Rolling Circle Amplification to Screen Yam Germplasm for Badnavirus Infections and to Amplify and Characterise Novel Badnavirus Genomes
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Author:
Date:
2018-01-05
[Abstract] Since the first discovery of badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem et al., 2009), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported (Phillips et al., 1999; Seal and Muller, 2007; Bömer et al., 2016 and 2017; Sukal et al., 2017; ...
[摘要] 自二十世纪七十年代山药(Dioscorea spp。)种质中首次发现坏病毒属(家庭花椰菜科,属于病毒属)之后(Harrison和Roberts, 1973),已经表征了数百个部分坏死病毒逆转录酶(RT) - 核糖核酸酶H(RNaseH)序列(Kenyon等人,2008; Bousalem等人,2009年),但仅有少数几种完整的Dioscorea杆状病毒(DBV)基因组序列已被报道(Phillips等,1999; Seal和Muller,2007;Bömer等, 2016和2017; Sukal等人,2017; Umber等人,2017)。我们优化了总核酸提取和滚环扩增(RCA)结合限制性酶分析的工作流程,以检测和扩增山药种质中存在的DBV。我们已经使用这种方法成功地揭示了三种新型附加体阴性坏死病毒(Bömer等人,2016年)。我们提出这是变性梯度凝胶电泳的补充方法,其能够快速指示坏死病毒多样性以及在宿主基因组中鉴定潜在整合的坏死病毒序列(Turaki等人,2017年) )。在这里,我们描述了一步一步的方案来筛选山药种质的坏死病毒感染使用RCA作为一个有效的研究工具,在扩增和表征的新型坏死病毒基因组。
【背景】RCA是经常用于扩增环状DNA病毒基因组的序列无关的策略(Rector等人,2004)。 Phi29聚合酶介导的RCA技术用于(i)检测新型病毒; ...
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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 ...
[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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Yeast DNA Replication 2D Gel Protocol
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Author:
Date:
2012-07-05
[Abstract] Two-dimensional agarose gel electrophoresis (2D gel) analysis is used extensively as a method to detect origins of replication. Here, I present a simplified method for the isolation of yeast genomic DNA for 2D gel analysis from a small number of yeast cells. This DNA isolation method is simpler and less time consuming than the traditional method that involves CsCl density gradient centrifugation. This method could be modified for 2D gel analysis in other organisms as well.
[摘要] 双向琼脂糖凝胶电泳是一种被广泛用来检测DNA复制起点的方法。在这里,我介绍一种从少量酵母细菌中提取其基因组DNA用于双向电泳分析的简便方法。与传统的提取DNA的方法相比较,这种涉及到氯化铯密度梯度离心的方法更加简便和省时。而且,对这种方法进行适当的改进,从而使其也可适用于其他有机物的双向电泳分析。
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