A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores
A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores
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DOI：
10.1007/pl00009996
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发表时间：
2001-01
期刊：
影响因子：
3.9
通讯作者：
Daniel Schwarzott;A. Schüßler
中科院分区：
文献类型：
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作者：
Daniel Schwarzott;A. Schüßler
我们描述了一种从丛枝菌根真菌中快速简便地扩增和克隆几乎完整的SSU rRNA基因的方法。对37个不同的球孢霉菌株的孢子进行了检测，方法是用动态磁珠磁选，然后用两对引物进行巢式聚合酶链式反应。所有试验都产生了预期大小的可见扩增产物。然后，利用拓扑异构酶激活的载体(pCR2.1-Topo)，可以快速高效地克隆出PCR片段。这项技术快速、简单，成本相对较低。使用单孢子提取DNA比多孢子制剂有一些优点，例如，它不太容易受到培养物中存在的其他微生物的污染。该方法可快速、可靠地制备大量样品，重现性好。它也可以用于SSU rRNA基因以外的基因。
We describe a method that allows quick and easy PCR amplification and cloning of nearly complete SSU rRNA genes from arbuscular mycorrhizal fungi. The procedure tested on spores from 37 different glomalean isolates was based on magnetic separation with Dynabeads, followed by nested PCR with two primer pairs. All trials led to visible amplification products of the expected size. Thereafter, the PCR fragments could be quickly and efficiently cloned by means of a topoisomerase-activated vector (pCR2.1-TOPO). The technique is rapid, uncomplicated and comparatively inexpensive. The use of single spores for DNA extraction has some advantages over multispore-preparations, e.g. it is less susceptible to contamination with other organisms present in the cultures. The method can be used for the quick and reliable preparation of a large number of samples and is highly reproducible. It could also be used for genes other than the SSU rRNA gene.