Abstract:
:Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Clark J,Shevchuk T,Swiderski PM,Dabur R,Crocitto LE,Buryanov YI,Smith SSdoi
10.2144/03353rr01subject
Has Abstractpub_date
2003-09-01 00:00:00pages
548-54issue
3eissn
0736-6205issn
1940-9818journal_volume
35pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全abstract::FoLT (formamide low temperature) PCR is a protocol for amplifying DNA directly from whole blood without any preparative steps. Up to 10% (vol/vol) whole blood can be added directly into the tube containing the PCR mixture. There is no need for transfers, centrifugations, pre-boiling or any preparative step. It involve...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1993-02-01 00:00:00
abstract::A method was developed for the detection of bacterial mRNAs using reverse transcriptase followed by the polymerase chain reaction (PCR) and Southern blot analysis. The method involves brief inhibition of protein synthesis with chloramphenicol, followed by reverse transcription, PCR amplification of cDNA and Southern b...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1991-01-01 00:00:00
abstract::Here we demonstrate the use of a mammalian two-hybrid system to study protein-protein interactions. Like the yeast two-hybrid system, this is a genetic, in vivo assay based on the reconstitution of the function of a transcriptional activator. In this system, one protein of interest is expressed as a fusion to the Gal4...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97222pf02
更新日期:1997-02-01 00:00:00
abstract::We report a straightforward methodology for purification of recombinant proteins by incorporating a short hydrophilic peptide marker segment at their N-termini. A calcium-dependent antibody that reacts primarily with the first three amino acids of this peptide segment was used to affinity purify the fusion proteins in...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1989-06-01 00:00:00
abstract::Address correspondence to Mart Speek, Department of Gene Technology, Akadeemia tee 15, Room 129, Tallinn University of Technology, Tallinn 19086, Estonia. E-mail: mart.speek@ttu.ee. ...
journal_title:BioTechniques
pub_type: 信件
doi:10.2144/000114441
更新日期:2016-08-01 00:00:00
abstract::Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114377
更新日期:2016-02-01 00:00:00
abstract::The reliability of the PCR technique used to type two human variable number tandem repeats, that is, 3' to apolipoprotein B gene and locus D17S30, was examined using DNA samples of mixed human and microbial origin. Mixtures of human and microbial DNA were amplified, choosing microbes found commonly in the vagina. Tota...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1992-08-01 00:00:00
abstract::Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor m...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112169
更新日期:2006-05-01 00:00:00
abstract::Here we describe a convenient method to generate homologous recombinant baculoviral genomes in E. coli. The recombination takes place with the aid of recombination enzymes provided by the phage lambda Red system between a bacmid (a baculoviral genome that can replicate in bacteria) and a linear fragment. Proof of conc...
journal_title:BioTechniques
pub_type:
doi:10.2144/02324st04
更新日期:2002-04-01 00:00:00
abstract::The use of quantitative PCR is recommended to monitor the level of residual hematological malignancies. The proposed multiplex IgH/ras PCR uses a co-amplification of the clonal CDR3 rearrangement of the immunoglobulin heavy chain gene (IgH) as a disease marker and a segment of the Hras 1 gene containing codon 61 (ras)...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/00284st08
更新日期:2000-04-01 00:00:00
abstract::A fluorescent in situ DNA hybridization assay employing a biotinylated DNA probe was used to visualize single copies of human immunodeficiency virus (HIV) proviral DNA in the nuclei and metaphase chromosomes of infected cells. In clonal cell lines that contain either one or two copies of proviral DNA, the efficiency o...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1990-08-01 00:00:00
abstract::The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids th...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/02335st03
更新日期:2002-11-01 00:00:00
abstract::We report a modified method of screening for point mutations using a PCR approach based upon the sensitivity of PCR to the 3' terminus of the primer. This method provides a sensitive screen when using either plasmid DNA or bacterial cell lysates. We have optimized the technique for general use to allow rapid screening...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1992-01-01 00:00:00
abstract::Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA. This DNA was transfected into E. coli without phosphorylation of primers, restriction enzyme digestion or ligation. Specifically, the polymerase ch...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1990-02-01 00:00:00
abstract::Human embryonic kidney (HEK293) cells were stably transduced with a retroviral vector containing an expression cassette for a short-lived green fluorescent protein (d2EGFP) and the neomycin resistance gene (Neor). When Neor HEK293 clones were treated with proteasome inhibitors, lactacystin or MG132, an increase in the...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/01303dd03
更新日期:2001-03-01 00:00:00
abstract::Conformation-sensitive gel electrophoresis (CSGE) has been introduced as the most reliable method for the screening of large and multi-exon genes because of its simplicity, sensitivity and specificity. Based on heteroduplex formation and with the use of mildly denaturing solvents, it allows detection of single-base mu...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/00285rr08
更新日期:2000-05-01 00:00:00
abstract::Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, a...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/04361ST04
更新日期:2004-01-01 00:00:00
abstract::A complete image digitizing and processing system is described for capturing, enhancing and analyzing molecular fingerprints. The low-cost, high-resolution system features a Motorola 68000 processor, multi-tasking, a separate video coprocessor, and color or gray scale processing. Thousands of manipulations are possibl...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1990-10-01 00:00:00
abstract::MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114574
更新日期:2017-08-01 00:00:00
abstract::We have developed an automated purification method for dye-terminator-based DNA sequencing products using a magnetic bead approach. This 384-well protocol generates sequence fragments that are essentially free of template DNA, salt, and excess dye-terminator products. In comparison with traditional ethanol precipitati...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/02326st05
更新日期:2002-06-01 00:00:00
abstract::The ZEBRA protein is a transcriptional activator that induces expression of viral lytic genes in cells harboring latent Epstein-Barr virus (EBV). In this report it is shown that a derivative of ZEBRA that cannot activate transcription (Zd) can be used to detect and characterize activation domains. Three expression vec...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97225rr02
更新日期:1997-05-01 00:00:00
abstract::We have developed a novel method for the rapid preparation of large quantities of 3' end-labeled single-stranded (ss) DNA (ssDNA) probes for transcript mapping. A recombinant phagemid vector containing the probe sequence was used to raise large quantities of ssDNA. Based on the DNA sequence of the probe, an oligonucle...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1993-05-01 00:00:00
abstract::A nonradioactive functional assay was developed to quantitate DNA binding proteins. The assay was designed to allow the use of 96-well microplates for high sample throughput. We show that the assay can measure sequence-specific DNA binding of purified proteins as well as DNA binding activity present in whole cell extr...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1995-06-01 00:00:00
abstract::We describe a rapid, simple and inexpensive method for the isolation of DNA from blood clots suited for use in PCR. Our method is based on the lysing and nuclease-inactivating properties of guanidine thiocyanate together with the nucleic acid-binding properties of silica particles. Isolated DNA can be used for in vitr...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1993-02-01 00:00:00
abstract::The limiting detection signal for identification of human genetic markers, such as HLA-D and VNTR genes, was determined using DNA isolated from a series of decreasing numbers of lymphocytes carrying the target marker in the polymerase chain reaction (PCR). The PCR procedure was assembled by incorporating 32P-labeled d...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1993-07-01 00:00:00
abstract::We have developed a high-throughput, multiplex reverse transcription PCR (RTPCR) assay that is suitable for the analysis of medium-to low-copy cellular RNA transcripts from small numbers of cells (10(4)). High throughput was attained by utilizing microplate-based RNA extraction and RTPCR protocols, followed by PCR pro...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97226st02
更新日期:1997-06-01 00:00:00
abstract::Protein analysis is crucial to elucidating the function of proteins and understanding the impact of their presence, absence and alteration. This is key to advancing knowledge about diseases, providing the opportunity for biomarker discovery and development of therapeutics. In this issue of Tech News, Nawsheen Boodhun ...
journal_title:BioTechniques
pub_type: 新闻
doi:10.2144/btn-2018-0055
更新日期:2018-05-01 00:00:00
abstract::The high-order structure of human chromosomes is an important biological question that is still under investigation. Studies have been done on imaging human mitotic chromosomes using mostly 2-D microscopy methods. To image micron-sized human chromosomes in 3-D, we developed a procedure for preparing samples for serial...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114236
更新日期:2014-12-01 00:00:00
abstract::Extracellular vesicles (EVs) are spherical membrane structures released by most cells. These highly conserved mediators of intercellular communication carry proteins, lipids, and nucleic acids, and transfer these cellular components between cells by different mechanisms, such as endocytosis, macropinocytosis, or fusio...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114371
更新日期:2016-01-01 00:00:00
abstract::We investigated the ability of an amphipathic oligopeptide to carry a synthetic dsDNA oligonucleotide inside human cells. The oligonucleotide was designed as a decoy binding site for the transcriptional activator of the methylguanine-DNA methyltransferase (MGMT) gene. The complex oligopeptide and decoy were administer...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/02321dd03
更新日期:2002-01-01 00:00:00