Minimal Template Research Articles

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems

The study describes a protocol for creating large (µg-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product.

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems.

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 µg of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

High throughput screening for expanded CTG repeats in myotonic dystrophy type 1 using melt curve analysis.

BackgroundMyotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet‐primed PCR (TP‐PCR) is technically challenging and cost prohibitive for population surveys.MethodsHere, we present a high throughput, low‐cost screening tool for CTG repeat expansions using TP‐PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye.ResultsWe determined that multimodal melt profiles from the TP‐PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri‐modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles.ConclusionWe demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large‐scale testing programs such as population studies and newborn screening programs.

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours.

In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell-based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost-optimized, scalably-manufactured Escherichia coli extract and minimal supplement reagents to perform cell-free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti-GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1-7)M(2-9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications.

Rapid identification of Gekko gecko by loop-mediated isothermal amplification based on 12S rRNA sequence

Gekko gecko (Tokay Gecko) is a valuable traditional Chinese medicine. In this study, the loop-mediated isothermal amplification (LAMP) technique was introduced for visual rapid identification of G. gecko from adulterants. A total of sixty-five 12S rRNA sequences of fourteen species of G. gecko and its adulterants were obtained. The results showed that G. gecko could be identified from its adulterants through BLAST analysis based on 12S rRNA regions. The 12S rRNA sequences of ten batches of G. gecko were conserved. There were only two haplotypes and three variation sites in the available regions for primers design. Six specific LAMP primers were successfully designed online based on 12S rRNA sequences. The visual rapid detection of G. gecko could be achieved with the optimized conditions (64 °C for 1 h and 80 °C for 5 min). And the required minimal template concentration was 5 μg·L⁻¹ while conventional PCR with 0.5 mg·L⁻¹. Consequently, the LAMP method established from this study was rapid, specific, highly sensitive, and simple. It could be applied to detect G. gecko from its adulterants efficiently.

Update on SU(2) gauge theory with NF = 2 fundamental flavours.

We present a non perturbative study of SU(2) gauge theory with two fundamental Dirac flavours. This theory provides a minimal template which is ideal for a wide class of Standard Model extensions featuring novel strong dynamics, such as a minimal realization of composite Higgs models. We present an update on the status of the meson spectrum and decay constants based on increased statistics on our existing ensembles and the inclusion of new ensembles with lighter pion masses, resulting in a more reliable chiral extrapolation. Preprint: CP3-Origins-2017-048 DNRF90

Fundamental composite electroweak dynamics: Status at the LHC

We determine the current status of the fundamental composite electroweak dynamics paradigm after the discovery of the Higgs boson at the Large Hadron Collider experiments. Our analysis serves as universal and minimal template for a wide class of models with the two limits in parameter space being composite Goldstone Higgs models and Technicolor. This is possible because of the existence of a unified description, both at the effective and fundamental Lagrangian levels, of models of composite Higgs dynamics where the Higgs boson itself can emerge, depending on the way the electroweak symmetry is embedded, either as a pseudo-Goldstone boson or as a massive excitation of the condensate. We constrain the available parameter space at the effective Lagrangian level. We show that a wide class of models of fundamental composite electroweak dynamics, including Technicolor, are compatible with experiments. The results are relevant for future searches of a fundamental composite nature of the Higgs mechanism at the Large Hadron Collider.

Composite Higgs Dynamics on the Lattice

We investigate the spectrum of the SU(2) gauge theory with $N_f$ = 2 flavors of fermions in the fundamental representation, in the continuum, using lattice simulations. This model provides a minimal template which has been used for different strongly coupled extensions of the Standard Model ranging from composite (Goldstone) Higgs models to intriguing types of dark matter candidates, such as the SIMPs. Here we will focus on the composite Goldstone Higgs paradigm, for which this model provides a minimal UV complete realization in terms of a new strong sector with fermionic matter. After introducing the relevant Lattice methods used in our simulations, we will discuss our numerical results. We show that this model features a SU(4)/Sp(4) $\sim$ SO(6)/SO(5) flavor symmetry breaking pattern, and estimate the value of its chiral condensate. Finally, we present our results for the mass spectrum of the lightest spin one and zero resonances, analogue to the QCD $\rho$, $a_1$, $\sigma$, $\eta'$, $a_0$ resonances, which are relevant for searches of new, exotic resonances at the LHC.

SU(2) gauge theory with two fundamental flavors: A minimal template for model building

We investigate the continuum spectrum of the SU(2) gauge theory with $N_f=2$ flavours of fermions in the fundamental representation. This model provides a minimal template which is ideal for a wide class of Standard Model extensions featuring novel strong dynamics that range from composite (Goldstone) Higgs theories to several intriguing types of dark matter candidates, such as the SIMPs. We improve our previous lattice analysis [1] by adding more data at light quark masses, at two additional lattice spacings, by determining the lattice cutoff via a Wilson flow measure of the $w_0$ parameter, and by measuring the relevant renormalisation constants non-perturbatively in the RI'-MOM scheme. Our results for the lightest isovector states in the vector and axial channels, in units of the pseudoscalar decay constant, are $m_V/F_{\rm{PS}}\sim 13.1(2.2)$ and $m_A/F_{\rm{PS}}\sim 14.5(3.6)$ (combining statistical and systematic errors). In the context of the composite (Goldstone) Higgs models, our result for the spin-one resonances are $m_V > 3.2(5)$ TeV and $m_A > 3.6(9)$ TeV, which are above the current LHC constraints. In the context of dark matter models, for the SIMP case our results indicate the occurrence of a compressed spectrum at the required large dark pion mass, which implies the need to include the effects of spin-one resonances in phenomenological estimates.

Mapping of Pelvic Lymph Node Metastases in Prostate Cancer

BackgroundOpinions about the optimal lymph node dissection (LND) template in prostate cancer differ. Drainage and dissemination patterns are not necessarily identical. ObjectiveTo present a precise overview of the lymphatic drainage pattern and to correlate those findings with dissemination patterns. We also investigated the relationship between the number of positive lymph nodes (LN+) and resected lymph nodes (LNs) per region. Design, setting, and participantsSeventy-four patients with localized prostate adenocarcinoma were prospectively enrolled. Patients did not show suspect LNs on computed tomography scan and had an LN involvement risk of ≥10% but ≤35% (Partin tables) or a cT3 tumor. InterventionAfter intraprostatic technetium-99m nanocolloid injection, patients underwent planar scintigraphy and single-photon emission computed tomography imaging. Then surgery was performed, starting with a sentinel node (SN) procedure and a superextended lymphadenectomy followed by radical prostatectomy. Outcome measurements and statistical analysisDistribution of scintigraphically detected SNs and removed SNs per region were registered. The number of LN+, as well as the percentage LN+ of the total number of removed LNs per region, was demonstrated in combining data of all patients. The impact of the extent of LND on N-staging and on the number of LN+ removed was calculated. Results and limitationsA total of 470 SNs were scintigraphically detected (median: 6; interquartile range [IQR]: 3–9), of which 371 SNs were removed (median: 4; IQR: 2.25–6). In total, 91 LN+ (median: 2; IQR: 1–3) were found in 34 of 74 patients. The predominant site for LN+ was the internal iliac region. An extended LND (eLND) would have correctly staged 32 of 34 patients but would have adequately removed all LN+ in only 26 of 34 patients. When adding the presacral region, these numbers increased to 33 of 34 and 30 of 34 patients, respectively. ConclusionsStandard eLND would have correctly staged the majority of LN+ patients, but 13% of the LN+ would have been missed. Adding the presacral LNs to the template should be considered to obtain a minimal template with maximal gain. NoteThis manuscript was invited based on the 2011 European Association of Urology meeting in Vienna.

A novel extraction device for efficient clean-up of molecularly imprinted polymers

For the efficient usage of molecularly imprinted polymers (MIPs) with minimal template leaching, a comprehensive clean-up of the synthesized polymer particles is of critical importance. Predominantly the template molecules, and also the unreacted functional monomer and cross-linker should be exhaustively removed for ensuring reliable results, in particular if MIPs are used within quantitative analytical applications such as e.g., in binding assays or in solid phase extraction. However, an exhaustive clean-up is considered a tedious procedure, which is time-consuming and requires substantial amounts of extraction solvent. In order to significantly improve the efficiency of this crucial step during MIP synthesis, a novel extractor device was developed, which facilitates the rapid and efficient clean-up of particulate polymer matrices, and which is particularly tailored toward the extraction of template molecules from imprinted polymer particles. Compared to commonly applied cleaning procedures such as Soxhlet extraction or HPLC extraction methods, this device offers significant advantages including e.g., the usage of solvent mixtures, temperature-controlled extraction conditions, improved kinetics, and continuous control of the extraction process. First results demonstrating the functionality of the device are discussed for the extraction of molecularly imprinted polymers against the radio contrast agent iohexol.

A modified visual loop-mediated isothermal amplification method for diagnosis and differentiation of main pathogens from Mycobacterium tuberculosis complex

This study was aimed to rapidly identify and differentiate two main pathogens of the Mycobacterium tuberculosis complex: Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium bovis by a modified loop-mediated isothermal amplification (LAMP) assay. The reaction results could be evaluated by naked eye with two optimized closed tube detection methods as follows: adding the modified fluorescence dye in advance into the reaction mix so as to observe the color changes or putting a tinfoil in the tube and adding the SYBR Green I dye on it, then making the dye drop into the bottom of the tube by centrifuge after reaction. The results showed that the two groups of primers used jointly in this assay could successfully identify and differentiate Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium tuberculosis bovis. Sensitivity test displayed that the modified LAMP assay with the closed tube system could determine the minimal template concentration of 1 copy/μl, which was more sensitive than that of routine PCR. The advantages of this LAMP method for detection of the Mycobacterium tuberculosis complex included high specificity, high sensitivity, simplicity, and superiority in avoidance of aerosol contamination. The modified LAMP assay would provide a potential for clinical diagnosis and therapy of tuberculosis in the developing countries and the resource-limited areas.

Optimal Template Removal from Molecularly Imprinted Polymers by Pressurized Hot Water Extraction

An optimal extraction method for the removal of templates from molecularly imprinted polymers (MIPs) is presented. The extraction method is based on pressurized hot water extraction (PHWE). PHWE was evaluated by application to three distinctly colored MIPs for chlorophyll (green), quercetin (yellow) and phthalocynine (dark blue) with subsequent monitoring of template removal and template bleeding by an ultraviolet spectrophotometer. The templates were washed-off and the extraction efficiency (EE) was compared to that of soxhlet and ultrasonic extraction methods. PHWE employed hot water at an optimal temperature of 220 °C, pressure of 50 bars and flow rate of 2 mL min−1 to thoroughly wash-off the respective templates from their MIPs. The EE evaluated for PHWE was over 99.6% for all the MIPs with no subsequent or minimal template bleeding ( 99.6% in all cases). Soxhlet and ultrasonic had washing procedures that were slower (over 18 h) and employed large quantities (400 mL) of organic solvents modified with acids. The percentage relative standard deviations (%RSD) for the EE and recovery results were less than 2.3% in all cases indicating the high reproducibility of the method. Overall, the three methods performed comparably in extracting templates. PHWE seems to be the method of choice as it employed water which poses no environmental threat.

Structural and Functional Characterization of an Influenza Virus RNA Polymerase-Genomic RNA Complex

The replication and transcription of influenza A virus are carried out by ribonucleoproteins (RNPs) containing each genomic RNA segment associated with nucleoprotein monomers and the heterotrimeric polymerase complex. These RNPs are responsible for virus transcription and replication in the infected cell nucleus. Here we have expressed, purified, and analyzed, structurally and functionally, for the first time, polymerase-RNA template complexes obtained after replication in vivo. These complexes were generated by the cotransfection of plasmids expressing the polymerase subunits and a genomic plasmid expressing a minimal template of positive or negative polarity. Their generation in vivo was strictly dependent on the polymerase activity; they contained mainly negative-polarity viral RNA (vRNA) and could transcribe and replicate in vitro. The three-dimensional structure of the monomeric polymerase-vRNA complexes was similar to that of the RNP-associated polymerase and distinct from that of the polymerase devoid of template. These results suggest that the interaction with the template is sufficient to induce a significant conformation switch in the polymerase complex.

Artificial β-defensin based on a minimal defensin template

We have designed and chemically synthesized an artificial beta-defensin based on a minimal template derived from the comparative analysis of over 80 naturally occurring sequences. This molecule has the disulfide-bridged beta-sheet core structure of natural beta-defensins and shows a robust salt-sensitive antimicrobial activity against bacteria and yeast, as well as a chemotactic activity against immature dendritic cells. An SAR (structure-activity relationship) study using two truncated fragments or a Cys-->Ser point-mutated analogue, from which one or two of the three disulfide bridges were absent, indicated that altering the structure resulted in a different type of membrane interaction and a switch to different modes of action towards both microbial and host cells, and that covalent dimerization could favour antimicrobial activity. Comparison of the structural, aggregational and biological activities of the artificial defensin with those of three human beta-defensins and their primate orthologues provided useful information on how their mode of action may relate to specific structural features.

CHARACTERISTICS OF CHANGE OF THE STAGE FROM ‘CLINICALLY ADVANCED' TO ‘PATHOLOGICALLY LOCALIZED' IN PATIENTS WHO UNDERWENT RADICAL PROSTATECTOMY

Opinions about the optimal lymph node dissection (LND) template in prostate cancer differ. Drainage and dissemination patterns are not necessarily identical.To present a precise overview of the lymphatic drainage pattern and to correlate those findings with dissemination patterns. We also investigated the relationship between the number of positive lymph nodes (LN+) and resected lymph nodes (LNs) per region.Seventy-four patients with localized prostate adenocarcinoma were prospectively enrolled. Patients did not show suspect LNs on computed tomography scan and had an LN involvement risk of ≥10% but ≤35% (Partin tables) or a cT3 tumor.After intraprostatic technetium-99m nanocolloid injection, patients underwent planar scintigraphy and single-photon emission computed tomography imaging. Then surgery was performed, starting with a sentinel node (SN) procedure and a superextended lymphadenectomy followed by radical prostatectomy.Distribution of scintigraphically detected SNs and removed SNs per region were registered. The number of LN+, as well as the percentage LN+ of the total number of removed LNs per region, was demonstrated in combining data of all patients. The impact of the extent of LND on N-staging and on the number of LN+ removed was calculated.A total of 470 SNs were scintigraphically detected (median: 6; interquartile range [IQR]: 3–9), of which 371 SNs were removed (median: 4; IQR: 2.25–6). In total, 91 LN+ (median: 2; IQR: 1–3) were found in 34 of 74 patients. The predominant site for LN+ was the internal iliac region. An extended LND (eLND) would have correctly staged 32 of 34 patients but would have adequately removed all LN+ in only 26 of 34 patients. When adding the presacral region, these numbers increased to 33 of 34 and 30 of 34 patients, respectively.Standard eLND would have correctly staged the majority of LN+ patients, but 13% of the LN+ would have been missed. Adding the presacral LNs to the template should be considered to obtain a minimal template with maximal gain.This manuscript was invited based on the 2011 European Association of Urology meeting in Vienna.

Seminaphthofluorones are a family of water-soluble, low molecular weight, NIR-emitting fluorophores

A readily accessible new class of near infrared (NIR) molecular probes has been synthesized and evaluated. Specific fluorophores in this unique xanthene based regioisomeric seminaphthofluorone dye series exhibit a combination of desirable characteristics including (i) low molecular weight (339 amu), (ii) aqueous solubility, and (iii) dual excitation and emission from their fluorescent neutral and anionic forms. Importantly, systematic changes in the regiochemistry of benzannulation and the ionizable moieties afford (iv) tunable deep-red to NIR emission from anionic species and (v) enhanced Stokes shifts. Anionic SNAFR-6, exhibiting an unusually large Stokes shift of approximately 200 nm (5,014 cm(-1)) in aqueous buffer, embodies an unprecedented fluorophore that emits NIR fluorescence when excited in the blue/green wavelength region. The successful use of SNAFR-6 in cellular imaging studies demonstrates proof-of-concept that this class of dyes possesses photophysical characteristics that allow their use in practical applications. Notably, each of the new fluorophores described is a minimal template structure for evaluation of their basic spectral properties, which may be further functionalized and optimized yielding concomitant improvements in their photophysical properties.

RiboSubstrates: a web application addressing the cleavage specificities of ribozymes in designated genomes

BackgroundRNA-dependent gene silencing is becoming a routine tool used in laboratories worldwide. One of the important remaining hurdles in the selection of the target sequence, if not the most important one, is the designing of tools that have minimal off-target effects (i.e. cleaves only the desired sequence). Increasingly, in the current dawn of the post-genomic era, there is a heavy reliance on tools that are suitable for high-throughput functional genomics, consequently more and more bioinformatic software is becoming available. However, to date none have been designed to satisfy the ever-increasing need for the accurate selection of targets for a specific silencing reagent.ResultsIn order to overcome this hurdle we have developed RiboSubstrates . This integrated bioinformatic software permits the searching of a cDNA database for all potential substrates for a given ribozyme. This includes the mRNAs that perfectly match the specific requirements of a given ribozyme, as well those including Wobble base pairs and mismatches. The results generated allow rapid selection of sequences suitable as targets for RNA degradation. The current web-based RiboSubstrates version permits the identification of potential gene targets for both SOFA-HDV ribozymes and for hammerhead ribozymes. Moreover, a minimal template for the search of siRNAs is also available. This flexible and reliable tool is easily adaptable for use with any RNA tool (i.e. other ribozymes, deoxyribozymes and antisense), and may use the information present in any cDNA bank.ConclusionRiboSubstrates should become an essential step for all, even including "non-RNA biologists", who endeavor to develop a gene-inactivation system.

Minimal template requirements for initiation of minus-strand synthesis in vitro by the RNA-dependent RNA polymerase of turnip yellow mosaic virus.

From mutational analysis of the 3'-terminal hairpin of turnip yellow mosaic virus (TYMV) RNA and use of nonstructured C-rich RNA templates, we conclude that the main determinant in the tRNA-like structure of TYMV RNA for initiation of minus-strand synthesis by the viral RNA-dependent RNA polymerase (RdRp) is the non-base-paired 3' ACC(A) end. Base pairing of this 3' end reduces the transcription efficiency drastically, and deletion of only the 3'-terminal A residue results in a fivefold drop in efficiency. The two C residues of the 3' ACCA end are required for efficient transcription, as shown by substitution mutations. However, the 5' A residue is not specifically involved in initiation of transcription, as shown by substitution mutations. Furthermore, the hairpin stem and loop upstream of the 3' ACCA end also do not interact with the RdRp in a base-specific way. However, for efficient transcription, the hairpin stem should be at least five bp in length, while the calculated deltaG value should be less than -10.5 kcal/mol. Unexpectedly, the use of nonstructured C-rich RNA templates showed that the RdRp can start internally on an NCCN or NUCN sequence. Therefore, a possible function of the tRNA-like structure of TYMV RNA may be to prevent internal initiation of minus-strand synthesis.

A Simplified Amplification Procedure for Two Regions of the Glycosyl Transferase (ABO Blood Group) Gene

The ability to classify the ABO blood group of physiological stains has been an important tool for forensic scientists. A streamlined method for the determination of glycosyl transferase (ABO) genotypes using PCR amplification is described and validated here. Successful amplification and typing is possible with 1-2 ng template DNA. Concordance studies with samples of known types and nonprobative forensic casework samples were performed. Bloodstains from a single individual from one case produced results discrepant from those obtained by conventional serological ABO typing. All other samples produced results in complete agreement with expected ABO phenotypes. The method described here is relatively simple to perform, requires minimal template DNA and can be completed in less than one day.