Gaytán P, Yañez J, Sánchez F, Mackie H, Soberón X
Department of Molecular Recognition and Biostructure, Instituto de Biotecnología/UNAM, Cuernavaca, Mor. México.
Chem Biol. 1998 Sep;5(9):519-27. doi: 10.1016/s1074-5521(98)90007-2.
Synthetic DNA has been used to introduce variability into protein-coding regions. In protocols that produce a few mutations per gene, the sampling of amino-acid sequence space is limited by the bias imposed by the genetic code. It has long been apparent that the incorporation of trinucleotides in the synthetic regime would circumvent this problem and significantly enhance the usefulness of the technique.
A new method is described for the creation of codon-level degenerate oligodeoxyribonucleotides that combines conventional dimethoxytrityl (DMT) mononucleoside phosphoramidite chemistry with 9-fluorenylmethoxycarbonyl (Fmoc) trinucleotide phosphoramidites (whose synthesis is reported in the paper). The substoichiometric use of these Fmoc-trinucleotides in an automatable, solid-phase synthesis procedure afforded DNA fragments comprising the wild-type sequence and a controllable distribution of mutants within two- and three-codon stretches of DNA, within the multiple cloning site of the conventional cloning vector pUC19.
DMT and Fmoc are compatible protecting groups in conventional oligonucleotide synthesis methods, resulting in controllable levels of codon-based mutagenesis.
合成DNA已被用于在蛋白质编码区域引入变异性。在每个基因产生少量突变的方案中，氨基酸序列空间的采样受到遗传密码所施加偏差的限制。长期以来，很明显在合成体系中掺入三核苷酸将规避这个问题并显著提高该技术的实用性。
描述了一种创建密码子水平简并寡脱氧核糖核苷酸的新方法，该方法将传统的二甲氧基三苯甲基（DMT）单核苷亚磷酰胺化学与9-芴甲氧羰基（Fmoc）三核苷酸亚磷酰胺（其合成在本文中报道）相结合。在可自动化的固相合成过程中化学计量不足地使用这些Fmoc-三核苷酸，得到了包含野生型序列以及在常规克隆载体pUC19的多克隆位点内DNA的两个和三个密码子片段中具有可控分布的突变体的DNA片段。
在传统的寡核苷酸合成方法中，DMT和Fmoc是兼容的保护基团，可实现基于密码子的可控诱变水平。
