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 distinguishes single base mutations. Two oligonucleotides tethered to the solid support hybridize to the target nucleic acid, forming a tripartite substrate that can be recognized and cleaved by Cleavase, a structure-specific 5'-nuclease. Each cleavage event yields fluorescence signal on the surface. When microspheres serve as the solid-support surface, analysis by fluorometer imparts real-time information about change in the reaction signal over time. Flow cytometry provides an alternative detection technology that collects endpoint information about the reaction signal on individual microspheres. A reaction volume of 10 microL with as few as 3000 microspheres is sufficient to distinguish single nucleotide differences at target concentrations less than 200 fM. This sensitivity level is within the range required for analysis of SNPs in genomic DNA. In addition, the flow cytometry format has multiplexing potential, making the microsphere-based invasive cleavage assay attractive for high-throughput genomic applications.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Stevens PW,Rao KV,Hall JG,Lyamichev V,Neri BP,Kelso DMdoi
10.2144/03341dd09subject
Has Abstractpub_date
2003-01-01 00:00:00pages
198-203issue
1eissn
0736-6205issn
1940-9818journal_volume
34pub_type
相关文献
BIOTECHNIQUES文献大全abstract：:RNA interference (RNAi) is a recent advance that provides the possibility to reduce the expression of specific target genes in cultured mammalian cells with potential applications on a genome-wide scale. However, to achieve this, robust methodologies that allow automated and efficient delivery of small interfering RNA...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/04373RT01
更新日期：2004-09-01 00:00:00
abstract：:We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-medi...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2019-0104
更新日期：2020-06-01 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：: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
pub_type：
doi：10.2144/02323dd05
更新日期：2002-03-01 00:00:00
abstract：:Changes in cardiac structure that depart from normal have generally been termed "remodeling". Assessment of ventricular remodeling at the cellular level should include measurement of myocyte dimensions. A well-established and reliable method to assess myocyte remodeling uses isolated cells and the Coulter Counter/Chan...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/98253pf02
更新日期：1998-09-01 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：:Sequencing of the human genome in combination with computational annotation has provided tremendous data on predicted genes. However, for most of them, no corresponding cDNAs are available yet. Furthermore, even where cDNA clones were obtained, gene transcripts often have many different splice variants that are not co...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/04371DD02
更新日期：2004-07-01 00:00:00
abstract：:Based on the requirement of a glutathione-deficient mutant strain of Saccharomyces cerevisiae to take up external glutathione for growth on synthetic media, a simple agar diffusion test for quantitative detection of total glutathione from various sources was established. Glutathione concentrations can be reliably dete...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/96215st07
更新日期：1996-11-01 00:00:00
abstract：:High-throughput genotyping technologies such as DNA pooling and DNA microarrays mean that whole-genome screens are now practical for complex disease gene discovery using association studies. Because it is currently impractical to use all available markers, a subset is typically selected on the basis of required satura...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/04376BIN03
更新日期：2004-12-01 00:00:00
abstract：:A method was developed for the detection of bacterial mRNAs using reverse transcriptase followed by the polymerase chain reaction (PCR) and Southern blot analysis. The method involves brief inhibition of protein synthesis with chloramphenicol, followed by reverse transcription, PCR amplification of cDNA and Southern b...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1991-01-01 00:00:00
abstract：:Previous experiments have clearly demonstrated that microtubule dynamic instability is regulated in living cells, but the molecular mechanisms that are responsible for this regulation are not well understood. We describe two rapid, functional assays that can be used to screen cell extracts for regulators of microtubul...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/98245rr04
更新日期：1998-05-01 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
pub_type： 杂志文章
doi：
更新日期：1992-02-01 00:00:00
abstract：:A novel method for large-scale plasmid preparation is described. Crude extracts are subjected to acidic phenol extraction to remove any contaminants present in the aqueous phase. The supercoiled plasmid DNA, which preferentially remains in the organic phase and inter-phase, is extracted back into the aqueous phase wit...
journal_title：BioTechniques
pub_type：
doi：
更新日期：1994-03-01 00:00:00
abstract：:We describe a multipurpose eukaryotic expression vector that incorporates the following features: restriction sites for in-frame insertion of cDNAs of interest between sequences encoding the glutathione-S-transferase (GST) and an oligohistidine element, allowing expression of the corresponding fusion proteins; a phosp...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1995-01-01 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
pub_type：
doi：10.2144/03342st01
更新日期：2003-02-01 00:00:00
abstract：:One of the challenges of performing live-cell imaging in plants is establishing a system for securing the sample during imaging that allows for the rapid addition of treatments. Here we report how a commercially available device called a HybriWell™ can be repurposed to create an imaging chamber suitable for Arabidopsi...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2018-0044
更新日期：2018-06-01 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
pub_type： 杂志文章
doi：
更新日期：1992-01-01 00:00:00
abstract：:Millions of museum specimens are integral to biodiversity studies; however, DNA degradation may limit the ability to obtain DNA sequences. In this study, a degradation analysis model for Lepidoptera specimens was established. Based on this model, we revealed the characteristics of DNA fragment distribution caused by e...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/btn-2019-0166
更新日期：2020-03-01 00:00:00
abstract：:In this communication we describe the sequential use of standard and low-melting agarose in a single gel slab for the electrophoresis of DNA. This method has the advantages of high resolution and reproducibility characteristic of standard agarose and the ease of manipulation of DNA for direct cloning, sequential diges...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1991-02-01 00:00:00
abstract：:A rapid and highly sensitive technique (MAPPing: message amplification phenotyping) has been developed to simultaneously analyze the array of messenger RNAs made by small numbers of cells. The technique incorporates a micro-procedure for isolating RNA, reverse transcription of total cellular RNA to produce cDNA, and e...
journal_title：BioTechniques
pub_type： 杂志文章
doi：
更新日期：1989-11-01 00:00:00
abstract：:Microsatellite sequences are important markers for population genetics studies. In the past, the development of adequate microsatellite primers has been cumbersome. However with the advent of next-generation sequencing technologies, marker identification in genomes of non-model species has been greatly simplified. Her...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114104
更新日期：2013-11-01 00:00:00
abstract：:Comprehensive analysis of DNA methylation patterns is critical for understanding the molecular basis of many human diseases. While hundreds of PCR-based DNA methylation studies are published every year, the selection and implementation of appropriate methods for these studies can be challenging for molecular genetics ...
journal_title：BioTechniques
pub_type： 杂志文章,评审
doi：10.2144/000114087
更新日期：2013-10-01 00:00:00
abstract：:Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/04363RR01
更新日期：2004-03-01 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
pub_type： 杂志文章
doi：
更新日期：1993-03-01 00:00:00
abstract：:Current protocols for screening proliferative factors for β cells ex vivo are time-consuming, require cell lines or dissociated islets, and often entail expensive specialized screening equipment. Here we present an efficient and lower cost alternative that utilizes intact mouse islets for the initial screening of prol...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000114115
更新日期：2013-12-01 00:00:00
abstract：:Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to ...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/000112279
更新日期：2006-11-01 00:00:00
abstract：:Identification of nucleotides used for RNA chain initiation or for contacting DNA binding proteins is basic to our understanding of gene regulation. Normally, a radioactive primer is used to copy RNA or DNA. The polymerase extension stops at free ends of mRNA (as in promoter mapping) or at the position of template cle...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/03351rr01
更新日期：2003-07-01 00:00:00
abstract：:DNA-binding proteins can be purified by their affinity for probes with specific sequences that are tethered to magnetic beads. A restriction site at the end of the tether allows release of probe with factor still on it. This probe with bound factor can be bandshifted directly to compare to bandshifts in whole extracts...
journal_title：BioTechniques
pub_type：
doi：
更新日期：1994-05-01 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
pub_type：
doi：10.2144/99273st04
更新日期：1999-09-01 00:00:00
abstract：:The need for a primer hybridization step before sequencing has been eliminated using a stem-loop structure generated by PCR. The loop structure is obtained by careful design of the PCR primer or by cloning the target DNA into a dedicated vector (pRIT 28HP). After solid-phase capture of the PCR product, the loop is for...
journal_title：BioTechniques
pub_type： 杂志文章
doi：10.2144/98255rr02
更新日期：1998-11-01 00:00:00
