LIVIVO - The Search Portal for Life Sciences

zur deutschen Oberfläche wechseln
Advanced search

Search results

Result 1 - 2 of total 2

Search options

  1. Article ; Online: Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy.

    Baoutina, A / Bhat, S / Zheng, M / Partis, L / Dobeson, M / Alexander, I E / Emslie, K R

    Gene therapy

    2016  Volume 23, Issue 10, Page(s) 708–717

    Abstract: There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression ... ...

    Abstract There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression is typically performed by qPCR using plasmid-based calibrants incorporating transgenic sequences. These often undergo limited characterisation and differ between manufacturers, potentially leading to inaccurate quantification, inconsistent inter-laboratory results and affecting clinical outcomes. Contamination of negative samples with such calibrants could cause false positive results. We developed a design strategy for synthetic reference materials (RMs) with modified transgenic sequences to prevent false positives due to cross-contamination. When such RM is amplified in transgene-specific assays, the amplicons are distinguishable from transgene's amplicons based on size and sequence. Using human erythropoietin as a model, we produced certified RM according to this strategy and following ISO Guide 35. Using non-viral and viral vectors, we validated the effectiveness of this RM in vector genome analysis in blood in vitro. The developed design strategy could be applied to production of RMs for other transgenes, genes or transcripts. Together with validated PCR assays, such RMs form a measurement tool that facilitates standardised, accurate and reliable genetic analysis in various applications.
    MeSH term(s) DNA, Recombinant/genetics ; DNA, Recombinant/metabolism ; Erythropoietin/genetics ; Erythropoietin/metabolism ; Genetic Therapy/standards ; Humans ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA, Messenger/standards ; Reference Standards ; Transgenes
    Chemical Substances DNA, Recombinant ; RNA, Messenger ; Erythropoietin (11096-26-7)
    Language English
    Publishing date 2016-10
    Publishing country England
    Document type Journal Article
    ZDB-ID 1191036-7
    ISSN 1476-5462 ; 0969-7128
    ISSN (online) 1476-5462
    ISSN 0969-7128
    DOI 10.1038/gt.2016.47
    Database MEDical Literature Analysis and Retrieval System OnLINE

    More links

    Kategorien

    In stock of ZB MED Cologne/Königswinter

    Zs.A 3991: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (2.OG)
    ab Jg. 2022: Lesesaal (EG)

    Order via subito

    This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.

  2. Article: International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus sequence comparisons.

    Olson, Nathan D / Lund, Steven P / Zook, Justin M / Rojas-Cornejo, Fabiola / Beck, Brian / Foy, Carole / Huggett, Jim / Whale, Alexandra S / Sui, Zhiwei / Baoutina, Anna / Dobeson, Michael / Partis, Lina / Morrow, Jayne B

    Biomolecular detection and quantification

    2015  Volume 3, Page(s) 17–24

    Abstract: This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification ... ...

    Abstract This study presents the results from an interlaboratory sequencing study for which we developed a novel high-resolution method for comparing data from different sequencing platforms for a multi-copy, paralogous gene. The combination of PCR amplification and 16S ribosomal RNA gene (16S rRNA) sequencing has revolutionized bacteriology by enabling rapid identification, frequently without the need for culture. To assess variability between laboratories in sequencing 16S rRNA, six laboratories sequenced the gene encoding the 16S rRNA from Escherichia coli O157:H7 strain EDL933 and Listeria monocytogenes serovar 4b strain NCTC11994. Participants performed sequencing methods and protocols available in their laboratories: Sanger sequencing, Roche 454 pyrosequencing(®), or Ion Torrent PGM(®). The sequencing data were evaluated on three levels: (1) identity of biologically conserved position, (2) ratio of 16S rRNA gene copies featuring identified variants, and (3) the collection of variant combinations in a set of 16S rRNA gene copies. The same set of biologically conserved positions was identified for each sequencing method. Analytical methods using Bayesian and maximum likelihood statistics were developed to estimate variant copy ratios, which describe the ratio of nucleotides at each identified biologically variable position, as well as the likely set of variant combinations present in 16S rRNA gene copies. Our results indicate that estimated variant copy ratios at biologically variable positions were only reproducible for high throughput sequencing methods. Furthermore, the likely variant combination set was only reproducible with increased sequencing depth and longer read lengths. We also demonstrate novel methods for evaluating variable positions when comparing multi-copy gene sequence data from multiple laboratories generated using multiple sequencing technologies.
    Language English
    Publishing date 2015-03-05
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 2821770-6
    ISSN 2214-7535
    ISSN 2214-7535
    DOI 10.1016/j.bdq.2015.01.004
    Database MEDical Literature Analysis and Retrieval System OnLINE

    More links

    Kategorien

    Order via subito

    This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.

To top