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BioTechniques

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https://www.readbyqxmd.com/read/28625161/recombinase-polymerase-amplification-rpa-an-isothermal-technique-suitable-for-a-range-of-applications-evaluation-of-rpa-volume-tolerance
#1
(no author information available yet)
No abstract text is available yet for this article.
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625160/blotcycler-consistently-shows-an-increase-in-western-blot-signal-intensity
#2
(no author information available yet)
No abstract text is available yet for this article.
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625159/dna-extraction-for-streamlined-metagenomics-of-diverse-environmental-samples
#3
Clarisse Marotz, Amnon Amir, Greg Humphrey, James Gaffney, Grant Gogul, Rob Knight
A major bottleneck for metagenomic sequencing is rapid and efficient DNA extraction. Here, we compare the extraction efficiencies of three magnetic bead-based platforms (KingFisher, epMotion, and Tecan) to a standardized column-based extraction platform across a variety of sample types, including feces, oral, skin, soil, and water. Replicate sample plates were extracted and prepared for 16S rRNA gene amplicon sequencing in parallel to assess extraction bias and DNA quality. The data demonstrate that any effect of extraction method on sequencing results was small compared with the variability across samples; however, the KingFisher platform produced the largest number of high-quality reads in the shortest amount of time...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625158/a-minimally-invasive-method-for-sampling-human-petrous-bones-from-the-cranial-base-for-ancient-dna-analysis
#4
Kendra A Sirak, Daniel M Fernandes, Olivia Cheronet, Mario Novak, Beatriz Gamarra, Tímea Balassa, Zsolt Bernert, Andrea Cséki, János Dani, József Zsolt Gallina, Gábor Kocsis-Buruzs, Ivett Kővári, Orsolya László, Ildikó Pap, Róbert Patay, Zsolt Petkes, Gergely Szenthe, Tamás Szeniczey, Tamás Hajdu, Ron Pinhasi
Ancient DNA (aDNA) research involves invasive and destructive sampling procedures that are often incompatible with anthropological, anatomical, and bioarcheological analyses requiring intact skeletal remains. The osseous labyrinth inside the petrous bone has been shown to yield higher amounts of endogenous DNA than any other skeletal element; however, accessing this labyrinth in cases of a complete or reconstructed skull involves causing major structural damage to the cranial vault or base. Here, we describe a novel cranial base drilling method (CBDM) for accessing the osseous labyrinth from the cranial base that prevents damaging the surrounding cranial features, making it highly complementary to morphological analyses...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625157/purification-of-single-stranded-dna-by-co-polymerization-with-acrylamide-and-electrophoresis
#5
Tulsi Ram Damase, Andrew D Ellington, Peter B Allen
Single-stranded DNA (ssDNA) oligonucleotides are useful as aptamers, hybridization probes and for emerging applications in DNA nanotechnology. Current methods to purify ssDNA require both a strand-separation step and a separate size-separation step but may still leave double-stranded DNA (dsDNA) impurities in the sample. Here, we use commercially available acrydite DNA primers to immobilize one strand of a PCR product within a polyacrylamide matrix. Electrophoresis moves the non-crosslinked DNA into the gel where the single-stranded product of desired size can be recovered...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625156/heteroduplex-cleavage-assay-for-screening-of-probable-zygosities-resulting-from-crispr-mutations-in-diploid-single-cell-lines
#6
Kyle D Luttgeharm, Kit-Sum Wong, Steve Siembieda
The most common gene editing methods, such as CRISPR, involve random repair of an induced double-stranded DNA break through the non-homologous end joining (NHEJ) repair pathway, resulting in small insertions/deletions. In diploid cells, these mutations can take on one of three zygosities: monoallelic, diallelic heterozygous, or diallelic homozygous. While many advances have been made in CRISPR delivery systems and gene editing efficiency, little work has been done to streamline detection of gene editing events...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625155/applications-of-optical-dna-mapping-in-microbiology
#7
Diana Bogas, Lena Nyberg, Rui Pacheco, Nuno F Azevedo, Jason P Beech, Margarita Gomila, Jorge Lalucat, Célia M Manaia, Olga C Nunes, Jonas O Tegenfeldt, Fredrik Westerlund
Optical mapping (OM) has been used in microbiology for the past 20 years, initially as a technique to facilitate DNA sequence-based studies; however, with decreases in DNA sequencing costs and increases in sequence output from automated sequencing platforms, OM has grown into an important auxiliary tool for genome assembly and comparison. Currently, there are a number of new and exciting applications for OM in the field of microbiology, including investigation of disease outbreaks, identification of specific genes of clinical and/or epidemiological relevance, and the possibility of single-cell analysis when combined with cell-sorting approaches...
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625154/expanding-the-genetic-alphabet
#8
Janelle Weaver
Synthetic biologists have engineered a semisynthetic organism that can store increased genetic information using an alphabet consisting of six letters that form three base pairs. Janelle Weaver learns how they did it.
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28625153/stories-in-ancient-dna
#9
Nathan Blow
Technology developments are leading to rapid advances in ancient DNA analysis. Nathan Blow talks to researchers peering into the past through ancient DNA.
June 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528577/single-step-pcr-based-genetic-sex-determination-of-rat-tissues-and-cells
#10
Pramod Dhakal, Michael J Soares
The advent of genome editing strategies has expanded the range of animal models available for gene manipulation and renewed research interest in the rat. Gender is a key variable for in vivo gene function analyses. Here, we present a simple PCR-based method to determine genetic sex in the rat.
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528576/using-non-thermal-irreversible-electroporation-to-create-an-in-vivo-niche-for-exogenous-cell-engraftment
#11
Tammy T Chang, Vivian X Zhou, Boris Rubinsky
The critical shortage of donor organs has spurred investigation of alternative approaches to either generate replacement organs or implant exogenous cells for treatment of end-stage organ failure. Non-thermal irreversible electroporation (NTIRE), which uses brief high electric field pulses to induce irreversible permeabilization of cell membranes, has emerged as a technique for tumor ablation. Here, we introduce a new application for NTIRE that employs in situ cell ablation to create a niche within a solid organ for engraftment of exogenous cells in vivo...
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528575/characterization-of-a-cyclic-olefin-polymer-microcentrifuge-tube
#12
Lloyd Waxman, Rachel L Erwin, Vinod D Vilivalam
Here, we describe the properties of a prototype microcentrifuge tube made from the plastic cyclic olefin polymer (COP). This material has been used in the manufacture of primary containers including syringes and vials for the storage, shipment, and delivery of biotherapeutics, vaccines, and cell therapy products. Its low level of extractable substances and metals along with its glass-like clarity make COP an attractive material for the fabrication of microcentrifuge tubes and other consumable laboratory plasticware where contamination is an important consideration, such as in the storage and analysis of labile proteins, nucleic acids, and metabolites...
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528574/esc-track-a-computer-workflow-for-4-d-segmentation-tracking-lineage-tracing-and-dynamic-context-analysis-of-escs
#13
Laura Fernández-de-Manúel, Covadonga Díaz-Díaz, Daniel Jiménez-Carretero, Miguel Torres, María C Montoya
Embryonic stem cells (ESCs) can be established as permanent cell lines, and their potential to differentiate into adult tissues has led to widespread use for studying the mechanisms and dynamics of stem cell differentiation and exploring strategies for tissue repair. Imaging live ESCs during development is now feasible due to advances in optical imaging and engineering of genetically encoded fluorescent reporters; however, a major limitation is the low spatio-temporal resolution of long-term 3-D imaging required for generational and neighboring reconstructions...
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528573/a-sensitive-semi-quantitative-mammalian-two-hybrid-assay
#14
Elisabeth Riegel, Thomas Heimbucher, Thomas Höfer, Thomas Czerny
Protein-protein interactions critically determine the function of a protein within the cell. Several methods have been developed for the analysis of protein interactions, including two-hybrid assays in yeast and mammals. Mammalian two-hybrid systems provide the ideal physiological environment to study the interactions of mammalian proteins; however, these approaches are limited in sensitivity and their ability to quantify interaction strength. Here, we present an inducible mammalian two-hybrid (iM2H) system using the small-molecule dimerizer rapalog for recruitment of multiple transactivation domains into the M2H system...
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28528572/going-with-the-flow
#15
Nathan Blow
From bacteria to circulating tumor cells, advances in flow cytometry technology are pushing the boundaries of cell biology research.
May 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403810/transient-dominant-host-range-selection-using-chinese-hamster-ovary-cells-to-generate-marker-free-recombinant-viral-vectors-from-vaccinia-virus
#16
Liang Liu, Tamara Cooper, Preethi Eldi, Pablo Garcia-Valtanen, Kerrilyn R Diener, Paul M Howley, John D Hayball
Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses...
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403809/effective-dna-fragmentation-technique-for-simple-sequence-repeat-detection-with-a-microsatellite-enriched-library-and-high-throughput-sequencing
#17
Keisuke Tanaka, Rumi Ohtake, Saki Yoshida, Takashi Shinohara
Two different techniques for genomic DNA fragmentation before microsatellite-enriched library construction-restriction enzyme (NlaIII and MseI) digestion and sonication-were compared to examine their effects on simple sequence repeat (SSR) detection using high-throughput sequencing. Tens of thousands of SSR regions from 5 species of the plant family Myrtaceae were detected when the output of individual samples was >1 million paired-end reads. Comparison of the two DNA fragmentation techniques showed that restriction enzyme digestion was superior to sonication for identification of heterozygous genotypes, whereas sonication was superior for detection of various SSR flanking regions with both species-specific and common characteristics...
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403808/imagej-macros-for-the-user-friendly-analysis-of-soft-agar-and-wound-healing-assays
#18
João Paulo Silva Nunes, Adriana Abalen Martins Dias
Recent advances in biological imaging techniques and the enormous amount of data they generate call for the development of computational tools for efficient and reliable high-throughput analysis. Several software applications with this functionality are available, and one of the most commonly used is ImageJ. Here, we present two independent macros (WH_NJ and SA_NJ) for automating and facilitating the analysis of images acquired from two in vitro assays frequently used in cancer studies and drug screening: the wound-healing and soft-agar assays...
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403807/generating-stable-cell-lines-with-quantifiable-protein-production-using-crispr-cas9-mediated-knock-in
#19
Chiu-An Lo, Alexander W Greben, Brian Edwin Chen
Cell lines expressing foreign genes have been widely used to produce a variety of recombinant proteins. However, generating recombinant protein-expressing cell lines is usually a lengthy process and the resulting protein expression levels are often inconsistent. Here, we describe an efficient method for making stable cell lines expressing any recombinant protein of interest in a controllable and quantifiable manner. We integrate transgenes into specific genomic loci using CRISPR/Cas9 such that transgene expression is driven by endogenous promoters to ensure consistent and predictable expression of the recombinant protein...
April 1, 2017: BioTechniques
https://www.readbyqxmd.com/read/28403806/two-color-fluorescent-cytosine-extension-assay-for-the-determination-of-global-dna-methylation
#20
Gu Zhou, Craig Parfett, Cathy Cummings-Lorbetskie, Gong-Hua Xiao, Daniel Desaulniers
Here, we present a DNA restriction enzyme-based, fluorescent cytosine extension assay (CEA) to improve normalization and technical variation among sample-to-sample measurements. The assay includes end-labeling of parallel methylation-sensitive and methylation-insensitive DNA restriction enzyme digests along with co-purification and subsequent co-measurement of incorporated fluorescence. This non-radioactive, two-color fluorescent CEA (TCF-CEA) was shown to be a relatively rapid and accurate, with 3-fold greater precision than the one-color CEA...
April 1, 2017: BioTechniques
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