大规模酶促合成寡核糖核苷酸的简化方法。
Simplified methods for large scale enzymatic synthesis of oligoribonucleotides.
作者信息
Shum B W, Crothers D M
出版信息
Nucleic Acids Res. 1978 Jul;5(7):2297-311. doi: 10.1093/nar/5.7.2297.
Abstract
We report simplified methods for large scale enzymatic synthesis of oligoribonucleotides using polynucleotide phosphorylase. The main features of the method are use of RPC-5 chromatography, including chromatography at two pH values to deal with the problem of primer phosphorolysis, rapid dialysis for large scale desalting, simplified methods for enzyme removal, and high resolution 1H and 31P NMR for product identification and demonstration of purity. The capacity of the method is adequate to allow beginning with grams of material in the first polymerization step, so that product yields of several milligrams, sufficient for many physical studies, are possible after as many as three separate polymerization reactions.
摘要
我们报告了使用多核苷酸磷酸化酶大规模酶促合成寡核糖核苷酸的简化方法。该方法的主要特点包括使用RPC-5色谱法，其中包括在两个pH值下进行色谱分离以解决引物磷酸解问题，采用快速透析进行大规模脱盐，简化的酶去除方法，以及使用高分辨率1H和31P NMR进行产物鉴定和纯度验证。该方法的能力足以在第一步聚合反应中从克级材料开始，因此在多达三个单独的聚合反应后，有可能获得数毫克的产物产量，这对于许多物理研究来说已经足够。
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本文引用的文献
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2
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Biochemistry. 1969 Aug;8(8):3269-78. doi: 10.1021/bi00836a021.
3
Improved separation of transfer RNA's on polychlorotrifuoroethylene-supported reversed-phase chromatography columns.在聚氯三氟乙烯支持的反相色谱柱上改进转运RNA的分离。
Biochim Biophys Acta. 1971 Feb 11;228(3):770-4. doi: 10.1016/0005-2787(71)90748-9.
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