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 specimens (n = 34) subjected to the 12-min BBB method yielded 0.40 ± 0.17 (mean ± SD) μg of gDNA per milligram of tissue, sufficient for many downstream applications, and 3- and 6-min extensions resulted in an additional 0.43 ± 0.23 μg and 0.48 ± 0.43 μg per milligram of tissue, respectively. The BBB method provides a simple and rapid method for gDNA extraction from mammalian tissue that is applicable to time-sensitive clinical applications.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Wei S,Levy B,Hoffman N,Cujar C,Rodney-Sandy R,Wapner R,D'Alton M,Williams Zdoi
10.2144/btn-2019-0172subject
Has Abstractpub_date
2020-05-01 00:00:00pages
240-244issue
5eissn
0736-6205issn
1940-9818journal_volume
68pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全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
pub_type: 杂志文章
doi:
更新日期:1991-07-01 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::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::A labeled peptide nucleic acid (PNA) antisense probe was used to study the spatial distribution of triplet repeats (CTG) in human myotonic dystrophy (DM) cells by high-resolution fluorescence in situ hybridization (FISH). It was found that transcripts containing triplet repeats were present as a number of discrete foc...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/98243rr02
更新日期:1998-03-01 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::We have developed a high-throughput, multiplex reverse transcription PCR (RTPCR) assay that is suitable for the analysis of medium-to low-copy cellular RNA transcripts from small numbers of cells (10(4)). High throughput was attained by utilizing microplate-based RNA extraction and RTPCR protocols, followed by PCR pro...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97226st02
更新日期:1997-06-01 00:00:00
abstract::We describe here a simple and efficient transfection method for transient expression of cloned genes in cell lines and primary cultured cells. The method involves the use of DEAE-dextran to target DNA to the cellular endocytotic pathway and the use of a human adenovirus to ensure efficient lysis of endosomal vesicles....
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1994-08-01 00:00:00
abstract::A rapid and inexpensive method for isolating bacterial DNA for use in PCR is described. The method is based on the guanidinium thiocyanate (GuSCN)-lysis method of Boom et al. (J. Clin. Microbiol. 28:495-503, 1990) and enables a multiple of 96 samples to be prepared in only one hour. We use Multiscreen plates and a vac...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1995-08-01 00:00:00
abstract::The type IIS/IIC restriction endonuclease TspGWI recognizes the sequence 5'-ACGGA-3', cleaving DNA 11/9 nucleotides downstream. Here we show that sinefungin, a cofactor analog of S-adenosyl methionine, induces a unique type of relaxation in DNA recognition specificity. In the presence of sinefungin, TspGWI recognizes ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113685
更新日期:2011-06-01 00:00:00
abstract::Ribosome profiling is a powerful tool for characterizing in vivo protein translation at the genome scale, with multiple applications ranging from detailed molecular mechanisms to systems-level predictive modeling. Though highly effective, this intricate technique has yet to become widely used in the microbial research...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114302
更新日期:2015-06-01 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
abstract::Recombinant alphaviruses have been used as vehicles for delivery and expression of heterologous genes in mammalian, avian and insect cell lines. We have used a Sindbis replicon virus (Sinreplac) able to express the E. coli lacZ gene to compare the efficiency of transduction in one insect, six mammalian cell lines and ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/96213rr02
更新日期:1996-09-01 00:00:00
abstract::We have demonstrated that efficient polymerase chain reaction amplifications from chromosomal DNA can be carried out using whole bacterial cells as the starting material. Cells from the liquid or solid cultures can be used directly, without any pre-treatment, thus eliminating the need for DNA isolation. ...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1991-01-01 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
pub_type:
doi:10.2144/01313dd05
更新日期:2001-09-01 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
pub_type: 杂志文章
doi:
更新日期:1992-08-01 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
pub_type: 杂志文章
doi:
更新日期:1991-04-01 00:00:00
abstract::We have developed a new strategy for differential screening of genes that are expressed in two or more tissues, and have used it to identify genes that are preferentially expressed in both testis and ovary. In this approach, testis-specific cDNAs were first generated using the suppression subtractive hybridization tec...
journal_title:BioTechniques
pub_type:
doi:10.2144/97236st05
更新日期:1997-12-01 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::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
pub_type: 杂志文章
doi:
更新日期:1993-09-01 00:00:00
abstract::Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114377
更新日期:2016-02-01 00:00:00
abstract::High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinit...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114473
更新日期:2016-11-01 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::Primary human fibroblasts and a series of cell lines (A549, BNL CL.2, H225, NIH 3T3 and Rat-1) are efficiently transfected by using positively charged complexes of plasmid DNA and transferrin-polylysine or polylysine in the presence of glycerol (1 molar to 1.8 molar, depending on the cell type). An increase in gene ex...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/96205rr04
更新日期:1996-05-01 00:00:00
abstract::Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor m...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112169
更新日期:2006-05-01 00:00:00
abstract::Biopharmaceutical products are of great importance in the treatment or prevention of many diseases and represent a growing share of the global pharmaceutical market. The usual technology for protein synthesis (cell-based expression) faces certain obstacles, especially with 'difficult-to-express' proteins. Cell-free pr...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2020-0155
更新日期:2021-01-20 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
pub_type: 杂志文章
doi:10.2144/01315st03
更新日期:2001-11-01 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
pub_type: 杂志文章
doi:
更新日期:1992-12-01 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
pub_type: 杂志文章
doi:10.2144/00285rr08
更新日期:2000-05-01 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
pub_type: 杂志文章
doi:10.2144/00291rr02
更新日期:2000-07-01 00:00:00
abstract::Rapid identification of viruses is needed to monitor the blood supply for emerging threats. Here we present a method that meets these criteria and allows for the shotgun sequencing of novel, uncultured DNA viruses directly from human blood. This method employs selection based on the physical properties of viruses comb...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112019
更新日期:2005-11-01 00:00:00