A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta
A rapid and versatile combined DNA/RNA extraction protocol and its application to the analysis of a novel DNA marker set polymorphic between Arabidopsis thaliana ecotypes Col-0 and Landsberg erecta
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DOI：
10.1186/1746-4811-1-4
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发表时间：
2005-01-01
期刊：
影响因子：
5.1
通讯作者：
Uelker, Bekir
中科院分区：
文献类型：
--
作者：
Berendzen, Kenneth;Searle, Iain;Uelker, Bekir
背景：在植物分子生物学中，许多已建立的基于PCR的方法依赖于冗长且昂贵的核酸分离方法。虽然有几种快速DNA分离方案，但它们尚未被测试用于RT-PCR应用的同时RNA分离。此外，传统的基于图位的克隆技术往往使用不成比例的标记区域，即使在与模式植物拟南芥，其中的可用性的完整基因序列，现在可以利用创建一个高密度的标记systems.Results：我们设计了一个高密度的多态性标记集之间的两个常用的生态型。这种新的多态性标记集允许PCR产物在琼脂糖凝胶上的大小分离，并在连锁作图实验中提供10 cM的初始分辨率，通过使用最少量的A.拟南芥组织使用该提取方案，我们还表征了分离的T-DNA插入突变。此外，我们已经表明，我们的快速核酸提取方案也可用于通过RT-PCR扩增监测转录物水平。最后，我们已经证明，我们的核酸分离方法也适用于其他植物物种，如烟草和大麦。结论：为了促进高通量连锁图谱和其他genornic应用，我们的核酸分离协议产生足够的质量的DNA和RNA模板的PCR和RT-PCR反应，分别。与其他纯化方法相比，这种新技术需要相当少的时间，并且与新的多态性PCR标记集相结合，大大减少了A.利用Col-0和兰茨贝格直立（Ler）生态型之间的杂交的拟南芥。
Background: Many established PCR-based approaches in plant molecular biology rely on lengthy and expensive methods for isolation of nucleic acids. Although several rapid DNA isolation protocols are available, they have not been tested for simultaneous RNA isolation for RT-PCR applications. In addition, traditional map-based cloning technologies often use ill-proportioned marker regions even when working with the model plant Arabidopsis thaliana, where the availability of the full genorne sequence can now be exploited for the creation of a high-density marker systems.Results: We designed a high-density polymorphic marker set between two frequently used ecotypes. This new polymorphic marker set allows size separation of PCR products on agarose gels and provides an initial resolution of 10 cM in linkage mapping experiments, facilitated by a rapid plant nucleic acid extraction protocol using minimal amounts of A. thaliana tissue. Using this extraction protocol, we have also characterized segregating T-DNA insertion mutations. In addition, we have shown that our rapid nucleic acid extraction protocol can also be used for monitoring transcript levels by RT-PCR amplification. Finally we have demonstrated that our nucleic acid isolation method is also suitable for other plant species, such as tobacco and barley.Conclusion: To facilitate high-throughput linkage mapping and other genornic applications, our nucleic acid isolation protocol yields sufficient quality of DNA and RNA templates for PCR and RT-PCR reactions, respectively. This new technique requires considerably less time compared to other purification methods, and in combination with a new polymorphic PCR marker set dramatically reduces the workload required for linkage mapping of mutations in A. thaliana utilizing crosses between Col-0 and Landsberg erecta (Ler) ecotypes.