Fred E. Gouker, Yonghong Guo, Harlan T. Svoboda, Margaret R. Pooler
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引用次数: 2
摘要
本研究的目的是优化现有的顽固植物类群DNA提取方案，以从保存的植物标本组织中获得适合下游PCR应用的高质量DNA。方法与结果从美国国家植物标本室获得30种不同植物的叶片组织。我们之前的DNA提取方案(Gouker et al.， 2020, Applications in Plant Sciences 8: e11403)通过使用10X Tris-EDTA缓冲液，添加聚乙烯聚吡咯烷酮，以及在均质化过程中省略样品加热来改进，并导致总DNA产量在60-2460 ng之间。使用通用植物引物优化的PCR扩增对trnL (UAA)叶绿体内含子的ts -p3/u4区和P6环在大多数标本中都是成功的。该方法简单、快速，使用标准的实验室级化学品，从植物标本馆标本中获得的DNA质量与市售试剂盒相当，并且具有足够的质量和数量用于其他应用。
Optimizing efficient PCR-amplifiable DNA extraction from herbarium specimens
Premise
The objective of this study was to optimize an existing DNA extraction protocol for recalcitrant plant taxa to obtain high-quality DNA from preserved herbarium tissue suitable for downstream PCR applications.
Methods and Results
Leaf tissue from 30 diverse plant species was obtained from the U.S. National Arboretum Herbarium. Our previous DNA extraction protocol (Gouker et al., 2020, Applications in Plant Sciences 8: e11403) was improved by use of 10X Tris-EDTA buffer, addition of polyvinylpolypyrrolidone, and omission of sample heating during homogenization, and resulted in total DNA yields ranging from 60–2460 ng. Optimized PCR amplification using universal plant primers for the ITS-p3/u4 region and the P6 loop of the trnL (UAA) chloroplast intron was successful for most specimens.
Conclusions
This protocol, which is simple, fast, and uses standard laboratory-grade chemicals, yields DNA from herbarium specimens that is comparable in quality to that from commercially available kits, and is of sufficient quality and quantity for other applications.
期刊介绍：
Applications in Plant Sciences (APPS) is a monthly, peer-reviewed, open access journal promoting the rapid dissemination of newly developed, innovative tools and protocols in all areas of the plant sciences, including genetics, structure, function, development, evolution, systematics, and ecology. Given the rapid progress today in technology and its application in the plant sciences, the goal of APPS is to foster communication within the plant science community to advance scientific research. APPS is a publication of the Botanical Society of America, originating in 2009 as the American Journal of Botany''s online-only section, AJB Primer Notes & Protocols in the Plant Sciences.
APPS publishes the following types of articles: (1) Protocol Notes describe new methods and technological advancements; (2) Genomic Resources Articles characterize the development and demonstrate the usefulness of newly developed genomic resources, including transcriptomes; (3) Software Notes detail new software applications; (4) Application Articles illustrate the application of a new protocol, method, or software application within the context of a larger study; (5) Review Articles evaluate available techniques, methods, or protocols; (6) Primer Notes report novel genetic markers with evidence of wide applicability.
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