A new randomization assay reveals unexpected elements of sequence bias in model 'randomized' gene libraries: implications for biopanning
A new randomization assay reveals unexpected elements of sequence bias in model 'randomized' gene libraries: implications for biopanning
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DOI：
10.1016/s0378-1119(00)00206-7
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发表时间：
2000-06-13
期刊：
影响因子：
3.5
通讯作者：
Hine, AV
中科院分区：
文献类型：
--
作者：
Palfrey, D;Picardo, M;Hine, AV
尽管生物扫描等技术在很大程度上依赖于随机基因文库的筛选，但关于这些文库的构建，可用的信息令人惊讶地很少。一般说来，它是基于‘随机化’的合成寡核苷酸的克隆，其中给定的位置(S)包含所有四个碱基的相等混合。然而，许多被认为是“随机”的文库包含了显著的偏见和/或遗漏因素。在这里，我们报告了一种新的、基于PCR的分析方法的开发和验证，该方法能够在克隆之前和之后快速检查文库组成。通过使用我们的分析方法来分析模型文库，我们证明了对给定分布的序列的克隆并不一定会导致类似组成的克隆库。因此，虽然随机合成寡核苷酸混合物中的偏差可以通过使用四种不同比例的亚磷酰胺几乎消除，但使用这种混合物并不能确保检索到真正的随机化文库。我们认为，在没有控制克隆频率的技术的情况下，克隆后分析文库组成的能力将显著提高从这些文库获得的信息的质量。(C)2000由爱思唯尔科学公司出版。保留所有权利。
Although techniques such as biopanning rely heavily upon the screening of randomized gene libraries, there is surprisingly little information available on the construction of those libraries. In general, it is based on the cloning of 'randomized' synthetic oligonucleotides, in which given position(s) contain an equal mixture of all four bases. Yet, many supposedly 'randomized' libraries contain significant elements of bias and/or omission. Here, we report the development and validation of a new, PCR-based assay that enables rapid examination of library composition both prior to and after cloning. By using our assay to analyse model libraries, we demonstrate that the cloning of a given distribution of sequences does not necessarily result in a similarly composed library of clones. Thus, while bias in randomized synthetic oligonucleotide mixtures can be virtually eliminated by using unequal ratios of the four phosphoramidites, the use of such mixtures does not ensure retrieval of a truly randomized library. We propose that in the absence of a technique to control cloning frequencies, the ability to analyse the composition of libraries after cloning will enhance significantly the quality of information derived from those libraries. (C) 2000 Published by Elsevier Science B.V. All rights reserved.