Abstract：
:Recent advances in long reverse transcription (RT)-PCR technology allow the copying of full-length coding regions of large mRNAs in one step. Using long RT-PCR, one can be certain that a given cDNA is derived from a single mRNA. In what to our knowledge is a novel application, we can isolate and characterize splice variants for any given mRNA in a systematic manner. We optimized long RT-PCR to copy the full-length coding region of human multidrug resistance (MDR1) mRNA or the major vault protein (MVP) mRNA in one step, so that only one full-length PCR product was synthesized in each case. Such stringent conditions are necessary to ensure that smaller than full-length products derived from total cell RNA are true splice variants. Twenty MDR1 double-stranded (ds) cDNAs, isolated from either the full-length or one prominent splice-variant DNA band, visualized on agarose gels, were cloned and sequenced. Two were full-length, wild-type in sequence as expected, and the rest were splice-variant mRNAs. Fourteen of the clones were identical and encoded a prominent splice-variant mRNA that can be detected in two tumor cell lines. This approach is shown to be generally applicable to the systematic analysis of splice-variant mRNAs derived from any gene.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Hu Y,Tanzer LR,Cao J,Geringer CD,Moore REdoi
10.2144/98252st01subject
Has Abstractpub_date
1998-08-01 00:00:00pages
224-9issue
2eissn
0736-6205issn
1940-9818journal_volume
25pub_type
相关文献
BIOTECHNIQUES文献大全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
pub_type： 杂志文章,评审
doi：
更新日期：2002-10-01 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
pub_type：
doi：
更新日期：1993-06-01 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
pub_type： 杂志文章
doi：
更新日期：1992-10-01 00:00:00
abstract：:A method allowing the evaluation of the structure and the calculation of the volume of a biofilm, using an optical microscope, is proposed based on the linear relation between the intensity of a pixel in biofilm images grabbed on the x-y plane and the corresponding number of cells in the z direction, which allows the ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112403
更新日期：2007-05-01 00:00:00
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
pub_type： 杂志文章
doi：10.2144/01306bc01
更新日期：2001-06-01 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
pub_type： 杂志文章
doi：
更新日期：1991-12-01 00:00:00
abstract：:Conventional genomic DNA (gDNA) extraction methods can take hours to complete, may require fume hoods and represent the most time-consuming step in many gDNA-based molecular assays. We systematically optimized a bead bashing-based (BBB) approach for rapid gDNA extraction without the need for a fume hood. Human tissue ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2019-0172
更新日期：2020-05-01 00:00:00
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
pub_type：
doi：10.2144/03344st04
更新日期：2003-04-01 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
pub_type： 杂志文章
doi：
更新日期：1993-08-01 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
pub_type： 杂志文章
doi：
更新日期：1991-05-01 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
pub_type： 杂志文章
doi：10.2144/01301bc01
更新日期：2001-01-01 00:00:00
abstract：:A modification of the TRI Reagent procedure has been elaborated for isolation of RNA from polysaccharide- and proteoglycan-rich material. In the modified procedure, RNA is precipitated from the aqueous phase by the combined action of isopropanol and a high-salt concentration. Under these conditions, RNA is effectively...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1995-12-01 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：: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：:Polyethyleneimine (PEI) is a flocculent that is widely used in the downstream purification of monoclonal antibodies. It is an in-process residual that is carried through the drug purification process and strongly inhibits residual DNA quantitation by real-time quantitative PCR assay. Very high sample dilutions (e.g., ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2020-0011
更新日期：2020-06-01 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
pub_type：
doi：10.2144/03342mt02
更新日期：2003-02-01 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
pub_type： 杂志文章
doi：
更新日期：1992-11-01 00:00:00
abstract：:A 200-year-old observation might provide a new way to eliminate tumors by infecting cancer patients with bacteria. Kristie Nybo explores a new approach that could transform cancer treatment. ...
journal_title：BioTechniques
pub_type： 新闻
doi：10.2144/000114624
更新日期：2018-01-01 00:00:00
abstract：:Chromosome walking in mammalian DNA by vectorette PCR is not always very specific, and the walks have been limited to distances <1 kb. To improve the method, we have designed new vectorettes, which unlike the currently used ones have very little repetitive sequences or homology with known DNA sequences of various orig...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/04374DD03
更新日期：2004-10-01 00:00:00
abstract：:Here we present a procedure for preparing amino acid mixtures--having both the desired composition and a physiological pH--at high concentrations for cell-free expression systems. Up to 2.1 mg/mL of active protein was synthesized in batch mode reactions with an all Escherichia coli cell-free expression system. Our met...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114249
更新日期：2015-01-01 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：:A method for the determination of prostaglandin G/H synthase and lipoxygenase activities in tissues was developed and employed with rat gastric mucosa samples. Tissues and microsomes were incubated in a buffer containing nonionic detergent and 1.32 mM arachidonic acid for 10 min. Following extraction with ethyl acetat...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1991-02-01 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
pub_type： 杂志文章
doi：10.2144/02321dd02
更新日期：2002-01-01 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：:Here, we present a rapid and reproducible procedure based on square-wave pulse electroporation that allows efficient penetration of synthetic oligonucleotides into intact yeast cells. This procedure was successfully used to modify the yeast genome with small amounts of oligonucleotide. ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/98252gt04
更新日期：1998-08-01 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
pub_type： 杂志文章
doi：10.2144/01315rr03
更新日期：2001-11-01 00:00:00
abstract：:A simple spectrophotometric method for measuring DNA in proliferating cells is described. The method is an adaptation of the widely used diphenylamine (DPA) reaction to examine DNA in cells grown in a 96-well plate. This assay was capable of detecting as little as 10 ng DNA and could be used to measure DNA in stored a...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1994-07-01 00:00:00
abstract：:The CRISPR/Cas9 system is an efficient gene-editing method, but it is difficult to obtain mutants for some specific species and special genome structures. A previously reported multiplexed, semi-nested PCR target-enrichment approach, which does not rely on transgenic technology, has been shown to be an effective and a...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2019-0001
更新日期：2019-12-01 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
pub_type：
doi：10.2144/99266st02
更新日期：1999-06-01 00:00:00
abstract：:As more and more researchers are examining proteins that are available only in extremely limited quantities, i.e., cellular extracts or genetic engineering products, it is critical to utilize staining methods that maximize sensitivity. The protocol we describe here--double staining of polyacrylamide electrophoresis ge...
journal_title：BioTechniques
pub_type：
doi：
更新日期：1990-11-01 00:00:00
