A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy
|
Author:
Date:
2021-04-05
[Abstract] The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis (Chang et al., 2020). This protocol was used in the 3D ...
[摘要] [摘要]自由生活的线虫秀丽隐杆线虫是研究发育生物学的流行模型系统。在这里,我们描述了详细的协议,以高压冷冻线虫的胚胎(解剖后离体,或完整的蠕虫内),然后快速冷冻替代。经过处理的样品适合通过常规电子显微镜(EM)或更新的体积EM(vEM)方法(如Focuse d离子束扫描电子显微镜(FIB-SEM))进行超微结构分析。的细胞特征,例如超微结构的NUCL耳信封,染色体,内质网和线粒体保存良好这些实验程序后,并产生精确的三维模型用于可视化和分析(张等人,2020)。在秀丽隐杆线虫合子的前核相遇后,该方案被用于膜和染色体的3D重建(Rahman等,2020)。
[背景技术]线虫是自由生活线虫具有许多特性,使其适合于科学的研究:(1)将蠕虫是〜1毫米长; ...
|
|
Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome
|
Author:
Date:
2021-03-05
[Abstract] Next generations sequencing (NGS) has become an important tool in biomedical research. The Primer ID approach combined with the MiSeq platform overcomes the limitation of PCR errors and reveals the true sampling depth of population sequencing, making it an ideal tool to study mutagenic effects of potential broad-spectrum antivirals on RNA viruses. In this report we describe a protocol using Primer ID sequencing to study the mutations induced by antivirals in a coronavirus genome from an in vitro cell culture model and an in vivo mouse model. Viral RNA or total lung tissue RNA is tagged with Primer ID-containing cDNA primers during the initial reverse transcription step, followed by two rounds of PCR to amplify viral sequences and incorporate sequencing adaptors. Purified and pooled ...
[摘要] [摘要]下一代测序(NGS)已成为生物医学研究的重要工具。结合MiSeq平台的Primer ID方法克服了PCR错误的局限性,并揭示了群体测序的真实采样深度,使其成为研究潜在的广谱抗病毒剂对RNA病毒的诱变作用的理想工具。在本报告中,我们描述了一种使用引物ID测序的方案,用于研究体外细胞培养模型和体内小鼠模型中冠状病毒基因组中抗病毒药诱导的突变。在最初的反转录步骤中,病毒RNA或总肺组织RNA用含Primer ID的cDNA引物标记,然后进行两轮PCR扩增病毒序列并整合测序适配器。使用MiSeq平台对纯化和合并的文库进行测序。测序数据使用模板共有序列(TCS)网络应用处理。引物ID方法提供了一种精确的测序方案,可以测量病毒RNA基因组和宿主mRNA中的突变错误率。测序结果表明,β-D-N4-羟基胞嘧啶核苷(NHC)大大提高了病毒RNA基因组中的过渡取代率,但并未显着提高颠覆取代率,并且发现胞嘧啶(C)至尿苷(U)是最常见的突变。
[背景]下一代测序(NGS)已被广泛应用在生物医学研究中使用在过去十年。当应用NGS研究宿主内病毒种群的RNA病毒时,需要考虑对文库制备和测序方案的修改。样本之间的病毒滴度(或病毒载量)差异很大。传统的NGS平台在测序运行中需要1-500 ng的DNA(或RNA),但在大多数情况下,临床样品中的病毒RNA少于100 ...
|
|
Nonenzymatic RNA-templated Synthesis of N3′→P5′ Phosphoramidate DNA
|
Author:
Date:
2020-09-05
[Abstract] The RNA world hypothesis describes a scenario where early life forms relied on RNA to govern both inheritance and catalyze useful chemical reactions. Prior to the emergence of enzymes capable of replicating the RNA genome, a nonenzymatic replication process would have been necessary to initiate Darwinian Evolution. However, the one-pot nonenzymatic RNA chemical copying of templates with mixed-sequences is insufficient to generate strand products long enough to encode useful function. The use of alternate (RNA-like) genetic polymers may overcome hurdles associated with RNA copying, and further our understanding of nonenzymatic copying chemistry. This protocol describes the nonenzymatic copying of RNA templates into N3′→P5′ phosphoramidate DNA (3′-NP-DNA). We describe, in detail, the ...
[摘要] [摘要 ] RNA世界假说描述了一种场景,即早期生命形式依赖于RNA来控制遗传和催化有用的化学反应。在能够复制RNA基因组的酶出现之前,必须进行非酶复制过程以启动达尔文进化论。然而,具有混合序列的模板的一锅法非酶RNA化学复制不足以产生足够长的链产物以编码有用的功能。使用替代的(类似RNA的)遗传聚合物可以克服与RNA复制相关的障碍,并进一步我们对非酶复制化学的理解。该协议描述了将RNA模板非酶复制到N3'→P5' 氨基磷酸酯中的过程DNA(3'-NP-DNA)。我们详细描述了被2-氨基咪唑(3'-NH 2 -2AIpddN)活化的3'-氨基-2',3'-二脱氧核糖核苷酸单体的合成及其在模板指导的聚合反应中的用途。
[背景 ] 的Prim itive生命形式可能包括能够复制和功能的基因组中,一空间限定的隔室中包封的(绍斯塔克等人,2001;绍斯塔克,2012;布莱恩和Szostak,2014) 。在能够复制RNA基因组的酶出现之前,非酶复制过程可能是启动达尔文进化所必需的(Szostak,2012和2017)。由于RNA具有催化重要的化学反应(例如蛋白质合成)并在现代生物学环境中充当遗传信息载体的能力,因此它是原始遗传聚合物的逻辑候选物。
...
|
|