Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710, USA.
J Virol Methods. 2010 Aug;167(2):146-51. doi: 10.1016/j.jviromet.2010.03.025. Epub 2010 Mar 31.
Characterization of multiple sites in a single gene that are important in biological phenotypes is challenging due to the difficulty to generate many mutants representing all or a majority of combinations of mutations in the gene. Using the HIV-1 env and pol genes as templates, four random libraries were generated representing different combinations of mutations introduced by up to 36 mutagenesis primers in a single assay. Over 86% of the clones contained mutations and the mutants tended to have single or fewer mutations in the libraries. When protein size was used as a screening marker, all identified clones contained at least 2 mutations and up to 12 mutations were detected in a single clone. Nearly all mutant clones in each library contained unique mutations, indicating that mutants in the library were generated at random. Closely related mutations which were overlapped by neighboring mutagenesis primers were often introduced in this system. Analysis of the env library showed that some potential N-linked glycosylation sites did not increase the Env molecular mass significantly, suggesting they were not used for glycosylation or only limited carbohydrate moieties were added at these sites. This novel method can serve as a powerful tool to study the biological phenotypes of genes whose functions are determined by multiple sites.
由于难以生成代表基因中所有或大多数突变组合的许多突变体，因此对单个基因中多个对生物表型重要的位点进行特征描述具有挑战性。使用 HIV-1 env 和 pol 基因作为模板，在单个测定中使用多达 36 个诱变引物，生成了四个随机文库，代表了不同的突变组合。超过 86%的克隆包含突变，并且突变体在文库中倾向于具有单个或更少的突变。当蛋白大小用作筛选标记时，所有鉴定的克隆均包含至少 2 个突变，并且在单个克隆中检测到多达 12 个突变。每个文库中的几乎所有突变体克隆都包含独特的突变，表明文库中的突变体是随机生成的。在该系统中，经常引入由相邻诱变引物重叠的密切相关的突变。对 env 文库的分析表明，一些潜在的 N 连接糖基化位点不会使 Env 分子质量显著增加，这表明它们未被用于糖基化，或者仅在这些位点添加有限的碳水化合物部分。这种新方法可以作为研究由多个位点决定功能的基因的生物表型的有力工具。
