| Coupling Exonuclease Digestion with Selective Chemical Labeling for Base-resolution Mapping of 5-Hydroxymethylcytosine in Genomic DNA
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Author:
Date:
2018-03-05
[Abstract] This protocol is designed to obtain base-resolution information on the level of 5-hydroxymethylcytosine (5hmC) in CpGs without the need for bisulfite modification. It relies on (i) the capture of hydroxymethylated sequences by a procedure known as ‘selective chemical labeling’ (see Szulwach et al., 2012) and (ii) the digestion of the captured DNA by exonucleases. After Illumina sequencing of the digested DNA fragments, an ad hoc bioinformatic pipeline extracts the information for further downstream analysis.
[摘要] 该协议旨在获得CpGs中5-羟甲基胞嘧啶（5hmC）水平的碱基分辨率信息，而无需亚硫酸氢盐修饰。 它依赖于（i）通过称为“选择性化学标记”（参见Szulwach等人，2012）的方法捕获羟甲基化序列和（ii）通过外切核酸酶消化捕获的DNA。 在消化的DNA片段的Illumina测序之后，特设的生物信息学管道提取信息用于进一步的下游分析。
【背景】基因组DNA中胞嘧啶的甲基化可以被蛋白质读取，并且主要被翻译成基因沉默。基因组中的大多数CpG二核苷酸是甲基化的，包括位于基因调控区如增强子的那些。然而，当需要时，这些CpG可以通过Ten Eleven Translocation（TET）酶将甲基氧化并且通过碱基切除修复系统用未甲基化的胞嘧啶置换来去甲基化。 5-羟甲基胞嘧啶（5hmC）是5-甲基胞嘧啶的第一个氧化衍生物，并且在基因组中绘制该修饰的碱基提供了关于正在进行活性去甲基化的区域的信息。尽管选择性化学标记（SCL）可以非常特异地检测5hmC，但该技术的分辨率受DNA片段大小的限制，特别是当捕获的DNA中存在多个CpG时。为了提高分辨率，我们引入了使用外切核酸酶的消化步骤，所述核酸外切酶将DNA分子修剪成靠近羟甲基化的胞嘧啶（Sérandour et。，2016）。然后对测序读数进行适当的生物信息学处理，然后将羟甲基化评分赋予捕获的CpG。
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| 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 telomeric G-overhang is an important indicator of telomere maintenance and functionality. Here we describe a method for analysis of G-overhangs in a model plant Arabidopsis thaliana by in-gel ...
[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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| Generation and Screening of a Non-typeable Haemophilus influenzae Tn-seq Mutant Library
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Author:
Date:
2014-03-05
[Abstract] The genome-wide screen Tn-seq (van Opijnen et al., 2009) is very valuable tools to identify bacterial genes with a conditionally essential function, for instance genes involved in bacterial virulence. These techniques are based on the generation of a random mutant library, which is grown in a control of challenge situation (Figure 1). The advantage of using a mariner transposon for the generation of a random transposon mutant library is its insertion into TA sites, which makes the insertion in the genome highly random. In addition, an MmeI restriction site can be introduced in the inverted repeat of the transposon, without affecting the recognition by HimarC9 transposase.
[摘要] 全基因组筛选Tn-seq（van Opijnen等人，2009）是鉴定具有条件必需功能的细菌基因（例如涉及细菌毒力的基因）的非常有价值的工具。 这些技术基于产生随机突变体文库，其在挑战情况的对照中生长（图1）。 使用水手转座子产生随机转座子突变体文库的优点是其插入TA位点，这使得在基因组中的插入高度随机。 此外，可以在转座子的反向重复中引入MmeI限制位点，而不影响HimarC9转座酶的识别。
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