cDNA Display Mediated Immuno-PCR (cD-IPCR): A Novel PCR-based Antigen Detection Method
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Author:
Date:
2019-12-20
[Abstract] Immuno-PCR (IPCR) is a powerful method in antigen detection where a PCR-amplifiable DNA reporter is conjugated to a specific antibody or an aptamer for the target molecule. In the development and application of IPCR, successful conjugation of a protein (an antibody) with a reporter DNA becomes challenging. To address this issue, we recently demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level. The cDNA display molecule for IPCR is generated first by transcribing the DNA that encodes the detection antibody into an mRNA by in vitro transcription. A puromycin DNA linker is then ligated to the mRNA and then in vitro translation and reverse-transcription are performed to generate ...
[摘要] 免疫PCR(IPCR)是抗原检测中的一种强大方法,其中可PCR扩增的DNA报道分子与目标分子的特异性抗体或适体偶联。在IPCR的开发和应用中,蛋白质(抗体)与报告DNA的成功偶联变得具有挑战性。为了解决这个问题,我们最近证明了基于cDNA展示,多肽的1:1共价复合物及其单分子编码cDNA的IPCR的可行性。首先,通过体外转录将编码检测抗体的DNA转录为mRNA,从而生成用于IPCR的cDNA展示分子。然后将嘌呤霉素DNA接头连接到mRNA,然后进行体外翻译和逆转录生成cDNA展示分子。然后将该分子直接用于抗原检测和后续qPCR。如果已知其单结构域抗体(VHH)或肽适体的序列,则该方法可用于检测生物样品中的各种抗原。
【背景】免疫-PCR,通常缩写为IPCR,是一种检测和定量生物样品(例如,血清和尿液)中存在的低丰度生物标志物的强大方法。它充当免疫反应和信号放大之间的桥梁。在大多数情况下,IPCR依赖于已与报道寡核苷酸偶联的检测抗体的使用,然后使用报道者的实时PCR定量分析物(Sano et al。, 1992)。然而,在IPCR的开发和应用中,与寡核苷酸适当结合抗体的困难已经成为最具挑战性的问题(van ...
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Measuring Nucleosome Assembly Activity in vitro with the Nucleosome Assembly and Quantification (NAQ) Assay
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Author:
Date:
2018-02-05
[Abstract] Nucleosomes organize the eukaryotic genome into chromatin. In cells, nucleosome assembly relies on the activity of histone chaperones, proteins with high binding affinity to histones. At least a subset of histone chaperones promotes histone deposition in vivo. However, it has been challenging to characterize this activity, due to the lack of quantitative assays.
Here we developed a quantitative nucleosome assembly (NAQ) assay to measure the amount of nucleosome formation in vitro. This assay relies on a Micrococcal nuclease (MNase) digestion step that yields DNA fragments protected by the deposited histone proteins. A subsequent run on the Bioanalyzer machine allows the accurate quantification of the fragments (length and amount), relative to a loading ...
[摘要] 核小体将真核生物基因组组装成染色质。在细胞中,核小体装配依赖于组蛋白分子伴侣的活性,对组蛋白具有高结合亲和力的蛋白。至少有一部分组蛋白伴侣促进组蛋白在体内的沉积。然而,由于缺乏定量分析,鉴定这种活性一直是一个挑战。
在这里,我们开发了一种定量核小体装配(NAQ)测定来测量体外核小体形成的量。该测定依赖于微球菌核酸酶(MNase)。随后在生物分析仪上运行,可以准确量化相对于加样对照的片段(长度和数量)。这使我们能够测量约150bp的DNA长度。该测定最终实现了不同组蛋白分子伴侣的核小体装配活性的表征,这是理解这些蛋白体内功能作用的一个步骤。
【背景】真核生物基因组被组织成核小体。核小体是由组蛋白八聚体核心组成的模块化和动态结构,由147bp的DNA包裹(Luger等人,1997)。核小体组装始于一个(H3-H4)2四聚体沉积到DNA上以形成四体体。随后的H2A-H2B二聚体结合形成六聚体,最后形成核小体。组蛋白高度带电,因为它们以生理盐浓度存在于组蛋白二聚体中。由于它们的作用,组蛋白需要分子伴侣将它们从细胞质穿梭到细胞核,然后辅助它们沉积到DNA上或从DNA上去除(Gurard-Levin等人,2014)。
组蛋白分子伴侣分组在结构上不相关的蛋白质家族中,所有这些蛋白质的特征在于对组蛋白的高度结合亲和力(Laskey等,1978)。通过这种方式,他们屏蔽了组蛋白的电荷,阻止了与DNA和其他细胞因子的非特异性相互作用(Elsässerand ...
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In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
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Author:
Date:
2017-06-20
[Abstract] We previously reported that oxidized nucleotide insertion by DNA polymerase β (pol β) can confound the DNA ligation step during base excision repair (BER) (Çağlayan et al., 2017). Here, we describe a method to investigate pol β nucleotide insertion coupled with DNA ligation, in the same reaction mixture including dGTP or 8-oxo-dGTP, pol β and DNA ligase I. This in vitro assay enables us to measure the products for correct vs. oxidized nucleotide insertion, DNA ligation, and ligation failure, i.e., abortive ligation products, as a function of reaction time. This protocol complements our previous publication and describes an efficient way to analyze activities of BER enzymes and the functional interaction between pol β and DNA ligase I in vitro.
[摘要] 我们以前报告说,通过DNA聚合酶β(polβ)的氧化的核苷酸插入可能会在碱基切除修复(BER)(Çağlayan等,2017)期间混淆DNA连接步骤。 在这里,我们描述了在与dGTP或8-氧代-dGTP,polβ和DNA连接酶I相同的反应混合物中研究与DNA连接相结合的polβ核苷酸插入的方法。该体外测定使得我们能够测量产物的正确性 与氧化核苷酸插入,DNA连接和连接失败,即流产结扎产物,作为反应时间的函数。 该协议补充了我们以前的出版物,并描述了一种有效的方法来分析BER酶的活性和polβ和DNA连接酶I在体外的功能相互作用。 【背景】该方案是观察BER路径的最后两个步骤:通过连接酶I通过polβ进行核苷酸插入和DNA连接。使用该方案,在体外在同一反应混合物中测量两种反应作为时间的函数。原始实体是在模拟BER中间体的单核苷酸缺口DNA底物上分析BER酶polβ和DNA连接酶I。这些BER酶与此BER中间体结合并起作用。该方案中使用的DNA底物包括在5'和3'端的荧光标记,使我们能够观察DNA底物的单核苷酸插入和DNA连接。与DNA连接偶联的polβ核苷酸插入模拟了切口DNA中间体从核苷酸插入步骤切换或引导到BER途径期间的连接步骤。使用该方案,也可以在polβ氧化的核苷酸(8-氧代-dGTP)插入后测量连接失败或者终止连接。这通过在BER中间体的5'-末端添加腺苷酸(AMP)基团进行定量来实现。该方案也可用于测量与其他DNA聚合酶和DNA连接酶连接的核苷酸插入。 ...
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