Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis.

We report two methods for isolation of plasmid DNA from the gram-positive bacterium Bacillus subtilis. The protoplast alkaline lysis procedure was developed for general use, and the protoplast alkaline lysis magic procedure was developed for isolation of DNA for sequencing. Both procedures yielded large amounts of high-quality DNA in less than 1 h, while current protocols require 4 to 7 h to perform and give lower yields and quality. Plasmid DNA was obtained from strains containing either high- or low-copy-number plasmids. In addition, the procedures were easily adapted to yield large amounts of plasmid DNA suitable for sequencing from another gram-positive organism, Staphylococcus aureus. Further, we demonstrated that neither chloramphenicol, used for plasmid selection, nor the mutation recE4 reduced plasmid DNA yield from the strains we examined.     

A simple procedure for isolation of cloning vectors and endogenous plasmids from viridans group streptococci and Staphylococcus aureus.

Isolation of plasmid DNA from viridans group streptococci is difficult, and preparations are often heavily contaminated with chromosomal DNA. We developed a simple protocol to isolate pure plasmid DNA for use in different molecular techniques, including automated sequencing. The protocol is also applicable for plasmid isolation from Staphylococcus aureus. In addition, the protocol allows isolation of pure endogenous plasmids from streptococci and S. aureus.     

Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol.

We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid.     

P51-T Optimizing Heat Lysis for High-Throughput Sequencing of Various Plasmid Libraries

Heat lysis is a very reliable method for high-throughput labs to decrease costs and increase throughput with little sacrifice in quality of sequence data compared to typically used two-plate or magnetic-bead DNA purification methods. Laboratories employing this procedure on high-copy plasmids, using a sequencing reaction with a 1/8^th Big Dye (Applied Biosystems) dilution, have reported generating data with 85% of all wells having read lengths of at least 600 bases with quality value (QV) 20 or above.¹We have adopted this procedure in our laboratory and optimized it for sequencing from various in-house cDNA and genomic shotgun libraries cloned into high-copy plasmids, as well as for libraries constructed outside of our sequencing center. To fully develop the procedure, we utilized a number of culture plates, testing different plate volumes, well shapes, and growth times. Also, we tested various volumes of resuspension buffer in order to generate the highest sequencing success rates and read lengths. To further cut costs, we optimized the sequencing reactions by testing various dilutions of Big Dye and template amounts.Based upon over 55,000 reads, we have been able to consistently generate sequencing results with average success rates of 90–95% and read lengths of over 700 bases with QV 20 or above. Our past protocol employed a standard two-plate DNA preparation method with a 1/16^th Big Dye dilution in the sequencing reaction. In contrast, we now use the optimized heat lysis protocol combined with a 1/32^nd Big Dye dilution. These changes have increased throughput and produced the high-quality sequencing results stated above, yet reduced our consumables cost by over 55%.     

Affinity purification of plasmid DNA by temperature-triggered precipitation

This protocol presents a new method to purify plasmid DNA using temperature-triggered precipitation. The principle is based on the specific DNA-binding affinity of a bacterial metalloregulatory (MerR) protein to its cognate DNA sequence and the temperature responsiveness of elastin-like protein (ELP). A bifunctional ELP-MerR fusion protein is created to enable the precipitation of plasmid DNA, designed to contain the MerR recognition sequence, by a simple temperature trigger. The protocol covers all stages of the process from the design of ELP-MerR fusion proteins and MerR-binding plasmids, to the isolation of plasmid DNA from Escherichia coli cultures after boiling lysis, the subsequent temperature-triggered precipitation of plasmid DNA-fusion protein complexes and final elution of plasmid DNA by mild heating. This protocol is well suited to laboratory research-scale applications, producing plasmid DNA of better purity and similar yield as one of the most commonly used laboratory methods, standard alkaline lysis (known as the midiprep procedure). The protocol takes approximately 30 min to obtain pure plasmid DNA from cell cultures using the temperature-triggered precipitation method.     

CircumVent thermal cycle sequencing and alternative manual and automated DNA sequencing protocols using the highly thermostable VentR (exo-) DNA polymerase.

CircumVent thermal cycle and standard DNA sequencing protocols utilizing the cloned and highly thermostable VentR (exo-) DNA polymerase are described. The thermal cycle sequencing procedures are advantageous because they allow fast and simple semiautomation of the sequencing reaction; make possible the direct DNA sequencing of PCR products, bacterial colonies and phage plaques; require only femtomoles of template DNA; eliminate the requirement of an independent primer annealing step; remove the requirement of denatured plasmids for sequencing double-stranded templates; and use a highly thermostable DNA polymerase for sequencing through potential recalcitrant secondary structure domains and large linear double-stranded DNA templates such as lambda derivatives. More standard methods of DNA sequencing (i.e., a one-step protocol and a labeling-termination protocol) are also presented. For each protocol, alternatives for choice of label and method of labeling are presented, including the use of 5' biotinylated primers for chemiluminescent DNA sequencing and fluorinated primers for automated sequencing using the BaseStation Automated DNA Sequencer.     

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.     

Flexprep: Scale-flexible rapid plasmid preparation for analysis of recombinant clones

A scale-flexible and cost-efective protocol for plasmid preparation is described to cover miniprep and midiprep scale work in a microcentriguge format for analysis of recombinant clones. this protocol relies on a modified alkaline lysis of Escherichia coli cells and subsequent purification of plasmid DNA with no organic extraction and alcohol precipitation. It can process up to 20 mL of E. coli cells carrying 3–10 kbp plasmid vectors in <10 min. Flexprep delivers sufficient yield and purity of plasmid DNA for routine applications including restriction enzyme digestion and fluorescent automated sequencing.     

A procedure for large-scale plasmid isolation without using ultracentrifugation.

An expedient procedure for large-scale plasmid isolation from Escherichia coli strains without using ultracentrifugation or special setups or reagents is described. The protocol, which utilizes a modified alkaline extraction procedure as well as differential precipitations by isopropanol and lithium chloride, is simple and rapid and yet produces plasmid DNA with a yield of about 2 mg/liter culture. The isolated plasmids consisted of mostly monomeric and dimeric covalently closed circular DNA. The plasmids could be digested by various restriction endonucleases and were compatible with gene cloning, transfection-gene expression, and viral production.     

Magnetic, microplate-format plasmid isolation protocol for high-yield, sequencing-grade DNA

We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.     

Rapid and reliable fluorescent cycle sequencing of double-stranded templates.

Automated DNA sequencing is an extremely valuable technique which requires very high quality DNA templates to be carried out successfully. While it has been possible to readily produce large numbers of such templates from M13 or other single-stranded vectors for several years, the sequencing of double-stranded DNA templates using the ABI 373 DNA Sequencer has had a considerably lower success rate. We describe how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure. From a single five milliliter culture enough DNA can be isolated (up to 20 micrograms) to do 4-8 sequencing reactions, each of which yields 400-500 bases of high quality sequence data. These procedures make the routine use of double-stranded DNA templates a viable strategy in automated DNA sequencing projects.     

Purification of RNA-free plasmid DNA using alkaline extraction followed by Ultrogel A2 column chromatography

A procedure for extracting RNA-free plasmid DNA from bacterial cells is described. The method is simple and rapid enough to obtain pure plasmid DNA in 8 to 10 h after plasmid amplification. The protocol uses the alkaline extraction procedure described by Birnboim and Doly (1979, Nucl. Acid Res. 7, 1513-1523). Plasmid DNA is then separated from high-molecular-weight RNA by ammonium acetate precipitation and from low-molecular-weight RNA contaminants by Ultrogel A2 column chromatography. The plasmid DNA obtained by this inexpensive technique is sufficiently pure to be used for restriction endonuclease analysis, 5'-end labeling, S1 mapping, DNA sequencing, and colony hydridization.     

Plasmid sonication improves sequencing efficiency and quality in the Beckman Coulter CEQ system

We report on an unexpectedly high rate of unreadable chromatograms from plasmid sequencing using Beckman Coulter's protocols, chemistry, and CEQ8000 instrument. Failed or poor quality plasmid sequence chromatograms were accompanied by a sharp drop, fluctuation, or steady decline in the current and a corresponding delay in signal counts beyond the time of capillary injection. We observe a correlation between the presence of supercoiled DNA and these sequencing problems. Herein we demonstrate that plasmid sonication, which is known to fragment supercoiled DNA, is an effective way to improve sequence phred20 read lengths to the point that they are not significantly different from Beckman Coulter's control template or enzymatically linearized plasmids.     

Simple method for extracting plasmid DNA from lactic acid bacteria

Rapid screening and large-scale plasmid DNA isolation procedures are described for lactic acid bacteria, using glass beads to break cells. The rapid screening procedure allows one to obtain plasmid DNA pellets in less than 1 h. This method has been successfully tested on various bacteria from the genera Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Streptococcus, Enterococcus and Propionibacterium. This procedure yields plasmid DNA with minor chromosomal and plasmid DNA-degraded form contaminations.     

Molecular cloning of DNA complementary to a mouse α-fetoprotein mRNA sequence

DNA complementary to mouse yolk sac messenger RNA has been inserted at the PstI site of the plasmid pBR322 by annealing of the oligo(dG)-tailed plasmid DNA with the oligo(dC)-tailed mouse DNA. Transformation of Escherichia coli strain RRI with this annealed DNA yielded clones bearing recombinant plasmids. The clones were screened for DNA complementary to mouse a-fetoprotein (AFP) messenger RNA sequences by hybridization with a cDNA probe transcribed from an AFP mRNA of over 90% purity. Out of nine plasmids that were isolated and analyzed by restriction mapping, all had homologous insert DNA of various lengths. The plasmid with the longest insert, pAF6, contained 1.65 kb of added DNA, which is about 70% of the AFP mRNA. This clone was positively identified by a hybridization-translation procedure to contain a cDNA sequence for AFP. A restriction map of this clone and the orientation of the message are presented.     

Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

IncX plasmids are narrow host range plasmids of Enterobactericeae that have been isolated for over 50years. They are known to encode type IV fimbriae enabling their own conjugative transfer, and to provide accessory functions to their host bacteria such as resistance towards antimicrobial agents and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid diversity and prevalence is underappreciated. To address these possible shortcomings, we generated additional plasmid sequences of IncX plasmids of interest and compared them to the genomes of all sequenced IncX-like plasmids. These comparisons revealed that IncX plasmids possess a highly syntenic plasmid backbone, but that they are quite divergent with respect to nucleotide and amino acid similarity. Based on phylogenetic comparisons of the sequenced IncX plasmids, the IncX plasmid group has been expanded to include at least four subtypes, IncX1-IncX4. A revised IncX plasmid replicon typing procedure, based upon these sequences and subtypes, was then developed. Use of this revised typing procedure revealed that IncX plasmid occurrence among bacterial populations is much more common than had previously been acknowledged. Thus, this revised procedure can be used to better discern the occurrence of IncX type plasmids among enterobacterial populations.     

Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle

Bovine papilloma virus (BPV) replicates as a multicopy nuclear plasmid in mouse fibroblasts. Using fluorescence activated cell sorting and mitotic selection procedures, we show that the replication of BPV occurs throughout S phase of the cell cycle and that replication is confined to S phase. After one round of chromosomal DNA replication, almost one quarter of BPV plasmids have replicated more than once, while a similar number of plasmids have not replicated at all. While multiple forms of BPV exist in the cell, all forms show the same pattern of replication. These results are consistent with a model in which BPV plasmids are chosen at random for replication throughout, and only during, S phase and support the view that the completion of S phase is a specifically activated event in the cell cycle rather than simply the end of one round of chromosomal DNA replication.     

A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis

A simple and rapid microscale technique is described for the isolation of plasmid DNA which involves cell lysis with phenol, centrifugation, phenol extraction, ethanol precipitation, and RNase digestion. The plasmid DNA is of suitable purity and quantity for multiple restriction endonuclease digestions and bacterial transformations. This “miniprep” procedure is applicable for a variety of types of plasmids ranging in size from 2900 to 18,400 base pairs (bp) and for a number of Escherichia coli strains. The plasmids are rapidly cleaved by all restriction enzymes (total of 14) tested to date. Recombinant clones have been screened for insertions as small as 10 bp and as large as 5000 bp. The procedure takes ~3 h and has been routinely used to simultaneously analyze 24 candidate clones. This procedure is reliable and useful for rapid screening of recombinant DNA candidates where analysis by restriction endonuclease digestion is necessary.     

Increased Throughput by Parallelization of Library Preparation for Massive Sequencing

Background

Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing.

Methodology/Principal Findings

In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling.

Conclusions/Significance

We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.