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    • 9. 发明授权
      US09068972B2 GFP mutagenesis amplification: use of a fluorescence-antibiotic resistance fusion dual reporter construct to provide quantitative and highly sensitive detection of mutations 有权
    • GFP mutagenesis amplification: use of a fluorescence-antibiotic resistance fusion dual reporter construct to provide quantitative and highly sensitive detection of mutations
    • GFP诱变扩增：使用荧光抗生素抗性融合双重报道构建体提供定量和高度灵敏的突变检测
    • US09068972B2
    • 2015-06-30
    • US13504911
    • 2010-11-02
    • Manel Camps , Jennifer Allen
    • Manel Camps , Jennifer Allen
    • G01N21/64 , C12N15/63 , C40B30/06 , G01N33/50
    • G01N33/5023 , C12N15/1086 , C12Q1/6897 , G01N2500/10
    • A reversion mutation assay that is unique in providing a quantitative readout for mutagenesis. This assay is based on the creation of a functional GFP-β-lactamase fusion protein as a reporter providing both antibiotic resistance and fluorescence. This dual reporter is placed in a multicopy plasmid to increase the number of targets, with a reversion site at the N-terminus. Rare mutations at the reversion site allow read-through of the fusion protein, producing both beta-lactamase (providing antibiotic resistance) and GFP (emitting fluorescence). In the presence of carbenicillin, beta-lactamase production confers a selective advantage that allows amplification of mutant plasmids, raising the level of fluorescence emitted by GFP to levels that are detectable by fluorimetry. A window of time can be found where fluorescence is proportional to the number of mutation events at the reversion site, making fluorescence a quantitative measure of mutagenesis. Quantitative (as opposed to binary) detection of mutations allows substantial savings in test sample. This has applications in drug discovery, allowing high-throughput screening for DNA-targeting compounds and early pre-screening of leads for potential carcinogenic activity. The increased sensitivity of this assay also facilitates monitoring complex environmental samples.
    • 在提供定量读数用于诱变方面独特的逆转突变测定法。 该测定基于产生功能性GFP-＆bgr-β-内酰胺酶融合蛋白作为提供抗生素抗性和荧光的报告物。 将该双重报告物置于多拷贝质粒中以增加靶的数目，其N-末端具有逆转位点。 在反转位点的罕见突变允许融合蛋白的读取，产生β-内酰胺酶（提供抗生素抗性）和GFP（发射荧光）。 在羧苄青霉素存在下，β-内酰胺酶的产生具有允许扩增突变质粒的选择性优势，将GFP发射的荧光水平提高到通过荧光测定可检测的水平。 可以发现一个时间窗口，其中荧光与反转位点处的突变事件的数量成比例，使荧光成为诱变的定量测量。 定量（而不是二元）检测突变可以大大节省测试样品。 这在药物发现中有应用，允许对DNA靶向化合物进行高通量筛选和对潜在致癌活性进行早期预筛选。 该测定的灵敏度增加也有助于监测复杂的环境样品。
    • 基本信息 添加关注 加入工作空间 PDF全文 PDF下载 全文下载 相似专利 双屏查看 权利要求书 说明书全文 Espacenet 法律信息 同族专利 专利引用
    • 10. 发明申请
      US20120302461A1 GFP MUTAGENESIS AMPLIFICATION: USE OF A FLUORESCENCE-ANTIBIOTIC RESISTANCE FUSION DUAL REPORTER CONSTRUCT TO PROVIDE QUANTITATIVE AND HIGHLY SENSITIVE DETECTION OF MUTATIONS 有权
    • GFP MUTAGENESIS AMPLIFICATION: USE OF A FLUORESCENCE-ANTIBIOTIC RESISTANCE FUSION DUAL REPORTER CONSTRUCT TO PROVIDE QUANTITATIVE AND HIGHLY SENSITIVE DETECTION OF MUTATIONS
    • GFP MUTAGENESIS放大：使用荧光抗性抗性融合双重报告器结构提供定量和敏感性检测突变
    • US20120302461A1
    • 2012-11-29
    • US13504911
    • 2010-11-02
    • Manel Camps , Jennifer Allen
    • Manel Camps , Jennifer Allen
    • G01N21/64 , C12N15/63 , C40B30/06
    • G01N33/5023 , C12N15/1086 , C12Q1/6897 , G01N2500/10
    • A reversion mutation assay that is unique in providing a quantitative readout for mutagenesis. This assay is based on the creation of a functional GFP-β-lactamase fusion protein as a reporter providing both antibiotic resistance and fluorescence. This dual reporter is placed in a multicopy plasmid to increase the number of targets, with a reversion site at the N-terminus. Rare mutations at the reversion site allow read-through of the fusion protein, producing both beta-lactamase (providing antibiotic resistance) and GFP (emitting fluorescence). In the presence of carbenicillin, beta-lactamase production confers a selective advantage that allows amplification of mutant plasmids, raising the level of fluorescence emitted by GFP to levels that are detectable by fluorimetry. A window of time can be found where fluorescence is proportional to the number of mutation events at the reversion site, making fluorescence a quantitative measure of mutagenesis. Quantitative (as opposed to binary) detection of mutations allows substantial savings in test sample. This has applications in drug discovery, allowing high-throughput screening for DNA-targeting compounds and early pre-screening of leads for potential carcinogenic activity. The increased sensitivity of this assay also facilitates monitoring complex environmental samples.
    • 在提供定量读数用于诱变方面独特的逆转突变测定法。 该测定基于产生功能性GFP-＆bgr-β-内酰胺酶融合蛋白作为提供抗生素抗性和荧光的报告物。 将该双重报告物置于多拷贝质粒中以增加靶的数目，其N-末端具有逆转位点。 在反转位点的罕见突变允许融合蛋白的读取，产生β-内酰胺酶（提供抗生素抗性）和GFP（发射荧光）。 在羧苄青霉素存在下，β-内酰胺酶的产生具有允许扩增突变质粒的选择性优势，将GFP发射的荧光水平提高到通过荧光测定可检测的水平。 可以发现一个时间窗口，其中荧光与反转位点处的突变事件的数量成比例，使荧光成为诱变的定量测量。 定量（而不是二元）检测突变可以大大节省测试样品。 这在药物发现中有应用，允许对DNA靶向化合物进行高通量筛选和对潜在致癌活性进行早期预筛选。 该测定的灵敏度增加也有助于监测复杂的环境样品。
    • 基本信息 添加关注 加入工作空间 PDF全文 PDF下载 全文下载 相似专利 双屏查看 权利要求书 说明书全文 Global Dossier Espacenet 法律信息 同族专利 专利引用
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