abstract：:Oligonucleotide primers used to amplify target DNA regions via PCR should meet certain design criteria to maximize the potential for efficient priming. The Random Oligonucleotide Construction Kit (ROCK), a spreadsheet-based program that runs under Microsoft Excel 97 or later version for Microsoft Windows, was develope...

journal_title：BioTechniques

authors： Strain SR,Chmielewski JG

更新日期：2001-06-01 00:00:00

abstract：:The polymorphism of the fourth component of human serum complement (C4) is well established at the proteinic level; at the DNA level in the analysis of C4A and C4B gene polymorphism, the PCR technique is not widely and routinely used because it is time consuming and still presents reproducibility problems. This is a s...

journal_title：BioTechniques

authors： Zorzetto M,Campo I,Cortelazzo AG,Panelli S,Cuccia M

更新日期：2001-05-01 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

authors： Heuermann K,Cosgrove J

更新日期：2001-05-01 00:00:00

abstract：:Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions. In this report, we describe a nonradioactive band shift assay using a fluorescent DNA target and an ALFexpress automatic DNA sequencer in place of the current method that utilizes radioac...

journal_title：BioTechniques

authors： Filée P,Delmarcelle M,Thamm I,Joris B

更新日期：2001-05-01 00:00:00

abstract：:Reducing the scale of biochemical reactions is becoming commonplace. Examples include the screening of large libraries of chemical compounds or gene sequences. These applications demand the ability to transfer sub-microliter volumes of fluid. To this end, we have modified a Hamilton MICROLAB 2200 with high-speed solen...

journal_title：BioTechniques

authors： Hicks JS,Harker BW,Beattie KL,Doktycz MJ

更新日期：2001-04-01 00:00:00

abstract：:beta-galactosidase (beta-gal), the product of the E. coli LacZ gene, has been used extensively as a reporter in numerous systems. Until recently, the most commonly used method of detecting beta-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the beta-gal substrate 5-bromo-4-chloro-3-in...

journal_title：BioTechniques

authors： Nazarenko DA,Dertinger SD,Gasiewicz TA

更新日期：2001-04-01 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

authors： Andreatta C,Nahreini P,Hovland AR,Kumar B,Edwards-Prasad J,Prasad KN

更新日期：2001-03-01 00:00:00

abstract：:We describe a convenient PCR-based protocol for in vitro recombination of homologous genes, thereby minimizing the rate of associated point mutations. High-fidelity recombination conditions were obtained using Vent DNA polymerase, which, in contrast to Taq DNA polymerase, shows significant proofreading activity and ra...

journal_title：BioTechniques

authors： Ninkovic M,Dietrich R,Aral G,Schwienhorst A

更新日期：2001-03-01 00:00:00

abstract：:Display of functional antibody fragments on the surface of filamentous bacteriophages allows fast selection of specific phage antibodies against a variety of target antigens. However, enrichment of single chain variable fragment (scFv)-displaying phages is often hampered by the abundance of bacteriophages lacking anti...

journal_title：BioTechniques

authors： Tur MK,Huhn M,Sasse S,Engert A,Barth S

更新日期：2001-02-01 00:00:00

abstract：:The resolution of complex protein mixtures by discontinuous buffer SDS-PAGE is accomplished by their concentration into thin bands in the stacking gel, followed by their separation during migration through the resolving gel. Recombinant human interferon-inducible protein-10 (IP-10), a 10-kDa C-X-C chemokine with four ...

journal_title：BioTechniques

authors： Crow MK,Karasavvas N,Sarris AH

更新日期：2001-02-01 00:00:00

abstract：:Vascular targeting agents (VTAs) can be produced by linking antibodies or antibody fragments directed against endothelial cell markers to effector moieties. So far, it has been necessary to produce the components of VTAs (antibody, antibody fragment, linker, and effector) separately and, subsequently, to conjugate the...

journal_title：BioTechniques

authors： Gottstein C,Wels W,Ober B,Thorpe PE

更新日期：2001-01-01 00:00:00

abstract：:Methylation of DNA in CpG dense regions of gene promoters (CpG islands) is important for transcriptional inactivation of selective genes in normal and neoplastic cells. Here, we present a spreadsheet-based program adapted from Microsoft Excel that is useful for identifying CpG islands and for assisting in the laborato...

journal_title：BioTechniques

authors： Anbazhagan R,Herman JG,Enika K,Gabrielson E

更新日期：2001-01-01 00:00:00

abstract：:Gene expression profiles may offer more or additional information than classic morphologic- and histologic-based tumor classification systems. Because the number of tissue samples examined is usually much smaller than the number of genes examined, efficient data reduction and analysis methods are critical. In this rep...

journal_title：BioTechniques

authors： Xiong M,Jin L,Li W,Boerwinkle E

更新日期：2000-12-01 00:00:00

abstract：:To measure mutation load or to detect minimal residual disease, a robust method for identifying one mutant allele in the range of 10(6)-10(9) wild-type alleles would be advantageous. Herein, we present evidence that pyrophosphorolysis-activated polymerization (PAP) has the potential to provide a highly specific and ro...

journal_title：BioTechniques

authors： Liu Q,Sommer SS

更新日期：2000-11-01 00:00:00

abstract：:Recombinant retroviruses are a common vehicle to deliver an exogenous gene to a target cell, which makes them a useful tool in the field of gene therapy. A major drawback to using recombinant retroviruses is the low titer achieved, resulting in a limited number of target cells infected and subsequently poor expression...

journal_title：BioTechniques

authors： Tobias CA,Kim D,Fischer I

更新日期：2000-10-01 00:00:00

abstract：:A nascent protein remains in a complex with its ribosome and mRNA if the stop codon is deleted from the mRNA. In the same manner, mRNA forms a stable complex with DNA if the transcription termination is blocked. In principle, if both mRNA translation and DNA transcription termination are prevented, the protein should ...

journal_title：BioTechniques

authors： Liu S,Shivashankar GV,Sano T,Libchaber A

更新日期：2000-10-01 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

authors： Hegde P,Qi R,Abernathy K,Gay C,Dharap S,Gaspard R,Hughes JE,Snesrud E,Lee N,Quackenbush J

更新日期：2000-09-01 00:00:00

abstract：:The yeast two-hybrid system has been used to characterize many protein-protein interactions. A two-hybrid system for E. coli was constructed in which one hybrid protein bound to a specific DNA site recruits another to an adjacent DNA binding site. The first hybrid comprises a test protein, the bait, fused to a chimeri...

journal_title：BioTechniques

authors： Hays LB,Chen YS,Hu JC

更新日期：2000-08-01 00:00:00

abstract：:We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tub...

journal_title：BioTechniques

authors： Tahir SK,Kovar P,Rosenberg SH,Ng SC

更新日期：2000-07-01 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

authors： Battaglia C,Salani G,Consolandi C,Bernardi LR,De Bellis G

更新日期：2000-07-01 00:00:00

abstract：:Cycling probe technology (CPT) is a simple signal amplification method for the detection of specific target DNA sequences. CPT uses a chimeric DNA-RNA-DNA probe that is cut by RNase H when bound to its complementary target sequence. In this study, a hybridization assay was developed to detect biotinylated CPT products...

journal_title：BioTechniques

authors： Warnon S,Zammatteo N,Alexandre I,Hans C,Remacle J

更新日期：2000-06-01 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

authors： Blesa JR,Hernández-Yago J

更新日期：2000-05-01 00:00:00

abstract：:A dot immunoblotting technique has been developed to estimate the relative expression levels of tagged recombinant human proteins in mammalian cell culture media. Variations in sample denaturation, blocking agents and membrane composition and treatment were used to optimize the signal-to-noise ratio of the defined pro...

journal_title：BioTechniques

authors： Ellsworth JL,Hamacher N,Bagwell N,West JW

更新日期：2000-05-01 00:00:00

abstract：:The isolation of a single DNA molecule for cloning is technically difficult, and the subsequent screening of colonies for recombinant DNA clones is time consuming. Ion pair-reversed phase HPLC (IP-RP-HPLC) analysis of nucleic acids improves the resolution and isolation of PCR products to be cloned. We demonstrate that...

journal_title：BioTechniques

authors： Shaw-Bruha CM,Lamb KA

更新日期：2000-04-01 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

authors： Slavícková A,Ullmannová V,Klener P

更新日期：2000-04-01 00:00:00

abstract：:The expression of foreign proteins in Saccharomyces cerevisiae is a powerful tool for basic research and the biotechnological industry. In spite of the potential of S. cerevisiae, only a few useful expression vectors have been developed for this yeast. These vectors are based on an increasing transcription rate in com...

journal_title：BioTechniques

authors： Mimran A,Marbach I,Engelberg D

更新日期：2000-03-01 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

authors： Hashemi FB,Spear GT,Madsen L,Mollenhauer J

更新日期：2000-03-01 00:00:00

abstract：:The technique described here is a fast and simple method of extracting chloroplast DNA (cpDNA). It overcomes the need for differential centrifugation using density gradients. The leaves do not have to be kept in the dark and lyophilized before extraction, but lyophilization is still possible. The chloroplasts are spec...

journal_title：BioTechniques

authors： Mariac C,Trouslot P,Poteaux C,Bezançon G,Renno JF

更新日期：2000-01-01 00:00:00

abstract：:The trend toward high-throughput techniques in molecular biology and the explosion of online scientific data threaten to overwhelm the ability of researchers to take full advantage of available information. This problem is particularly severe in the rapidly expanding area of gene expression experiments, for example, t...

journal_title：BioTechniques

authors： Tanabe L,Scherf U,Smith LH,Lee JK,Hunter L,Weinstein JN

更新日期：1999-12-01 00:00:00

abstract：:We describe a rapid and sensitive method for the detection of nucleotide sequence variation that can be used for large-scale screening of population markers. Denaturing gradient gel electrophoresis (DGGE) detects sequence variants of amplified fragments by the differences in their melting behavior. DGGE detects most s...

journal_title：BioTechniques

authors： Miller KM,Ming TJ,Schulze AD,Withler RE

更新日期：1999-11-01 00:00:00

abstract：:We present a fast, convenient and inexpensive method that allows the automated, large-scale screening of chemical libraries for compounds that are converted by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) into inhibitors of cell growth. The method is based on the use of budding yeast (Saccharomyces ce...

journal_title：BioTechniques

authors： Wera S,Degrève B,Balzarini J,De Clercq E,Thevelein JM,Neyts J

更新日期：1999-10-01 00:00:00

abstract：:Pairs of primers flanking known miniTn10 transposon insertion sites were used to confirm the presence of the transposon in DNA isolated from Legionella pneumophila mutants. It was expected that the polymerase chain reaction products derived from the mutant template would be larger than those from the wild-type (WT) te...

journal_title：BioTechniques

authors： Viswanathan VK,Krcmarik K,Cianciotto NP

更新日期：1999-09-01 00:00:00

abstract：:We describe a simple and efficient system for epitope mapping by cloning random gene fragments into a specially designed gIIIp-based phage display vector. DNA encoding the antigen of interest is PCR-amplified and partially digested with DNaseI to generate 50-150-bp-long fragments, which are polished with T4 DNA polyme...

journal_title：BioTechniques

authors： Gupta S,Arora K,Sampath A,Khurana S,Singh SS,Gupta A,Chaudhary VK

更新日期：1999-08-01 00:00:00

abstract：:A fully automated nucleic acid analysis system is described, which offers positive sample identification, improved sensitivity and reduced user interaction compared to conventional techniques. The system relies on the sequence-specific capture of DNA onto solid-phase particles, confirming product identity without the ...

journal_title：BioTechniques

authors： Brown A,Akinsanya AA,Barker SJ,Brophy M,Dobb AK,Doyle SM,Hudson IR,Minter SJ,Wraith MJ,Oultram JD

更新日期：1999-07-01 00:00:00

abstract：:The two-hybrid system in Saccharomyces cerevisiae is a genetic approach for the detection of of protein-protein interactions in vivo. This technology relies on the the activity of separated DNA-binding and transactivation domains of specific transcription factors to reconstitute an active transcription factor complex ...

journal_title：BioTechniques

authors： Mayer G,Launhardt H,Munder T

更新日期：1999-07-01 00:00:00

abstract：:Hereditary hemochromatosis (HHC) represents an autosomal recessive disease in which increased iron absorption causes iron overload and irreversible tissue damage. The recently detected association between two point mutations in the HFE gene on chromosome 6p and HHC has made it possible to screen for the disease before...

journal_title：BioTechniques

authors： Bosserhoff AK,Seegers S,Hellerbrand C,Schölmerich J,Büttner R

更新日期：1999-06-01 00:00:00

abstract：:We have developed an improved subtractive hybridization method that provides a fast, simple and reliable isolation of desired different sequences from two compared DNA libraries, one of which contains all unwanted homologues (subtracter) and another contains certain desired heterologues (tester). The DNA library can b...

journal_title：BioTechniques

authors： Ying SY,Lin S

更新日期：1999-05-01 00:00:00

abstract：:We present a simple method for sequential chemiluminescent detections of two different DNA loci on a single Southern blot. First, an enzyme-linked DNA probe for a unique sequence is detected with a horse-radish peroxidase (HRP) substrate followed by the detection of another enzyme-linked DNA probe for a different uniq...

journal_title：BioTechniques

authors： Reddy LV,DeSilva R,Handley RS,Schaap AP,Akhavan-Tafti H

更新日期：1999-04-01 00:00:00

abstract：:An easy, sensitive and direct fluorescent immunodetection method for proteins is described using the new fluorochrome PBXL-1 imaged with the FMBIO II Laser Scanning Imaging System. PBXL-1 is derived from a protein supra-molecular complex that contains a large number of chromophores. This complex, the phycobilisome, is...

journal_title：BioTechniques

authors： Morseman JP,Moss MW,Zoha SJ,Allnutt FC

更新日期：1999-03-01 00:00:00

abstract：:The concentration of proteins in cells is an important parameter that determines how a protein will interact with other proteins or pharmacological agents. Recent developments in Western blotting techniques have now made this a method of choice to measure protein concentration in complex solutions such as total cell e...

journal_title：BioTechniques

authors： Fradelizi J,Friederich E,Beckerle MC,Golsteyn RM

更新日期：1999-03-01 00:00:00