| 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; Umber et al., 2017). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection ...
[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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| Fluorescence-based CAPS Multiplex Genotyping on Capillary Electrophoresis Systems
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Author:
Date:
2015-05-20
[Abstract] Recent advances in next-generation sequencing techniques allow the detection of a large number of SNPs and their use in a high throughput manner. However, Cleaved Amplified Polymorphic Sequences (CAPSs) still play a significant role as complement to other high throughput methods for SNP genotyping. Therefore, new methods focusing on the acceleration of this type of markers are highly desirable. The combination of the classical CAPS technique and a M13-tailed primer multiplexing assay was used to develop an agarose gel free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected ...
[摘要] 新一代测序技术的最新进展允许以高通量方式检测大量的SNP及其使用。然而，裂解扩增多态性序列（CAPS）仍然作为补充其他高通量方法为SNP基因分型发挥重要作用。因此，非常需要关注于加速这种类型的标记的新方法。使用经典CAPS技术和M13尾引物多重测定的组合开发用于通过限制酶消化分析SNP的无琼脂糖凝胶方案。 PCR产物用通用M13引物进行荧光标记，随后用适当的限制性内切酶消化。在混合不同标记的产物后，在毛细管电泳系统上检测它们。该方法允许在短时间内以整体低成本以多路复用的方式进行成本有效的几种SNP的基因分型。此外，该方法可以有效地与在同一电泳运行中同时检测SSR组合，导致非常适合于基于标记的选择程序，绘图群体的基因分型和遗传多样性测定的程序。
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| Pyrosequencing Approach for SNP Genotyping in Plants Using a M13 Biotinylated Primer
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Author:
Date:
2015-05-20
[Abstract] Single Nucleotide Polymorphisms (SNPs), which constitute single base-pair variations in the DNA sequence, are the most abundant molecular markers in plant and animal genomes. They are becoming the markers of choice for genotyping in all fields of molecular biology, as they are easily prone to automation and high throughput, for example through pyrosequencing. This technology is accurate, flexible and can be easily automated. However, the need for primers labelled with biotin, promptly rise the cost of any methodology employing a pyrosequencing approach. In this protocol we described an improved, efficient, reliable and cost-effective pyrosequencing protocol, based on a universal M13 biotinylated primer, for SNP genotyping in plants.
[摘要] 单核苷酸多态性（SNP），其构成DNA序列中的单碱基对变异，是植物和动物基因组中最丰富的分子标记。 它们正成为分子生物学所有领域中基因分型的选择标记，因为它们易于自动化和高通量，例如通过焦磷酸测序。 这种技术是准确，灵活和可以很容易自动化。 然而，对用生物素标记的引物的需要迅速提高了使用焦磷酸测序方法的任何方法的成本。 在这个协议中，我们描述了基于通用M13生物素化引物，用于植物中的SNP基因分型的改进的，有效的，可靠的和成本有效的焦磷酸测序方案。
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