abstract：:In this study, we have developed an air-interface culture system in which guinea pig tracheobronchial epithelial (GPTE) cells rapidly form tight monolayers. Enzymatically isolated GPTE cells were plated on collagen-treated polycarbonate microporous cell culture inserts at a density of 10(6) cells/cm2 (day 0). Bioelect...

journal_title：BioTechniques

authors： Robison TW,Dorio RJ,Kim KJ

更新日期：1993-09-01 00:00:00

abstract：:DNA sequences containing tandem dinucleotide repeats represent an abundant source of DNA polymorphism in human and other eukaryotic genomes. Here we describe a novel technique for the identification and characterization of regions of DNA containing these repetitive elements. Using primers designed to recognize tandem ...

journal_title：BioTechniques

authors： Grist SA,Firgaira FA,Morley AA

更新日期：1993-08-01 00:00:00

abstract：:A method is described for preparing mutants with multiple, site-directed mutations by ordered coupling of PCR-generated fragments catalyzed by a thermostable DNA ligase. Annealing of the sense strands of the fragments to a single-stranded (antisense) template created a full-length sense strand leaving only nicks that ...

journal_title：BioTechniques

authors： Rouwendal GJ,Wolbert EJ,Zwiers LH,Springer J

更新日期：1993-07-01 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

authors： McDaniel DO,Naftilan J,Barber WH

更新日期：1993-07-01 00:00:00

abstract：:The two-hybrid system is a genetic method to identify proteins that interact with a specific target protein, which is expressed in yeast as a hybrid with a DNA-binding domain. Use of this method entails transforming yeast, both with this DNA-binding domain hybrid and with a library of activation domain hybrids, follow...

journal_title：BioTechniques

authors： Bartel P,Chien CT,Sternglanz R,Fields S

更新日期：1993-06-01 00:00:00

abstract：:This paper reports a novel method for the identification of nucleic acid target sequences when these targets have high sequence identity. Homologous genes are currently identified by sequencing. We hypothesize that by primer extension in the presence of selected nucleotides, genes with similar sequence can be identifi...

journal_title：BioTechniques

authors： Sandhu GS,Kline BC,Bolander ME,Sarkar G

更新日期：1993-06-01 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

authors： Silk GW,Wu M

更新日期：1993-05-01 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

authors： Chong KY,Chen CM,Choo KB

更新日期：1993-04-01 00:00:00

abstract：:Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports r...

journal_title：BioTechniques

authors： Finke J,Fritzen R,Ternes P,Lange W,Dölken G

更新日期：1993-03-01 00:00:00

abstract：:A general method for solid-phase gene assembly on streptavidin-coated magnetic beads has been developed. The introduction of biotin in the 5'-end of the initiation oligonucleotide enables anchoring to the bead by means of the streptavidin-biotin interaction. The immobilization of one oligonucleotide enables controlled...

journal_title：BioTechniques

authors： Ståhl S,Hansson M,Ahlborg N,Nguyen TN,Liljeqvist S,Lundeberg J,Uhlén M

更新日期：1993-03-01 00:00:00

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

authors： Panaccio M,Georgesz M,Lew AM

更新日期：1993-02-01 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

authors： Zeillinger R,Schneeberger C,Speiser P,Kury F

更新日期：1993-02-01 00:00:00

abstract：:An assay is described in which an oligonucleotide probe is specifically digested by lambda exonuclease only when it is annealed to its complementary sequence. In this assay, a cycling effect occurs whereby a small amount of target sequence acts as a specific co-factor in the enzymatic degradation of a larger number of...

journal_title：BioTechniques

authors： Copley CG,Boot C

更新日期：1992-12-01 00:00:00

abstract：:Tandemly polymerized regulatory elements, antisense RNA segments or ribozymes are potentially useful in selective gene silencing. However, existing methods of tandemly polymerizing short DNA segments are laborious. We present a procedure that can create cloned arrays of 40-70 monomer units in two steps. We have create...

journal_title：BioTechniques

authors： Graham GJ,Maio JJ

更新日期：1992-11-01 00:00:00

abstract：:Another method has been developed to attach synthetic peptides to solid supports for use in enzyme immunoassays. The method is based on passively adsorbing a synthetic peptide to a solid-phase support, then further attaching more of the same peptide by means of cross-linking to the previously adsorbed peptide. This me...

journal_title：BioTechniques

authors： Leahy DC,Shah DO,Todd JA

更新日期：1992-11-01 00:00:00

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 R...

journal_title：BioTechniques

authors： Logel J,Dill D,Leonard S

更新日期：1992-10-01 00:00:00

abstract：:The systematic preparation of synthetic peptide combinatorial libraries (SPCLs), each composed of tens of millions of peptides that can be screened in existing diagnostically or pharmacologically relevant in vitro assay systems, is reviewed. The identification of optimal peptide sequences has been achieved through the...

journal_title：BioTechniques

authors： Houghten RA,Appel JR,Blondelle SE,Cuervo JH,Dooley CT,Pinilla C

更新日期：1992-09-01 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

authors： Lienert K,Fowler JC

更新日期：1992-08-01 00:00:00

abstract：:We have generated a genomic P1 bacteriophage library using Monterey pine (Pinus radiata) DNA. We first developed a method for isolating from pine tissue the very high molecular weight DNA necessary for the preparation of libraries requiring large inserts. The method involves protoplasting the cells, isolating nuclei a...

journal_title：BioTechniques

authors： Gorman SW,Roberts-Oehlschlager SL,Cullis CA,Teasdale RD

更新日期：1992-05-01 00:00:00

abstract：:A streamlined protocol is described that allows high sensitivity antigen detection by Western blotting in a single day. The choice of membrane blotting matrix, as well as blocking reagents, has been optimized in order to allow rapid development of the blot with chemiluminescent reagents. The entire process, from gel t...

journal_title：BioTechniques

authors： Klapper A,MacKay B,Resh MD

更新日期：1992-05-01 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

authors： Fernández-Silva P,Enriquez JA,Montoya J

更新日期：1992-04-01 00:00:00

abstract：:Duracryl is a mechanically strong and elastic acrylamide-based matrix, useful for a wide variety of electrophoretic applications. The matrix is stable as a refrigerated solution for one year. Upon addition of appropriate catalysts, Duracryl forms a polymer-reinforced polyacrylamide gel matrix suitable for electrophore...

journal_title：BioTechniques

authors： Patton WF,Lopez MF,Barry P,Skea WM

更新日期：1992-04-01 00:00:00

abstract：:A modification of PCR-mediated gene synthesis strategy is introduced. This modification enables the synthesis of a gene from oligonucleotides comprising only one of the two strands. Bridging oligonucleotides (approximately 20-mers in length) complementary to the junctions of template strand oligonucleotides and two ou...

journal_title：BioTechniques

authors： Jayaraman K,Puccini CJ

更新日期：1992-03-01 00:00:00

abstract：:This report describes a new method for labeling PCR-generated short length (60-120 bp) double-stranded DNA fragments for use as hybridization probes. The method utilizes gene-specific primers identical to those for PCR generation of non-radioactive DNA fragments. Radioactive probes are synthesized by Taq DNA polymeras...

journal_title：BioTechniques

authors： Mizobuchi M,Frohman LA

更新日期：1992-03-01 00:00:00

abstract：:Perfluorocarbon emulsions were applied to hybridoma cultures grown in tissue culture tubes and column bioreactors. The oxygen transfer enhancement effect of perfluorocarbon emulsions was clearly demonstrated by the higher cell densities obtained in emulsion-supplemented systems. In addition, perfluorocarbon emulsions ...

journal_title：BioTechniques

authors： Ju LK,Armiger WB

更新日期：1992-02-01 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

authors： Whitmore FA

更新日期：1992-02-01 00:00:00

abstract：:We report a simple and inexpensive method to quantitate loading and transfer of RNA and to examine RNA integrity for use with Northern hybridization. This technique involves quantitation by computer-assisted video densitometry of a negative photograph of 28S and 18S rRNA from ethidium bromide-stained RNA fixed to nylo...

journal_title：BioTechniques

authors： Correa-Rotter R,Mariash CN,Rosenberg ME

更新日期：1992-02-01 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

authors： Major JG Jr

更新日期：1992-01-01 00:00:00

abstract：:A procedure for amplification by PCR of reproducible allele markers for amplified fragment length polymorphism (Amp-FLP) analysis is presented. We have prepared markers for the allelic products of the VNTR loci D1S80 (MCT118) and D17S30 (YNZ22) and for the hypervariable VNTR locus close to the 3' end of the apolipopro...

journal_title：BioTechniques

authors： Sajantila A,Puomilahti S,Johnsson V,Ehnholm C

更新日期：1992-01-01 00:00:00

abstract：:A technique, Replacement PCR Mutagenesis, was developed to replace one immunoglobulin variable region (V) in a M13 phage cassette with a different, homologous V. This allows the use of the same mutagenesis and subsequent expression vectors for many V regions or V segments. The method combines PCR of V fragments and in...

journal_title：BioTechniques

authors： Near RI

更新日期：1992-01-01 00:00:00

abstract：:In order to study quantitative gene expression with Northern blots, it is important to have an internal standard that can be used to verify even loading or to correct for uneven loading between lanes. In this study it is shown that two-dimensional quantitation of ethidium bromide-intercalated 28S rRNA fluorescence can...

journal_title：BioTechniques

authors： Bonini JA,Hofmann C

更新日期：1991-12-01 00:00:00

abstract：:We describe an expeditious method for the isolation of cDNA clones utilizing PCR-based amplification of target sequences from cDNA libraries. This method is rapid, less labor-intensive and inexpensive when compared with screening libraries with radiolabeled probes. This method can be applied to isolate multiple member...

journal_title：BioTechniques

authors： Isola NR,Harn HJ,Cooper DL

更新日期：1991-11-01 00:00:00

abstract：:Autoantibodies directed against the 68-kDa (U1) ribonucleoprotein antigen are mainly found in sera of patients with mixed connective tissue disease. The corresponding cDNA was fragmented into four regions coding for the major antigenic epitopes A', B', C' and D'. All the epitopes were subcloned and expressed as fusion...

journal_title：BioTechniques

authors： Frorath B,Scanarini M,Netter HJ,Abney CC,Liedvogel B,Lakomek HJ,Northemann W

更新日期：1991-09-01 00:00:00

abstract：:Antibodies specific to avian myeloblastosis virus envelope glycoprotein gp80 were raised. Immunoliposomes were prepared using anti-avian myeloblastosis virus envelope glycoprotein gp80 antibody. The antibody was palmitoylated to facilitate its incorporation into lipid bilayers of liposomes. The fluorescence emission s...

journal_title：BioTechniques

authors： Kalvakolanu DV,Abraham A

更新日期：1991-08-01 00:00:00

abstract：:We describe a method for rapid radioactive labeling of PCR product. The method, employing the Klenow fragment of DNA polymerase I, consumes little product, requires no product purification and takes under 30 minutes. ...

journal_title：BioTechniques

authors： McDaniel TK,Huang Y,Yin J,Needleman SW,Meltzer SJ

更新日期：1991-08-01 00:00:00

abstract：:Replacement of colorimetric substrates in Western blots by chemiluminescent reagents enhances the sensitivity of detection by more than an order of magnitude. Protein levels beyond the detection limit of colorimetric substrates can be consistently detected without modifying pre-existing laboratory protocols. ...

journal_title：BioTechniques

authors： Sandhu GS,Eckloff BW,Kline BC

更新日期：1991-07-01 00:00:00

abstract：:SCORE, a program for computer-assisted scoring of Southern blots of clone DNA, retains the use of expert human judgment while taking over much of the drudgery of the scoring task. The primary functions of the program are to help make an aligned overlay of the fluorescence gel image and the autoradiogram blot image, to...

journal_title：BioTechniques

authors： Cannon TM,Koskela RJ,Burks C,Stallings RL,Ford AA,Hempfner PE,Brown HT,Fickett JW

更新日期：1991-06-01 00:00:00

abstract：:A simplified method for producing recombinant baculovirus for expression of foreign genes is described. The method utilizes insect cells infected with the wild-type virus before transfection with the plasmid transfer vector, instead of the standard procedure utilizing cotransfection with a plasmid and viral DNA. Recom...

journal_title：BioTechniques

authors： Goswami BB,Glazer RI

更新日期：1991-05-01 00:00:00

abstract：:A simple and rapid method for permanently marking autoradiographs is described. This procedure is based on the phosphorescence of light-activated zinc sulfide and the subsequent exposure of x-ray films by this light emission. A lacquer-based carrier allows the zinc sulfide to remain in suspension and permits permanent...

journal_title：BioTechniques

authors： Seto D,Rohrabacher C

更新日期：1991-05-01 00:00:00

abstract：:Haplotypes are useful in population genetics and medicine. Determining haplotypes in the absence of DNA samples from appropriate family members can be difficult and laborious. We have developed a rapid and reproducible method for haplotyping an individual in the absence of relatives. The method utilizes pairs of allel...

journal_title：BioTechniques

authors： Sarkar G,Sommer SS

更新日期：1991-04-01 00:00:00