Ohuchi S, Nakano H, Yamane T
Laboratory of Molecular Biotechnology, Department of Biological Mechanisms and Functions, Graduate School of Biological and Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Nucleic Acids Res. 1998 Oct 1;26(19):4339-46. doi: 10.1093/nar/26.19.4339.
A novel in vitro method for the generation of a protein library has been developed using the polymerase chain reaction (PCR) amplification of a single DNA molecule followed by in vitro coupled transcription/translation. DNA template encoding green fluorescent protein of a jellyfish Aequorea victoria was extensively diluted to one molecule per well, and then amplified by a total of 80 cycles of PCR with nested primers. The exact number of origins in the amplified DNA fragment was then estimated by directly sequencing a part of the fragment, at which an individual template molecule was marked by PCR with a primer containing three randomized bases. Since the sequences obtained in 91 independent amplifications were diversified statistically, each amplified fragment was likely originated from a single DNA molecule. In addition, the amplified fragments served as a template for in vitro coupled transcription/translation using T7 RNA polymerase and Escherichia coli S30 extract. These results suggest that the library obtained by the PCR amplification of a single DNA molecule diluted from a variety of DNA pools is potentially useful in high-throughput generation of protein libraries.
一种用于生成蛋白质文库的新型体外方法已经开发出来，该方法利用聚合酶链反应（PCR）对单个DNA分子进行扩增，随后进行体外偶联转录/翻译。将编码维多利亚多管水母绿色荧光蛋白的DNA模板大量稀释至每孔一个分子，然后用巢式引物进行总共80个循环的PCR扩增。然后通过直接对片段的一部分进行测序来估计扩增DNA片段中的起始位点的确切数量，在这一过程中，通过使用含有三个随机碱基的引物进行PCR来标记单个模板分子。由于在91次独立扩增中获得的序列在统计学上是多样化的，每个扩增片段可能都源自单个DNA分子。此外，扩增片段用作使用T7 RNA聚合酶和大肠杆菌S30提取物进行体外偶联转录/翻译的模板。这些结果表明，通过对从各种DNA库中稀释得到的单个DNA分子进行PCR扩增所获得的文库，在高通量生成蛋白质文库方面可能是有用的。
