Plant DNA flow cytometry and estimation of nuclear genome size
Plant DNA flow cytometry and estimation of nuclear genome size
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DOI：
10.1093/aob/mci005
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发表时间：
2005-01-01
期刊：
影响因子：
4.2
通讯作者：
Bartos, J
中科院分区：
文献类型：
--
作者：
Dolezel, J;Bartos, J
背景DNA流式细胞术描述了使用流式细胞术来估计细胞核中的DANA量。该方法涉及制备完整细胞核的水悬浮液，其DNA使用DNA荧光染料染色。细胞核根据其相对荧光强度或DNA含量进行分类。由于DNA流式细胞术具有样品制备和分析方便、快速等优点，已成为染色体倍性筛选、混倍体和非整倍体检测、细胞周期分析、多体性程度评估等的热门方法。生殖途径的确定和绝对DNA量或基因组大小的估计。虽然前者的应用程序是相对简单的，绝对DNA量的估计需要特别注意的样品制备和analysis.Scope中可能出现的错误-文章回顾了目前的程序，估计绝对DNA量的植物使用流式细胞术，特别强调制备的细胞核悬浮液，化学计量的DNA染色和使用的DNA参考标准。此外，在估计种内基因组大小的变化遇到的方法陷阱进行了讨论，以及与使用DNA流式细胞术fieldwork.Conclusions可靠的估计绝对DNA量在植物中使用流式细胞术的问题是不是一个微不足道的任务。虽然有几个经过充分验证的协议，并已确定了一些因素控制的精度和再现性。若干问题仍然存在：（1）对新鲜组织的需要使得将样品从现场转移到实验室和/或其储存变得复杂;（2）干扰定量DNA染色的胞质化合物的作用还没有被很好地理解;以及（3）使用一组4种国际公认的DNA参考标准仍然是一个未实现的目标。(C)2005年，公司成立。
Background DNA flow cytometry describes the use of flow cytometry for estimation of DANA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry, has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty. determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount require special attention to possible errors in sample preparation and analysis.Scope The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork.Conclusions Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified. several problems persist: (1) the need for fresh tissues complicates the transfer of samples home field to the laboratory and/or their storage: (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood: and (3) the use of a set 4 internationally agreed DNA-reference standards still remains an unrealized goal. (C) 2005 Annals of Botany Company.