带清晰,无模糊带及“鬼带”出现,重复性及稳定性较好。     

Construction of a recombinant bacterial plasmid containing pro-alpha 1(I) collagen DNA sequences

A partially purified mRNA preparation enriched for chick collagen messenger RNA activity was used as template for the synthesis of double stranded cDNA. The cDNA was ligated into the HindIII site of the plasmid vector pBR322 and used to transform Escherichia coli x1776. One plasmid with an 800-base pair insert was shown to contain DNA sequences corresponding to Type I pro-alpha 1 collagen.     

Use of a chemically modified T7 DNA polymerase for manual and automated sequencing of supercoiled DNA

Procedures are presented for reliable and accurate nucleotide sequence analysis using as template supercoiled DNA prepared by a modified rapid boiling minipreparation protocol. This method yields DNA templates suitable for sequencing within 1 h of bacterial harvest. We describe optimal reaction conditions for supercoiled miniprep DNA sequencing using a modified T7 DNA polymerase (Sequenase) in dideoxynucleotide chain termination reactions. We demonstrate that under these conditions, the sequencing data obtained with miniprep DNA is indistinguishable from that obtained with CsCl purified supercoiled DNA or from that obtained using single stranded DNA templates. We further show that the supercoiled DNA sequencing reactions can be analyzed on a commercially available automated DNA sequencing system that detects 32P labeled DNA during its electrophoretic separation. Taken together, these developments represent a significant improvement in the process of nucleotide sequence analysis.     

A one-step miniprep for the isolation of plasmid DNA and lambda phage particles

Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.     

An improved filamentous helper phage for generating single-stranded plasmid DNA

Gene cloning in plasmid vectors that contain a filamentous phage intergenic region presents several advantages. However, technical difficulties have been a problem, primarily low yields of packaged single stranded (ss) plasmid DNA from the rapid, small scale procedures usually employed, and ambiguities in sequencing reactions attributed to the contamination by helper phage ss DNA. We report here the construction and some properties of a new f1 helper phage. Using this phage, R408, plasmid ss DNA is packaged and exported preferentially over phage ss DNA, and the absolute yield of plasmid ss DNA is usually increased.     

Circular single stranded phage M13-DNA as a template for DNA synthesis in protein extracts from Xenopus laevis eggs: evidence for a eukaryotic DNA priming activity.

Unfractionated protein extracts from activated Xenopus laevis eggs contain all functions required for the chain elongation reactions in replicative DNA synthesis (A.Richter, B.Otto and R.Knippers, 1981, Nucl.Ac.Res. 9, 3793-3807). In order to further explore the DNA synthesizing capacity of this in vitro system and to obtain information on the DNA priming activity in these extracts single stranded phage M13-DNA was used as template for in vitro DNA synthesis. The main results of this investigation are: (i) single stranded circular template DNA is converted to a double stranded DNA form in an alpha-amanitin-insensitive reaction which is absolutely dependent on ribonucleoside triphosphates; (ii) the in vitro synthesized complementary strands are DNA fragments of 1000-2000 nucleotides lengths; (iii) the DNA primase activity copurifies through several column steps and sucrose gradient centrifugation with a DNA polymerase alpha. These activities may therefore be closely associated in a quarternary enzyme complex.     

Colorimetric quantification of in vitro-amplified template DNA to be used for solid phase sequencing.

A protocol for colorimetric determination of DNA amplified by the polymerase chain reaction (PCR) and subsequently immobilized to a solid support is described. The protocol consists of three steps: (i) binding of PCR amplified lac operator-containing DNA to magnetic beads; (ii) binding of a Lac repressor-beta-galactosidase fusion protein to the lac operator and (iii) colorimetric detection of the immobilized beta-galactosidase. In practice, steps (i) and (ii) are performed concurrently. The protocol is well suited both for manual and automated procedures and the immobilized template can, after melting, be used directly for solid phase sequencing. The assay is used to demonstrate that template concentration is important for the quality of sequence data obtained from an automated DNA sequencer.     

Isothermal strand-displacement amplification applications for high-throughput genomics

Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using random hexamer primers with 29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.     

A Method for Preparing DNA Sequencing Templates Using a DNA-Binding Microplate

A DNA-binding matrix was immobilized on the surface of a 96-well microplate and used for plasmid DNA preparation for DNA sequencing. The same DNA-binding plate was used for bacterial growth, cell lysis, DNA purification, and storage. In a single step using one buffer, bacterial cells were lysed by enzymes, and released DNA was captured on the plate simultaneously. After two wash steps, DNA was eluted and stored in the same plate. Inclusion of phosphates in the culture medium was found to enhance the yield of plasmid significantly. Purified DNA samples were used successfully in DNA sequencing with high consistency and reproducibility. Eleven vectors and nine libraries were tested using this method. In 10 μl sequencing reactions using 3 μl sample and 0.25 μl BigDye Terminator v3.1, the results from a 3730xl sequencer gave a success rate of 90–95% and read-lengths of 700 bases or more. The method is fully automatable and convenient for manual operation as well. It enables reproducible, high-throughput, rapid production of DNA with purity and yields sufficient for high-quality DNA sequencing at a substantially reduced cost.     

Non-radioactive automated sequencing of oligonucleotides by chemical degradation

A non-radioactive sequencing of fluorescently labelled oligonucleotides by solid-phase chemical degradation is described. Although non-radioactive methods have been reported for the dideoxy chain termination technique, such a method has not yet been developed for the chemical degradation sequencing of DNA fragments. A 21-mer fluorescein labelled M13 sequencing primer was sequenced in an on-line automated system in about 30 minutes. The fluorescent dye and its bond to the oligonucleotide were stable during the chemical reactions used for the base specific degradations. As the sequence is determined on-line during electrophoresis, reloading and running 10 fragments simultaneously allows us to use one gel for sequencing of about 50 different oligonucleotides.