abstract：:Large genes present particular cloning difficulties, especially when expressed at relatively low levels. We describe a novel method, termed 3' rapid amplification of cDNA ends (RACE) walking, for the rapid determination of unknown 3' flanking sequence of a large cDNA. The technique is a derivative of the anchored PCR ...

journal_title：BioTechniques

authors： Park DJ,Pask AJ,Renfree MB,Graves JA

更新日期：2003-04-01 00:00:00

abstract：:Gene expression analysis using high-density cDNA or oligonucleotide arrays is a rapidly emerging tool for transcriptomics, the analysis of the transcriptional state of a cell or organ. One of the limitations of current methodologies is the requirement of a relatively large amount of total or polyadenylated RNA as star...

journal_title：BioTechniques

authors： Scherer A,Krause A,Walker JR,Sutton SE,Serón D,Raulf F,Cooke MP

更新日期：2003-03-01 00:00:00

abstract：:Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of dev...

journal_title：BioTechniques

authors： Rupp PA,Rongish BJ,Czirok A,Little CD

更新日期：2003-02-01 00:00:00

abstract：:Piezoelectric dispensing of proteins from borosilicate glass capillaries is a popular method of protein biochip fabrication that offers the advantages of sample recovery and noncontact with the printing substrate. However, little regard has been given to the quantitative aspects of dispensing minute volumes (1 nL or l...

journal_title：BioTechniques

authors： Delehanty JB,Ligler FS

更新日期：2003-02-01 00:00:00

abstract：:A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguish...

journal_title：BioTechniques

authors： Stevens PW,Rao KV,Hall JG,Lyamichev V,Neri BP,Kelso DM

更新日期：2003-01-01 00:00:00

abstract：:A consensus peptide sequence, QSYP, appears as an artifact during the mapping of monoclonal antibodies (MAbs) using a random peptide phage display library. Phage bearing this QSYP sequence were independently selected by four different laboratories screening separate MAb preparations with the same phage library. In eac...

journal_title：BioTechniques

authors： Jacobs JM,Bailey BW,Burritt JB,Morrison SG,Morrison RP,Dratz EA,Jesaitis AJ,Teintze M

更新日期：2003-01-01 00:00:00

abstract：:High-throughput approaches for gene cloning and expression require the development of new nonstandard tools for molecular biologists and biochemists. We introduce a Web-based tool to design primers specifically for the generation of expression clones for both laboratory-scale and high-throughput projects. The applicat...

journal_title：BioTechniques

authors： Yoon JR,Laible PD,Gu M,Scott HN,Collart FR

更新日期：2002-12-01 00:00:00

abstract：:Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme f...

journal_title：BioTechniques

authors： Wang QS,Unrau PJ

更新日期：2002-12-01 00:00:00

abstract：:The possibility to miniaturize and parallelize biological assays has a great impact on the development of biomedical technologies. Here, we describe a simple, miniaturized, and parallelized method employing entire cells from different cell lines displaying a protein of interest on their surface, which were immobilized...

journal_title：BioTechniques

authors： Schwenk JM,Stoll D,Templin MF,Joos TO

更新日期：2002-12-01 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

authors： Miyazaki K,Takenouchi M

更新日期：2002-11-01 00:00:00

abstract：:Expanding applications of cDNA microarrays such as fine needle aspiration biopsy and laser capture microdissection necessitate the ability to perform arrays with minute starting amounts of RNA. While methods for amplifying RNA have been advocated, the fidelity of array results using amplified material has not been ful...

journal_title：BioTechniques

authors： Feldman AL,Costouros NG,Wang E,Qian M,Marincola FM,Alexander HR,Libutti SK

更新日期：2002-10-01 00:00:00

abstract：:SNPCEQer identifies and reports SNPs in sequences obtained from the Beckman CEQ2000 DNA Analysis System. SNPCEQer aligns sequences obtained using CEQ2000 heterozygote detection analysis and reports discrepancies between individual sequences and the consensus sequence it generates from this set as SNPs when the individ...

journal_title：BioTechniques

authors： Flood EM,Tang F,Horvath MM,Pertsemlidis A,Garner HR

更新日期：2002-10-01 00:00:00

abstract：:Radiolabeled proteins are used in a variety of laboratory applications as well as in radioimmunotherapy. This review focuses on methods that utilize genetic engineering to introduce exogenous phosphorylation sites into proteins. Protein kinase substrate sites can be introduced into target proteins to serve as tags for...

journal_title：BioTechniques

authors： Clark WA,Izotova L,Philipova D,Wu W,Lin L,Pestka S

更新日期：2002-10-01 00:00:00

abstract：:The extraction of high-quality nucleic acid may be problematic in formalin-fixed tissues because of cross-linking between proteins and DNA. Old fixed tissue specimens do produce fragmented DNA (<1.2 kb), which is only used for PCR amplification. Here we show that high molecular weight DNA (>194 kb) can be successfully...

journal_title：BioTechniques

authors： Fang SG,Wan QH,Fujihara N

更新日期：2002-09-01 00:00:00

abstract：:Array technology is a widely used tool for gene expression profiling in various biological systems. However, the application of this method to mammalian preimplantation embryos is limited by the small amount of mRNA that can be extracted from a single embryo, which is not sufficient for array analysis. Here we report ...

journal_title：BioTechniques

authors： Brambrink T,Wabnitz P,Halter R,Klocke R,Carnwath J,Kues W,Wrenzycki C,Paul D,Niemann H

更新日期：2002-08-01 00:00:00

abstract：:A critical issue in transfection or co-transfection experiments is to define the appropriate controls. In most cases, a corresponding empty vector is used as one control. We report a paradoxical effect of empty mammalian expression vectors on different reporters. We have found that different empty vectors can inhibit ...

journal_title：BioTechniques

authors： Hu Q,Suzuki K,Hirschler-Laszkiewicz I,Rothblum LI

更新日期：2002-07-01 00:00:00

abstract：:When PCR is carried out in a polyacrylamide gel, each target molecule forms a molecular colony that comprises many copies of the original template. By counting the number of colonies, one can directly determine the target titer, with 100% of the DNA molecules and approximately 15% of the RNA molecules being detected. ...

journal_title：BioTechniques

authors： Chetverina HV,Samatov TR,Ugarov VI,Chetverin AB

更新日期：2002-07-01 00:00:00

abstract：:The increase of information in biology makes it difficult for researchers in any field to keep current with the literature. The MEDLINE database of scientific abstracts can be quickly scanned using electronic mechanisms. Potentially interesting abstracts can be selected by matching words joined by Boolean operators. H...

journal_title：BioTechniques

authors： Perez-Iratxeta C,Keer HS,Bork P,Andrade MA

更新日期：2002-06-01 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

authors： Elkin C,Kapur H,Smith T,Humphries D,Pollard M,Hammon N,Hawkins T

更新日期：2002-06-01 00:00:00

abstract：:Impurity assays for recombinant protein therapeutics are essential to ensure batch-to-batch consistency and to meet the FDA's criteria for a well-characterized biopharmaceutical. For determination of residual host cell DNA, membrane hybridization assays utilizing radiolabeled DNA probes prepared from the host cell's g...

journal_title：BioTechniques

authors： Ji X,Lee K,DiPaolo B

更新日期：2002-05-01 00:00:00

abstract：:There are little independent data available about how well single nucleotide polymorphism (SNP) genotyping technologies perform in the typical molecular genetics laboratory. We evaluated the utility and accuracy of a widely used technology, template-directed dye-terminator incorporation with fluorescence-polarization ...

journal_title：BioTechniques

authors： Akula N,Chen YS,Hennessy K,Schulze TG,Singh G,McMahon FJ

更新日期：2002-05-01 00:00:00

abstract：:Microarray technologies have made possible comprehensive analyses of nucleic acid sequence and expression. However, the technology to obtain efficiently high-quality RNA and DNA suitable for array analysis from purified populations of neoplastic cells from human tissues has not been well addressed. Microdissection can...

journal_title：BioTechniques

authors： Barrett MT,Glogovac J,Prevo LJ,Reid BJ,Porter P,Rabinovitch PS

更新日期：2002-04-01 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

authors： Hou S,Chen X,Wang H,Tao M,Hu Z

更新日期：2002-04-01 00:00:00

abstract：:Cell-based immunizations are often used when membrane antigens are difficult to purify. To confirm that an antibody binding to the surface of a cell line is, in fact, binding to the desired antigen, FACS can be performed independently on two cell lines, a transfected cell line expressing the antigen of interest and a ...

journal_title：BioTechniques

authors： Yang XP,Gallo M,Ngan I,Nocerini M,Chen MM

更新日期：2002-03-01 00:00:00

abstract：:A simple, nondestructive PCR-based screening method has been developed for identifying putative transgenic soft white winter wheat (Triticum aestivum L.) carrying the coat protein gene of wheat streak mosaic virus. Removal of the endosperm end of individual seed provided sufficient material for DNA extraction and PCR....

journal_title：BioTechniques

authors： McCarthy PL,Hansen JL,Zemetra RS,Berger PH

更新日期：2002-03-01 00:00:00

abstract：:We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ...

journal_title：BioTechniques

authors： Zhang L,Uder S,Juehne T,Brizzard B,Song K

更新日期：2002-02-01 00:00:00

abstract：:A variety of technical errors have arisen in data analysis when using cDNA or oligonucleotide microarrays. One of the most insidious problems is the saturation of the hybridization signal of high-abundant transcripts. This problem arises from the truncation of the laser fluorescence signal. When the hybridization sign...

journal_title：BioTechniques

authors： Hsiao LL,Jensen RV,Yoshida T,Clark KE,Blumenstock JE,Gullans SR

更新日期：2002-02-01 00:00:00

abstract：:Cytokines are pivotal to a balanced innate or cell-mediated immune response, can be indicative of disease progression and/or resolution, and are being evaluated as therapeutics. There is a need to purify and/or to measure key cytokines rapidly with accuracy, precision, and sensitivity. The current assay technologies, ...

journal_title：BioTechniques

authors： Bai H,Buller RM,Chen N,Boyle MD

更新日期：2002-01-01 00:00:00

abstract：:Enhanced green fluorescent protein (EGFP) is the preferred reporter protein for real-time detection in individual cells, but its usefulness for gene expression quantification is limited by the sensitivity of standard detection techniques. We tested whether the unique feature of single-cell detection and quantification...

journal_title：BioTechniques

authors： Ferrer-Martínez A,Gomez-Foix AM

更新日期：2002-01-01 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

authors： Citti L,Rovero P,Colombo MG,Mariani L,Poliseno L,Rainaldi G

更新日期：2002-01-01 00:00:00

abstract：:Under-agarose chemotaxis has been used previously to assess the ability of neutrophils to respond to gradients of chemoattractant. We have adapted this assay to the chemotactic movement of Dictyostelium amoebae in response to folic acid. Troughs are used instead of wells to increase the area along which the cells can ...

journal_title：BioTechniques

authors： Laevsky G,Knecht DA

更新日期：2001-11-01 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

authors： Yang YS,Hughes TE

更新日期：2001-11-01 00:00:00

abstract：:Cell-imaging approaches using new laser-based technologies have a wide applicability to thefields of pathology and cell biology. Here, we present the application of several of these techniques, including confocal scanning laser microscopy (CSLM), laser scanning cytometry (LSC), and laser capture microdissection (LCM),...

journal_title：BioTechniques

authors： Taatjes DJ,Palmer CJ,Pantano C,Hoffmann SB,Cummins A,Mossman BT

更新日期：2001-10-01 00:00:00

abstract：:We describe a simple and cost-effective method for the synthesis of an internal fluorescently labeled DNA standard for fragment sizing using an automated DNA sequencer. A set of primer pairs labeled with ROX was developed to amplify 12 DNA fragments, 58-417 bp, derived from a conserved region of plant chloroplast DNA....

journal_title：BioTechniques

authors： Brondani RP,Grattapaglia D

更新日期：2001-10-01 00:00:00

abstract：:Immunization with naked DNA was used to elicit chicken egg yolk antibodies (IgY). Layer hens were inoculated with plasmid DNA encoding the enhanced green fluorescent protein, the fusion protein of Newcastle disease virus, and VP2 of African horse sickness virus. IgY was extracted from egg yolks by polyethylene glycol ...

journal_title：BioTechniques

authors： Romito M,Viljoen GJ,Du Plessis DH

更新日期：2001-09-01 00:00:00

abstract：:The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for th...

journal_title：BioTechniques

authors： Hsu TM,Chen X,Duan S,Miller RD,Kwok PY

更新日期：2001-09-01 00:00:00

abstract：:A new genetic marker was created in which sequences from enhanced green fluorescent protein were fused to those of puromycin N-acetyl transferase. The resulting fusion protein (EGFP-puro) conferred both green fluorescence and resistance to puromycin when expressed in mammalian cells. The utility of EGFP-puro as a sele...

journal_title：BioTechniques

authors： Abbate J,Lacayo JC,Prichard M,Pari G,McVoy MA

更新日期：2001-08-01 00:00:00

abstract：:The PCR method has proved to be an invaluable tool for the specific and sensitive detection of genetically modified material (e.g., Roundup Ready Soybean and Bt-176 "Maximizer" Maize) in foodstuffs. The first step in the procedure, namely the purification of nucleic acids from the sample, is often the deciding factor ...

journal_title：BioTechniques

authors： Tengel C,Schüssler P,Setzke E,Balles J,Sprenger-Haussels M

更新日期：2001-08-01 00:00:00

abstract：:Characterization of the thermodynamics of binding interactions is important in improving our understanding of bimolecular recognition and forms an essential part of the rational drug design process. Isothermal titration calorimetry (ITC) is rapidly becoming established as the method of choice for undertaking such stud...

journal_title：BioTechniques

authors： Holdgate GA

更新日期：2001-07-01 00:00:00

abstract：:Gene expression studies require analysis of RNA, but isolation of total RNA from very small samples by traditional methods can be difficult and inefficient. The Absolutely RNA microprep kit provides a convenient method for isolating total RNA from small numbers of cells such as those harvested by laser capture microdi...

journal_title：BioTechniques

authors： Dolter KE,Braman JC

更新日期：2001-06-01 00:00:00