Abstract:
:We have compared RNA polymerase promoter activities in PCR-generated DNA fragments for use in the in vitro transcription of cRNA probes. Sense oligonucleotide primers, specific for the mouse acidic fibroblast growth factor gene, were synthesized with 5' extensions containing promoter sequences for the T7, T3 and SP6 RNA polymerase promoters. A common antisense primer was used with each of the promoter/aFGF primers to prepare PCR-generated DNA fragments (minigenes). In vitro transcription efficiency for each of these constructs was evaluated by incorporation of radioactivity into the cRNA products. We find that both the T7 and T3 promoters can direct the synthesis of cRNA probes of high specific activity from a PCR-generated DNA fragment, but that SP6 cannot. No detectable cRNA product was obtained using either T7 polymerase on the T3/minigene or T3 on the T7/minigene. Antisense cRNA probes, transcribed from minigene constructs were used for both Northern and in situ hybridization studies. A PCR-generated DNA fragment with RNA polymerase promoter sequences at each end provides a single template for synthesis in vitro of either sense or antisense cRNA probes.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Logel J,Dill D,Leonard Ssubject
Has Abstractpub_date
1992-10-01 00:00:00pages
604-10issue
4eissn
0736-6205issn
1940-9818journal_volume
13pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全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::S-100B is used in melanoma follow-up. This serum biomarker is also present in adipocytes; therefore, subcutaneous adipocytes trapped in the needle before performing a venipuncture could contaminate the serum. The aim was to study the influence of adipocyte contamination on blood samples used for S-100B analysis, possi...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2019-0147
更新日期:2020-11-01 00:00:00
abstract::Microarray expression analysis has become one of the most widely used functional genomics tools. Efficient application of this technique requires the development of robust and reproducible protocols. We have optimized all aspects of the process, including PCR amplification of target cDNA clones, microarray printing, p...
journal_title:BioTechniques
pub_type: 杂志文章,评审
doi:10.2144/00293bi01
更新日期:2000-09-01 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::We previously reported that cervicovaginal lavages (CVL) contain a factor that enhances the replication of human immunodeficiency virus (HIV) by increasing virus transcription in T cells and monocytoid cells. This factor was named the HIV-inducing factor (HIF). To determine the molecular mass of HIF, we adapted a blot...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/00283st05
更新日期:2000-03-01 00:00:00
abstract::A head-to-tail trimer of the SV40 Bcl I-Bam H1 DNA fragment, specifying polyadenylation of RNA transcripts, was cloned as a cassette flanked by multiple restriction sites. Insertion of the trimer into several expression vectors efficiently prevented spurious expression of reporter genes resulting from transcriptional ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1989-03-01 00:00:00
abstract::We describe a new real-time fluorescence video microscope design for capturing intensified images of cells containing dual wavelength "ratio" dyes or multiple dyes. The microscope will perform real-time capture of two separate fluorescence emission images simultaneously, improving the time resolution of spatial distri...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1990-03-01 00:00:00
abstract::We describe a bead-based, multiplexed, oligonucleotide ligation assay (OLA) performed on the Luminex flow cytometer. Differences between this method and those previously reported include the use of far fewer beads and the use of a universal oligonucleotide for signal detection. These innovations serve to significantly...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112960
更新日期:2008-11-01 00:00:00
abstract::Chitin from crustacean shells has often been used to isolate and purify plant lectins that have an affinity for poly-N-acetylglucosamine (poly-GlcNAc). When we used washed chitin from crab shells as an affinity medium to isolate a lectin from Pinus strobus L. (eastern white pine) ovules, we found that a substance havi...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1992-02-01 00:00:00
abstract::We describe here a simple and rapid method for enzymatic DNA amplification using DNA template recovered from membrane filters previously used in hybridization analysis. This is done by first solubilizing membrane pieces carrying DNA of interest in dimethyl sulfoxide, followed by isopropanol precipitation and polymeras...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1993-04-01 00:00:00
abstract::New technologies are needed that can diagnose cancer more rapidly and accurately. These technologies must also have the ability to identify the particular cellular abnormalities contributing to the malignancy, thus directing the appropriate treatments. Such technologies should permit absolute quantitation of specific ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/05386su01
更新日期:2005-06-01 00:00:00
abstract::For a feasible and cost-effective impedance measurement of cellular alterations in real-time, we combined commercially available microelectrode arrays (MEAs), consisting of 60 microelectrodes, with a conventional impedance analyzer. For proof of principle, a breast carcinoma cell line (MCF-7) was cultured on MEAs, and...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112254
更新日期:2006-10-01 00:00:00
abstract::As a strategy to purify recombinant murine Interleukin (IL)-18, we cloned the mature coding region of this protein into the pFLAG-1 expression system. The intent was to use the FLAG peptide "tag" as an amino terminal addition to IL-18 so that purification of this fusion protein (FLAG-IL-18) on anti-FLAG antibody affin...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1998-09-01 00:00:00
abstract::A simple and rapid procedure for recovering the denaturing effect of methylmercuric hydroxide in agarose gel electrophoresis is described. The procedure consisted of the treatment of the commercial methylmercuric hydroxide solutions with Amberlite, a mixture of anion- and cation-exchange resins. This treatment greatly...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1992-04-01 00:00:00
abstract::Presepsin is a 13-kDa N-terminal glycoprotein of CD14. Previously, agitation-induced increases in presepsin levels have been reported; however, the mechanism remains poorly understood. In this study, we aimed to reveal the mechanism of presepsin increase. The agitated plasma or serum was separated using gel exclusion ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2020-0136
更新日期:2021-01-29 00:00:00
abstract::Microarray platforms require analytical pipelines with modules for data pre-processing including data normalization, statistical analysis for identification of differentially expressed genes, cluster analysis, and functional annotation. We previously developed the Automated Microarray Data Analysis (AMDA, version 2.3....
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/0000113889
更新日期:2012-07-01 00:00:00
abstract::The Cre/lox system is a powerful genetic tool with which to manipulate the genome. Here, we describe the development of a simple reporter system for Cre recombinase, called the Cre Stoplight. In the absence of Cre, the red fluorescent protein is expressed; when Cre catalyzes a recombination event, the green fluorescen...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/01315st03
更新日期:2001-11-01 00:00:00
abstract::Multiplex Manager 1.0 is a user-friendly cross-platform program that designs efficient combinations of existing genetic marker loci into multiplex polymerase chain reactions and optimizes using prior marker information. The program has the flexibility to solve two design problems: combining all markers into the smalle...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113156
更新日期:2009-06-01 00:00:00
abstract::Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized for resequencing applications in whi...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113913
更新日期:2012-09-01 00:00:00
abstract::The serum protein transferrin (Tf) is a valuable marker for genetic studies of primates, because it is usually polymorphic, exhibiting as many as 13 allelic forms with high heterozygosity. The standard procedure to detect the different phenotypes requires vertical electrophoresis on polyacrylamide gels for 18 h at 4 d...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1993-11-01 00:00:00
abstract::A simple, non-destructive procedure is described to determine the quality of DNA arrays before they are used. It consists of a preliminary staining step of the DNA microarray by using SYBR green II, a fluorophore with specific affinity for ssDNA, followed by a laser scan analysis. The surface quality, integrity and ho...
journal_title:BioTechniques
pub_type:
doi:10.2144/00291st01
更新日期:2000-07-01 00:00:00
abstract::The transcription factor GATA-1 is a zinc finger DNA-binding protein essential for the development of red blood cells. When we expressed different regions of the zinc finger domain in bacteria using an isopropyl-beta-D-thiogalactoside (IPTG) inducible system, growth of bacteria harboring the active DNA-binding domain ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/19962004684
更新日期:1996-04-01 00:00:00
abstract::Blue/white selection is the standard method for detecting a cloned DNA fragment. In the absence of an insert, uninterrupted expression of the vector-encoded alpha-complement of beta-galactosidase (beta-gal), results in the hydrolysis of X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) and the subsequent blue stai...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/01305pf01
更新日期:2001-05-01 00:00:00
abstract::We describe here a simple and efficient transfection method for transient expression of cloned genes in cell lines and primary cultured cells. The method involves the use of DEAE-dextran to target DNA to the cellular endocytotic pathway and the use of a human adenovirus to ensure efficient lysis of endosomal vesicles....
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1994-08-01 00:00:00
abstract::Here we introduce a modified antibody staining method that uses up to 80% less antibody for flow cytometry. We demonstrate this method for the detection of antigens expressed at high, moderate, or low levels in mouse and rat lymphocytes as well as mouse mammary epithelial cells. We obtained reproducibly accurate resul...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/0000113854
更新日期:2012-07-01 00:00:00
abstract::Plasmid pUC18rspL is a 3.788-kilobase pair vector, derived from pUC18, pKK232-8 and pHSG664, which identifies promoter-bearing DNA fragments functional in StrA E. coli by activation of a promoterless streptomycin-sensitive gene cartridge (rspL). Expression of the plasmid-borne rspL gene leads to sensitivity dominance ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1990-12-01 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 relative spatial distribution of cells in a solid tumor contributes to development of malignancy, yet the details of this process remain poorly understood. To elucidate these mechanisms, the ability to extract and analyze the entire DNA content of individual cells whose precise location in the tumor is known is re...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113860
更新日期:2012-04-01 00:00:00
abstract::We have designed and implemented a system to manage whole genome shotgun sequences and whole genome sequence assembly data flow. The Sequence Assembly Manager (SAM) consists primarily of a MySQL relational database and Perl applications designed to easily manipulate and coordinate the analysis of sequence information ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/05385ST01
更新日期:2005-05-01 00:00:00
abstract::Genomic analysis of circulating, cell-free DNA (cfDNA) is being used extensively for molecular diagnostics. Many approaches rely on the construction of cfDNA genomic libraries, targeted retrieval of specific genomic regions and analysis by next-generation DNA sequencing. Several steps during sample preparation require...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2019-0071
更新日期:2019-10-01 00:00:00