A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees
A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees
复制标题
DOI：
10.1007/bf02914048
复制
发表时间：
2006-03-01
影响因子：
2.1
通讯作者：
Bossinger, Gerd
中科院分区：
文献类型：
--
作者：
Tibbits, Josquin F. G.;McManus, Luke J.;Bossinger, Gerd
从成熟树种中收集组织并随后分离基因组 DNA 通常很困难。许多方案都建议从针、叶或芽中提取 DNA。如果要进行无损采样，从成熟树木中收集这些组织通常需要使用枪支或攀爬。因此，样本收集是许多基于树的项目的主要支出。树木（和植物）组织通常含有大量难以与 DNA 分离的多糖和酚类化合物。许多方法旨在克服这些问题，其中大多数涉及在含有非离子去污剂十六烷基三甲基溴化铵 (CTAB) 的缓冲液中进行提取，然后通过许多步骤使用有机溶剂和差异盐沉淀来清除 DNA 中的污染物。这些步骤非常耗时，因此 DNA 的分离成为许多分子研究的瓶颈。本文提出了一种新的、高效的树种形成层收集方法，以及基于 Doyle 和 Doyle (1987) 的 DNA 提取方案，并使用 Wizard 核裂解和蛋白质沉淀溶液 (Promega) 进行后续纯化。结果显示，与其他已发表的方法相比，产量和 DNA 纯度显着提高，纯基因组 DNA 的产量始终保持高水平，样品通量也很高。每次提取的成本相对较低、不需要使用液氮、不需要冷冻储存以及收集后样品的长期稳定性是重要的额外优势。
Collection Of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcome these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethylammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.