Purification Of Fragments Research Articles

Cloning and purification of large fragment of DNA polymerase I from Geobacillus stearothermophilus and it’s application in isothermal DNA amplification

Cloning and purification of large fragment of DNA polymerase I from Geobacillus stearothermophilus and it’s application in isothermal DNA amplification

Purification, concentration and recovery of small fragments of DNA from Giardia lamblia and their use for other molecular techniques

Purification of nucleic acids is an essential procedure for most experiments in molecular biology. In this paper, the freeze-squeeze method with some modifications is proposed as an alternative methodology for the purification, concentration and recovery of small DNA fragments from agarose gels. The advantage of this alternative methodology is that it enables the recovery of fragments that are less than 100bp in length and enables suspension of products in smaller volumes compared to several commercially available kits. In addition, the purified fragments were re-amplified by PCR and used for cloning and sequencing. Moreover, this protocol was used to perform the isolation and identification of microRNAs from Giardia lamblia, as previously reported. This protocol has the advantage of being inexpensive and easy and can be employed for various molecular applications. The advantages of this protocol include•A modified classical method was used for purification of small DNA fragments from G. lamblia.•The modified freeze-squeeze method was more efficient in cleaning up small DNA fragments from agarose gels compared to commercial kits.•The modified method allows concentration and recovery of fragments up to 60bp in length.•The modified freeze-squeeze method allows re-suspension of the products in volumes of up to 2.5μL.

Separation of human Fab fragments by negative chromatography on ω-aminohexyl- and poly(ethyleneimine)-agarose

Polyamines ω-aminohexyl and poly(ethyleneimine) (PEI) immobilized on agarose gel were evaluated as ligands for the purification of Fab fragments from papain-digested human immunoglobulin G (IgG) solution by negative chromatography. The adsorption of proteolytic fragments was investigated using different buffer systems and pH values. The results showed that the buffer system and pH had stronger effects on the adsorption of the proteolytic fragments onto ω-aminohexyl-agarose than onto PEI-agarose. Tris-HCl buffer pH 8.0 could be used to purify Fab fragments by negative chromatography using both adsorbents, achieving purity and recovery above 96% and 93%, respectively. Static binding experiments were conducted to determine the maximum binding capacity of ω-aminohexyl-agarose adsorbent (Qm). The experimental isotherm was best described by the Langmuir–Freundlich model, which indicated the positive cooperativity of adsorption (n = 1.68) and a Qm value of 44.32 mg mL−1 of gel. The results suggest that negative chromatography on ω-aminohexyl- and PEI-agarose is a potential technique for the purification of Fab fragments from papain-digested human IgG solution.

Single-Molecule Fluorescence Detection of a Synthetic Heparan Sulfate Disaccharide.

The first single‐molecule fluorescence detection of a structurally‐defined synthetic carbohydrate is reported: a heparan sulfate (HS) disaccharide fragment labeled with Alexa488. Single molecules have been measured whilst freely diffusing in solution and controlled encapsulation in surface‐tethered lipid vesicles has allowed extended observations of carbohydrate molecules down to the single‐molecule level. The diverse and dynamic nature of HS–protein interactions means that new tools to investigate pure HS fragments at the molecular level would significantly enhance our understanding of HS. This work is a proof‐of‐principle demonstration of the feasibility of single‐molecule studies of synthetic carbohydrates which offers a new approach to the study of pure glycosaminoglycan (GAG) fragments.

Cloning, expression and library construction for HIV-1 Tat Protein

Background: Designing novel therapeutic agents has been a critical challenge for HIV disease. Materials and Methods: In current study a DNA sequence which was encoded the Tat protein was synthesized and inserted in pET 28 vector. Vector was cloned in BL21-DE3 E. coli and cultured in TB media. After protein expression, recombinant Tat protein was purified by NTA affinity chromatography. The Tat purified protein efficiency and confirmed by SDS-PAGE and Western blot, respectively. We were immunized the camel against HIV-1 Tat recombinant protein to made a camelid antibody library. Total RNA was extracted from camel lymphocytes and VHH fragments synthesized and amplified using RT-PCR and Nested- PCR methods by special primers. Results: The 350- 450 bp VHH gene fragment was produced by RT-PCR and Nested- PCR and extracted from agarose gel 1%. Then gel extraction was performed and pure fragments were inserted in HEN-4 vector by T4 DNA ligase. Conclusion: The library can be applied for biopanning and isolation of nanobody against HIV-1 Tat Protein. Nanobody small size may be a useful drug for treatment of HIV disease because give them the potency of the recognizing the cryptic epitopes of tat and neutralized the virus.

An acid precipitation technique: A strip assay for the large-scale DNA polymerase activity screening

A new format of a very rapid, low-cost and high-productive analysis based on the acid precipitation of radiolabeled DNA was developed. By contrast to the conventional processing of a large number of GF/C discs, the method employs one GF/C strip containing samples on individual teeth. The strip assay was validated by comparison with the glass fiber disk technique; the efficiency was demonstrated by screening E. coli superproducers and fractions obtained at the steps of Bst DNA polymerase, Large Fragment purification by the protocol we developed. The principle proposed allows simultaneous assaying many samples for the activity of different polymerases.

Peptide Aβ(16-25) Forms Nanofilms in the Process of Its Aggregation.

A method for the synthesis and high purification of fragments of Aβ(1-42) peptide has been elaborated. We have synthesized the amyloidogenic fragment Aβ(16-25) predicted by us and studied the process of its aggregation by electron microscopy and X-ray analysis. Electron microscopy images show that the peptide forms a film, which is not characteristic of amyloid fibrils. At the same time, according to the X-ray diffraction data, its preparations display the presence of two main reflections (4.6-4.8 and 8-12 Å) characteristic of cross-β structure of amyloid fibrils. Thus, the fragment Aβ(16-25) that we predicted is a promising object not only for studying the process of polymerization of the peptides/proteins, but also for using it as a nanomaterial to study a number of biological processes.

Microfluidic chip for stacking, separation and extraction of multiple DNA fragments

A disposable integrated microfluidic device was developed for rapid sample stacking, separation and extraction of multiple DNA fragments from a relatively large amount of sample. Isotachophoresis hyphenated gel electrophoresis (ITP-GE) was used to pre-concentrate and separate DNA fragments, followed by extraction of pure DNA fragments with electroelution on-chip. DNA fragments of 200bp, 500bp and 1kbp were successfully separated and collected in the extraction chamber within 25min. The extraction efficiency obtained from the chip was 49.9%, 52.1% and 53.7% for 200bp, 500bp and 1kbp DNA fragments, respectively. The extracted DNA fragments exhibited compatibility with downstream enzymatic reactions, for example PCR. The chip was also used to extract DNA fragments with specific size range from sheared genomic DNA and demonstrated similar performance to that using traditional gel cutting method. The whole assay can finish in 32min, 6 times faster than traditional method.

Chromatin Immunoprecipitation: Application to the Study of Asthma.

Chromatin immunoprecipitation (ChIP) is a technique for studying interactions between proteins and DNA in living cells. A protein of interest is selectively immunoprecipitated from a chromatin preparation, to analyze the DNA sequences involved. ChIP can be used to determine whether a transcription factor interacts with a candidate target gene and to map the localization of histones with posttranslational modifications on the genome.The protein-DNA interactions are captured in vivo by chemical cross-linking. Cell lysis, DNA fragmentation, and immunoaffinity purification of the protein of interest allow to co-purify DNA fragments that are associated with that protein. The enriched protein-DNA population is ready to be quantified by PCR to detect precipitated DNA fragments. The combination of ChIP with DNA microarray analysis (ChIP-on-chip) and high-throughput sequencing (ChIP-seq) has enabled to obtain profiles of transcription factor occupancy sites and histone modifications throughout the genome.

Purification of Lamins and Soluble Fragments of NETs.

Lamins and associated nuclear envelope transmembrane proteins (NETs) present unique problems for biochemical studies. Lamins form insoluble intermediate filament networks, associate with chromatin, and are also connected via specific NETs to the cytoskeleton, thus further complicating their isolation and purification from mammalian cells. Adding to this complexity, NETs at the inner nuclear membrane function in three distinct environments: (a) their nucleoplasmic domain(s) can bind lamins, chromatin, and transcriptional regulators; (b) they possess one or more integral transmembrane domains; and (c) their lumenal domain(s) function in the unique reducing environment of the nuclear envelope/ER lumen. This chapter describes strategic considerations and protocols to facilitate biochemical studies of lamins and NET proteins in vitro. Studying these proteins in vitro typically involves first expressing specific polypeptide fragments in bacteria and optimizing conditions to purify each fragment. We describe parameters for choosing specific fragments and designing purification strategies and provide detailed purification protocols. Biochemical studies can provide fundamental knowledge including binding strengths and the molecular consequences of disease-causing mutations that will be essential to understand nuclear envelope-genome interactions and nuclear envelope linked disease mechanisms.

ChemInform Abstract: Glycosylation Chemistry of 3‐Deoxy‐D‐manno‐Oct‐2‐ulosonic Acid (Kdo) Donors

AbstractReview: 99 refs.

Purification and characterization of Fab fragments with rapid reaction kinetics against myoglobin.

Myoglobin is an early biomarker for acute myocardial infarction. Recently, we isolated the antibody IgG-Myo2-7ds, which exhibits unique rapid reaction kinetics toward human myoglobin antigen. Antibodies with rapid dissociation kinetics are thought to be premature IgG forms that are produced during the early stage of in vivo immunization. In the present study, we identified the epitope region of the IgG-Myo2-7ds antibody to be the C-terminal region of myoglobin, which corresponds to 144-154 aa. The Fab fragment was directly purified by papain cleavage and protein G affinity chromatography and demonstrated kinetics of an association constant of 4.02 × 10(4) M(-1) s(-1) and a dissociation constant of 2.28 × 10(-2) s(-1), which retained the unique reaction kinetics of intact IgG-Myo2-7ds antibodies. Because a rapid dissociation antibody can be utilized for antibody recycling, the results from this study would provide a platform for the development of antibody engineering in potential diagnostic areas such as a continuous monitoring system for heart disease.

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

Glycosylation Chemistry of 3‐Deoxy‐D‐manno‐Oct‐2‐ulosonic Acid (Kdo) Donors

Abstract3‐Deoxy‐D‐manno‐Oct‐2‐ulosonic acid (Kdo)‐containing oligosaccharides are a conserved carbohydrate component in lipopolysaccharides (LPSs) of the outer membrane of Gram‐negative bacteria, and they are also present in capsular polysaccharides (CPSs) of bacteria and plant cells. The association of the bacterial LPS with the pathophysiology of bacterial infection have long been recognized. Structurepathophysiology studies of bacterial infection need to use pure and homogeneous LPS fragments. Such a requirement can be fulfilled by the synthesis of Kdo glycosides and related Kdooligosaccharide conjugates. This article provides an overview of the glycosylation chemistries of the synthesis of Kdo α‐/β‐glycosides, oligosaccharides with Kdo units such as LPS core oligosaccharides, and lipid A derivatives.

Purification of restriction fragments containing replication intermediates from complex genomes for 2-D gel analysis.

In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a minute fraction of all of the restriction fragments in a standard DNA preparation-whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsorbed to benzoylated naphthoylated DEAE (BND)-cellulose in high salt concentrations. By combining matrix-enrichment and BND-cellulose chromatography, it is possible to obtain preparations that are enriched as much as 200-fold over the starting genomic DNA, and are thus suitable for analysis on 2-D gels.

Isolation of restriction fragments containing origins of replication from complex genomes.

The identification and isolation of origins of replication from mammalian genomes has been a demanding task owing to the great complexity of these genomes. However, two methods have been refined in recent years each of which allows significant enrichment of recently activated origins of replication from asynchronous cell cultures. In one of these, nascent strands are melted from the long template DNA, and the small, origin-centered strands are isolated on sucrose gradients. The second method involves the selective entrapment of bubble-containing fragments in gelling agarose and their subsequent recovery and isolation by molecular cloning. Libraries prepared by this method from Chinese hamster and human cells have been shown to be extremely pure, and provide a renewable resource of origins that can be used as probes on microarrays or sequenced by high-throughput techniques to localize them within the genomic source. The bubble-trapping method is described here for asynchronous mammalian cells that grow with reasonable doubling times and from which nuclear matrices can be reliably prepared. The method for nuclear matrix preparation and enrichment of replication intermediates is described in an accompanying chapter entitled "Purification of restriction fragments containing replication intermediates from mammalian cells for 2-D gel analysis" (Chapter 16 ).

Detection and Sequencing of Okazaki Fragments in S. cerevisiae.

We have previously demonstrated that lagging-strand synthesis in budding yeast is coupled with chromatin assembly on newly synthesized DNA. Using a strain of S. cerevisiae in which DNA ligase I can be conditionally depleted, we can enrich and purify Okazaki fragments. We delineate a method to extract, end label, and visualize Okazaki fragments using denaturing agarose gel electrophoresis. Furthermore, we describe an ion-exchange chromatographic method for purification of fragments and preparation of strand-specific sequencing libraries. Deep sequencing of Okazaki fragments generates a comprehensive, genomic map of DNA synthesis, starting from a single asynchronous culture. Altogether this approach represents a tractable system to investigate key aspects of DNA replication and chromatin assembly.

Effects of TC4 foil on the microstructures and mechanical properties of accumulatively roll-bonded aluminium-based metal matrix composites

In the present study, Ti–6Al–4V alloy (TC4) foils were initially used as raw materials to produce aluminum-based metal matrix composites via the warm accumulative roll-bonding process. A composite with homogeneously distributed TC4 fragments in Al matrix was produced after 12 ARB cycles were performed. Microstructure analyses revealed that the thickness and length of the TC4 phase and the Al grain size in the Al/TC4 composites were refined by increasing the number of ARB cycles. Compared with the pure metal fragments in Al/pure metal systems, the TC4 fragments in Al/TC4 were thick and long because of the low plasticity–high strength of TC4 phase. The tensile properties of the Al/TC4 composites were also investigated. Results showed that the tensile strength and elongation increased with the number of ARB cycles and reached the maximum values of 147MPa and 4.2% after 12 cycles were performed, respectively. Compared with the ARB monolithic, 1060-Al, Al/TC4 composites exhibited higher strength but lower elongation after 12 ARB cycles were conducted.

Aqueous micellar systems containing Triton X-114 and Pichia pastoris fermentation supernatant: A novel alternative for single chain-antibody fragment purification

The main goal of this work was to evaluate the feasibility of using aqueous micellar two-phase systems (AMTPS) of Triton X-114 to purify a single-chain antibody fragment (scFv) directly from yeast fermentation supernatant. The coexistence curves of aqueous micellar two-phase systems, highly loaded (up to 90% wt/wt) with the biological feedstock, were determined. Besides, the effect of several additives such as inorganic salts and affinity ligands on the phase separation behavior was investigated. The obtained coexistence curves demonstrated that the assayed surfactant/yeast broth solutions were able to separate into two phases at temperatures lower than 24°C. This information was then utilized to assay scFv partitioning and purification. In general, proteins present in the yeast broth, including the scFv, partitioned to the top, micelle-depleted phase due to its hydrophilic character. When affinity ligands were used, an opposite behavior was observed for scFv due to the uneven partitioning of ligands toward the micellar-rich phase. The best purification performances were attained for the system consisting of 4% wt/wt of Triton X-114, 60% wt/wt of yeast fermentation supernatant and the synthetic ligand Fabsorbent™ F1P HF, with a recovery percentage of 88% and a purification factor of 2. These results demonstrate the potential applicability of these systems for designing early steps for scFv purification directly from yeast broth. New perspectives are now open for the use of this methodology for recombinant antibody fragments purification.

Bioimaging based on fluorescent carbon dots

Nanosized fluorescent carbon particles, namely, carbon dots (CDs), are a kind of fluorescent material that has drawn increasing attention in recent years. CDs have size-, surface chemistry-, and wavelength-dependent luminescence emission, which is different from traditional semiconductor-based quantum dots. Moreover, with excellent chemical stability, good biocompatibility, low toxicity, up-conversion emission, resistance to photo bleaching, as well as easy chemical modifications, CDs are promising for substantial applications in numerous areas: bioimaging, sensors, and energy-related devices. Herein, three kinds of fluorescent dots are reviewed: graphene quantum dots (GQDs), carbon nanodots (CNDs) and polymer dots (PDs). After the first reported CDs prepared from electrophoretic analysis and purification of fluorescent carbon nanotube fragments, there were hundreds of publications focusing on fluorescent CDs. Bioimaging was one of the most common applications of the CDs; therefore, in this review, most of the chosen reference papers were related to bioimaging based on CDs.