Quantitative microdetermination of DNA by two-dimensional electron capture gas chromatography of the thymine constituent.
Quantitative microdetermination of DNA by two-dimensional electron capture gas chromatography of the thymine constituent.
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通过胸腺嘧啶成分的二维电子捕获气相色谱法定量微量测定 DNA。
DOI：
10.1016/0003-2697(78)90772-8
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发表时间：
1978
影响因子：
2.9
通讯作者：
J. Stadler
中科院分区：
文献类型：
--
作者：
J. Stadler
本文建立了一种高灵敏度、高特异性的二维电子捕获气相色谱法，通过测定DNA中胸腺嘧啶的含量来测定DNA。该方法也可用于基于尿嘧啶含量测量RNA。水解核酸，分离释放的成分，并检测其氯甲基二甲基甲硅烷基醚。检测到的最小量为每种碱基5 pg。标准曲线在5至200 pg范围内呈线性。该方法允许在甲酸中水解生物材料后定量测定2 ng DNA（常规可检测量），即使存在大量RNA和/或蛋白质。例如，该方法已被证明在测定人工分离的核酸（例如来自两栖动物卵母细胞的核酸）的DNA含量方面是成功的。除了是准确的，该程序是快速的：最大水解（通常约45分钟）后，衍生和气相色谱分析完成在另一个15分钟。所描述的程序代表了一个直接的生化替代细胞光度估计的核内容，并具有提供每个核的绝对DNA含量值的优势。
A highly sensitive and specific two-dimensional electron-capture gas chromatographic method has been developed for determining small amounts of DNA by measuring its thymine content. The method can also be used to measure RNA based on uracil content. The nucleic acids were hydrolyzed and released constituents were separated and detected as their chloromethyldimethylsilyl ethers. The minimal amount detected was 5 pg of each base. Standard curves were linear from 5 to 200 pg. This method allowed quantitative determination of 2 ng of DNA (routinely detectable quantity) after hydrolysis of biological material in formic acid, even in the presence of large amounts of RNA and/or protein. For example, this method has been shown to be successful in determination of the DNA contents of manually isolated nucleic such as from amphibian oocytes. Besides being accurate, the procedure was rapid: after maximal hydrolysis (usually about 45 min) the derivatization and gas chromatographic analysis was completed in another 15 min. The procedure described represents a direct biochemical alternative to cytophotometric estimation of nuclear contents and has the advantage of providing values for absolute DNA content per nucleus.