Cyclic enzymatic solid phase synthesis of isotopically labeled DNA oligonucleotides.
Cyclic enzymatic solid phase synthesis of isotopically labeled DNA oligonucleotides.
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同位素标记 DNA 寡核苷酸的循环酶促固相合成。
DOI：
10.1080/15257770903362263
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Germann,MarkusW
中科院分区：
文献类型：
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作者：
Khan,AhmedM;Mishra,SubrataH;Germann,MarkusW
使用标准固相合成法对 DNA 进行同位素标记需要大量过量使用的昂贵的亚磷酰胺。我们开发了一种方案，其中 DNA 的酶促、循环、固相合成有助于更经济地使用较便宜的标记 DNA 三磷酸 (dNTP)。在这种方法中，DNA 模板被固定在环氧活化的固体支持物上。 DNA 和树脂之间的支持物和连接对于高 pH 条件都是惰性的，这是该方案中产品释放所需的。当反应在 MgCl2/CAPS 中进行时，DNA 与树脂的有效共价连接得以实现。酶促填充反应以及产物释放和回收条件均经过优化，可有效重复使用 dNTP，无需任何纯化。开发的方案用于生成选择性[13C,15N]G 标记的10 聚体双链体。
Isotopic labeling of DNA using standard solid phase synthesis requires expensive phosphoramidites that are used in large excess. We have developed a protocol where enzymatic, cyclic, solid phase synthesis of DNA facilitates a more economical use of the less expensive labeled DNA triphosphates (dNTP). In this approach, the DNA template is immobilized on an epoxy-activated solid support. Both the support and the linkage between DNA and resin are inert to high pH conditions which are required for product release in this scheme. Efficient covalent attachment of the DNA to the resin was achieved when the reaction was carried out in MgCl2/CAPS. The enzymatic fill in reaction as well as product release and recycling conditions were optimized for efficient reuse of dNTPs without any purification. The developed protocol was used to generate a selectively [13C,15N] G labeled 10-mer duplex.