| Analysis of Telomeric G-overhangs by in-Gel Hybridization
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Author:
Date:
2016-04-05
[Abstract] Telomeric DNA in majority of eukaryotes consists of an array of TG-rich tandem repeats. The TG-rich DNA strand is oriented with its 3’ end towards chromosome termini and is usually longer than its complementary CA-rich strand, thus forming 3’ single stranded overhang (G-overhang). G-overhangs arise from incomplete replication of chromosome termini by the lagging strand mechanism and post-replicative nucleolytic processing. The G-overhang is important for telomere protection as it serves as a binding platform for specific proteins and is required for t-loop formation. Hence, structure of ...
[摘要] 在大多数真核生物中的端粒DNA由富含TG的串联重复阵列组成。富含TG的DNA链以其3'末端朝向染色体末端定向，并且通常比其互补的富含CA的链更长，从而形成3'单链突出端（G突出端）。 G突出由染色体末端的不完全复制通过滞后链机制和复制后核酸水解加工产生。 G突出端对于端粒保护是重要的，因为其用作特异性蛋白质的结合平台并且是t环形成所需的。因此，端粒G突出端的结构是端粒维持和功能的重要指标。在这里我们描述了通过凝胶内杂交技术在模拟植物拟南芥中分析G突出端的方法。该方法允许单链端粒DNA的量的相对定量。短端粒探针放射性标记并在非变性条件下与DNA杂交以特异性检测ssDNA。可以使用在相同凝胶中的变性条件测量总端粒DNA，并且该程序通常在非变性凝胶内杂交之后。 ssDNA的末端性质通过核酸外切酶处理来验证。这种技术最初是在酵母中开发的，现在被用作从人类到植物的多种生物体中的G突出端分析的主要工具。
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| Purification and Sequencing of DNA Guides from Prokaryotic Argonaute
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Author:
Date:
2014-11-20
[Abstract] Some proteins utilize nucleic acids to guide them to complementary nucleic acid targets. One example is prokaryotic Argonaute protein, which, binds small single stranded DNA molecules as guides (Swarts et al., 2014). This protocol describes a method to purify DNA guides from these proteins. It also describes a PCR-based method to enrich the guides by PCR amplification. This methods relies on addition of a poly-A tail at the 3’-end of the ssDNA molecules by Terminal Deoxynucleotidyl Transferase (TdT), followed by ligation of a oligonucleotide to the 5’-end of the ssDNA molecule using ...
[摘要] 一些蛋白利用核酸来将它们引导至互补核酸靶。 一个实例是原核Argonaute蛋白，其结合小的单链DNA分子作为指导（Swarts等人，2014）。 该协议描述了从这些蛋白质中纯化DNA指南的方法。 它还描述了基于PCR的方法以通过PCR扩增来富集指南。 该方法依赖于通过末端脱氧核苷酸转移酶（TdT）在ssDNA分子的3'末端添加聚腺苷酸尾，然后使用T4 RNA连接酶将寡核苷酸连接到ssDNA分子的5'末端， 和通过PCR扩增。 所产生的dsDNA产物适合于传统的克隆和测序以及高通量测序。 重要的是，与ssDNA分子匹配的链的信息在该过程中不会丢失。
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| 5-Hydroxymethylcytosine (5-hmC) Specific Enrichment
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Author:
Date:
2012-08-05
[Abstract] 5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA modification in mammalian genomes. This protocol is to be a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically, which could in turn be used for high-throughput mapping via next-generation sequencing.
[摘要] 该实验方案的中文版正在准备中．．．
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