Name: streamlined purification of plasmid dna from prokaryotic cultures
Uploaded: 2011-01-05T17:00:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

1. Lysate Clearance
<ol>
 <li>Grow E. coli cultures transformed with desired plasmid DNA in deep well plates (1 mL of culture/well) in Luria Broth with appropriate antibiotic overnight at 37 &deg;C with shaking.</li>
 <li>Pellet E. coli in culture plate at 5,000 x g for 10 minutes, then decant supernatant.</li>
 <li> Resuspend each pellet in 100 &mu;L Resuspension Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA,100 &mu;g/mL RNase A).</li>
	<li>Add 100 &mu;L/well Lysis Buffer (200 mM NaOH, 1% SDS) and shake using plate shaker for 2 minutes.</li>
 <li> Add 100 &mu;L/well Neutralization Buffer (3.0 M Potassium Acetate, pH 5.5) and shake for 2 minutes.</li>
	<li>Transfer cell lysate to lysate clarification plate.</li>
 <li> Place DNA binding plate on top of 350 &mu;L collection plate and place into bottom of vacuum manifold. Assemble vacuum manifold (see Figure 1).</li>
	<li>Place lysate clarification plate on top of vacuum apparatus.</li>
 <li> Apply vacuum at 10 in. Hg (0.34 bar) and collect filtrate into DNA binding plate. Do not use vacuum greater than 12 in. Hg. (0.41 bar).</li>
	<li>Disassemble vacuum manifold. Place 2 mL waste collection plate in bottom of apparatus.</li>
</ol>
2. DNA Binding
 <ol>
	<li>Move the DNA binding plate to the top of the manifold (see Figure 2).</li>
	<li>Add 300 &mu;L/well Binding Buffer (6 M Guanidine-HCl) and pipette up and down to mix.</li>
 <li>Apply vacuum [~ 5 in. Hg (0.17 bar) for slow vacuum] and discard filtrate. DNA is now bound to membrane.</li>
	<li>Wash with 400 &mu;L/well of Wash Buffer (80% Ethanol).</li>
	<li>Apply vacuum, then discard the filtrate. Discard unnecessary if using 2 mL collection plate or filtering directly to waste.</li>
	<li>Repeat wash once.</li>
	<li>Blot bottom of filter plate on absorbent towel to dry.</li>
	<li>Apply vacuum once more for 5-10 minutes to ensure removal of residual alcohol.</li>
	<li>Blot bottom to ensure removal of ethanol droplets.</li>
 </ol>
3. DNA Elution
<ol>
	<li> Add 70 &mu;L/well Elution Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). If higher DNA concentration is desired, volume can be reduced to 50 &mu;L/well, but the average total yield will be lower.</li>
	<li>Incubate the plate at room temperature for one minute.</li>
	<li>Purified DNA can be eluted by either vacuum or centrifugation.</li>
	<li>Vacuum method: Place clean collection plate (DNase, RNase free) into vacuum manifold. Place filter plate on top of vacuum manifold, apply vacuum at 15 in. Hg (0.5 bar) for 1 minute until all elution buffer has passed through the DNA binding plate. Collect purified DNA (see Figure 3).</li>
	<li>Centrifugation method: Place purification filter plate on top of clean collection plate and centrifuge at 1,000 x g for 5 minutes.</li>
</ol>
4. Quantitative and Qualitative Analyses
<ol>
	<li> Measure OD260/280, calculate concentration of purified DNA.</li>
	<li>Perform agarose gel analysis of the purified pDNA.</li>
</ol>
5. Representative Results
E. coli lysate clarified using traditional centrifugation and Pall AcroPrep Advance lysate clarification Filter Plates showed appreciable reduction of debris from starting material. (Figure 4).
Plasmid DNA at three concentrations was spiked into TE buffer and passed through the AcroPrep Advance lysate clarification filter plate. A comparison of plasmid DNA concentration, pre- and post-filtration, shows ~100% recovery (Figure 5) at all three concentrations.
To demonstrate the recovery of DNA from clarified E. coli lysate, plasmid DNA was spiked into crude lysate and then clarified by filtration. The same plasmid DNA was added to cell lysate following filtration. Electrophoresis was performed on aliquots from each sample (Figure 6). A single, clear band of similar intensity from each sample demonstrates little if any DNA loss during the filtration-based clarification step.
Cultures of E. coli containing pCAT plasmid DNA were purified from E. coli lysates using Pall's 350 &mu;L and 1 mL DNA binding Filter Plates, as well as two other brands of 350 &mu;L DNA binding plates. All purifications were performed as specified in the manufacturer's recommended protocol. The highest DNA concentration and total yield are seen from Pall's 1 mL AcroPrep Advance DNA purification filter plate (Figure 7 and Table 2).
Although Competitor W also shows high DNA concentration (155 ng/&mu;L), total DNA yield is the lowest (4.7 &mu;g/well) due to the low recovery volume, ~30 &mu;L. Competitor M gave the lowest concentration of DNA at 108 ng/&mu;L with a total yield of 5.6 &mu;g/well.
Agarose gel analysis of purified pCAT DNA from the three different DNA binding plates is shown in Figure 8. Each collected sample volume was adjusted to 65 &mu;L to correct for differences in recovery volume prior to electrophoresis. All samples show supercoiled plasmid DNA, but DNA yield from each of the Filter Plates varies.
Figure 9 shows the prepared DNA is all in supercoiled form and has similar concentration.
<img src="/files/ftp_upload/2407/2407fig1.jpg" alt="Figure 1" /> Figure 1. Assembly of Vacuum Manifold for Lysate Clarification.
<img src="/files/ftp_upload/2407/2407fig2.jpg" alt="Figure 2" /> Figure 2. Assembly of Vacuum Manifold for DNA Binding.
<img src="/files/ftp_upload/2407/2407fig3.jpg" alt="Figure 3" /> Figure 3. Assembly of Vacuum Manifold for Vacuum Elution.
<img src="/files/ftp_upload/2407/2407fig4.jpg" alt="Figure 4" /> Figure 4. Filtration simplifies sample clarification. Triplicate samples of 300 &mu;L of crude lysate clarified by vacuum filtration (350 &mu;L AcroPrep Advance lysate clarification filter plate) or traditional centrifugation. OD320 measured before and after clarification.
<img src="/files/ftp_upload/2407/2407fig5.jpg" alt="Figure 5" /> Figure 5. Full recovery of pDNA after passage through AcroPrep Advance lysate clarification filter plate. 300 &mu;L TE buffer spiked with pCAT at 25, 50 and 100 &mu;g/mL. DNA concentration and recovery calculated from OD260 before and after filtration, N = 2.
<img src="/files/ftp_upload/2407/2407fig6.jpg" alt="Figure 6" /> Figure 6. Recovery of pCAT DNA after clarification. Equal amounts of pUC19 spiked into lysate before or after clarification, then diluted 1 to 10 in TE buffer. Loaded 2 &mu;L/lane on 1.2% Agarose gel.
<img src="/files/ftp_upload/2407/2407fig7.jpg" alt="Figure 7" /> Figure 7. Higher plasmid DNA yield per well with pall Acroprep Advance DNA binding filter plate than competitor plates. pCAT plasmid DNA yield (OD260) using indicated DNA purification plates with 1 mL (Pall and Competitor M) or 1.5 mL (Competitor W) overnight culture of DH5&alpha;. Purification using plate manufacturer's recommended protocol. Error bars indicate standard error (n &gt; 6).
<img src="/files/ftp_upload/2407/2407fig8.jpg" alt="Figure 8" /> Figure 8. Agarose gel analysis of pCAT DNA purified on indicated DNA binding plate. 1.2% agarose gel electrophoresis of the purified pDNA. Pooled samples from three separate purifications, eluate adjusted to 65 &mu;L after purification, diluted 1:10. Loaded 2 &mu;L per lane.
<img src="/files/ftp_upload/2407/2407fig9.jpg" alt="Figure 9" /> Figure 9. Quality and recovery of pure dna similar when centrifuge or vacuum methods are used for final eluate collection agarose gel electrophoresis of the purified pDNA. The manufacturer's recommended protocol for AcroPrep Advance Filter Plates was followed for pDNA purification. Elution was performed by centrifuge at 1,000 x g for 5 minutes or vacuum with 15 in. Hg (0.5 bar) for 2 minutes.

The authors of this video-article are employees of Pall Life Sciences who produce the instrument and reagents featured in the video and text.

We describe a facile, streamlined protocol of AcroPrep Advance Filter Plates that affords several advantages. This method provides maximum plasmid DNA yields when processed in manual, semi-automated, or fully-automated AcroPrep Advance formats. An integrated prefilter on the lysate clearance plate provides consistent filtration of samples, even those containing high levels of particulates. The well geometry of the plate results in faster, more uniform filtration rates across the plate with minimal hold-up volume; and the outlet tip geometry provides direct flow of samples into receiver plate without concerns of cross contamination. Importantly, the high binding capacity membrane in DNA binding plate of AcroPrep Advance Filter Plates is optimized for maximum recovery of high quality plasmid DNA.

<table><tbody><tr><td>AcroPrep Advance Filter plate</td><td>Pall Corporation</td><td>PN 8175 (1 mL) or PN 8075 (350 &amp;#181;l)</td><td>3 &amp;#x03BC;m glass fiber 0.2 &amp;#x03BC;m Supor&amp;#174; membrane for clarification</td></tr><tr><td>AcroPrep Advance Filter Plate</td><td>Pall Corporation</td><td>PN 8132</td><td>for DNA Binding</td></tr><tr><td>Vacuum manifold</td><td>Pall Corporation</td><td>PN 5017</td><td></td></tr><tr><td>Vacuum pressure pump</td><td>Pall Corporation</td><td>PN 13157</td><td></td></tr></tbody></table>

streamlined purification of plasmid dna from prokaryotic cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

Watch this Scientific Journal Video about Streamlined Purification of Plasmid DNA From Prokaryotic Cultures at JoVE.com

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

streamlined-purification-of-plasmid-dna-from-prokaryotic-cultures

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

19.9K Views.  Pall Life Sciences.  中文 

Video: Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Biochemistry

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Immunology and Infection

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

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Plasmid Stability Analysis with Open-Source Droplet Microfluidics

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating the construction of biological circuits and pathways within and between cell populations. For many applications, maintaining functional plasmids without antibiotic selection is critical. This study introduces an open-hardware-based microfluidic workflow for analyzing plasmid retention by culturing single cells in gel microdroplets and quantifying microcolonies using fluorescence microscopy. This approach allows for the parallel analysis of numerous droplets and microcolonies, providing greater statistical power compared to traditional plate counting and enabling the integration of the assay into other droplet microfluidic workflows. By using plasmids expressing fluorescent proteins alongside a non-specific fluorescent DNA stain, single colonies can be identified and differentiated based on plasmid loss or fluorescent marker expression. Notably, this advanced workflow, implemented with open-source hardware, offers precise flow control and temperature management of both the sample and the microfluidic chip. These features enhance the workflow&#39;s ease of use, robustness, and accessibility. While the study focuses on Escherichia coli as the experimental model, the method&#39;s true potential lies in its versatility. It can be adapted for various studies requiring fluorescence signal quantification from plasmids or stains, as well as for other applications. The adoption of open-source hardware broadens the potential for conducting high-throughput bioanalyses using accessible technology in diverse research settings.

An accessible, open-source microfluidic workflow is presented for the parallelized analysis of plasmid retention in bacteria. By employing fluorescence microscopy to quantify plasmid presence in single-cell microcolonies within gel microdroplets, this method provides a precise, accessible, and scalable alternative to traditional plate counting.

Plasmids play a vital role in synthetic biology by enabling the introduction and expression of foreign genes in various organisms, thereby facilitating ...

Bioengineering

Use of In Vivo Assembly for High-efficiency Plasmid Construction

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA fragments to assemble plasmids. This method functions by transforming DNA fragments with regions of 15-50 bp of homology into commonly used laboratory Escherichia coli strains and the bacteria use the RecA-independent repair pathway to assemble the DNA fragments into a plasmid. This method is more rapid and cost-effective than many molecular cloning methods that rely on in vitro assembly of plasmids prior to transformation into E. coli strains. This is because in vitro methods require the purchase of specialized enzymes and the performance of sequential enzymatic reactions that require incubations. However, unlike in vitro methods, IVA has not been experimentally shown to assemble linear plasmids. Here we share the IVA protocol used by our laboratory to rapidly assemble plasmids and subclone DNA fragments between plasmids with different origins of replication and antibiotic resistance markers.

In vivo assembly is a ligation-independent cloning method that relies on intrinsic DNA repair enzymes in bacteria to assemble DNA fragments by homologous recombination. This protocol is both time and cost-effective, as few reagents are required, and cloning efficiency can be as high as 99 %.

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

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Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...