CONSTRUCTION AND CHARACTERIZATION OF A NORMALIZED CDNA LIBRARY
CONSTRUCTION AND CHARACTERIZATION OF A NORMALIZED CDNA LIBRARY
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DOI：
10.1073/pnas.91.20.9228
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发表时间：
1994-09-27
影响因子：
11.1
通讯作者：
EFSTRATIADIS, A
中科院分区：
文献类型：
--
作者：
SOARES, MB;BONALDO, MD;EFSTRATIADIS, A
我们开发了一种基于重关联动力学的简单程序，可以有效减少代表单个 mRNA 物种的 cDNA 文库克隆之间丰度的高度变化。为了进行这种标准化，我们使用人类婴儿大脑 cDNA 文库作为模型系统，该文库被定向克隆到噬菌粒载体中，因此可以很容易地转化为单链环。在受控引物延伸以在每个环状模板上合成短互补链、以相对低的 Cot 解链和重新退火部分双链体以及羟基磷灰石柱层析之后，从流经级分中回收未重联的环并电穿孔到细菌中，以繁殖标准化文库，而不需要亚克隆步骤。对标准化程度的评估表明，与原始文库中 4 个数量级的极端丰度范围相比，标准化文库中检查的任何克隆的出现频率都被控制在仅 1 个数量级的狭窄范围内。
We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low Cot, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.