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    • 6. 发明授权
    • Method for generating recombinant DNA library using unidirectional single-stranded DNA fragments
    • 使用单向单链DNA片段生成重组DNA文库的方法
    • US06955879B2
    • 2005-10-18
    • US10148724
    • 2001-06-16
    • Si-Hyoung LeeYong-Chul ShinYeong-Joong JeonKyung-Hwa JungEun-Jung RyuKo-Woon Lee
    • Si-Hyoung LeeYong-Chul ShinYeong-Joong JeonKyung-Hwa JungEun-Jung RyuKo-Woon Lee
    • C12N15/09C12N15/10C12Q1/68C12P19/34C12P21/06
    • C12N15/1027
    • The present invention relates to a method for producing a recombinant polynucleotides comprising the steps of generating a pool of unidirectional single-stranded polynucleotide fragments from two or more homologous double-stranded polynucleotides, conducting a polymerization process comprising multi-cyclic extension reactions using the unidirectional single-stranded polynucleotide fragments as templates and specific oligonucleotides as primers to obtain recombinant polynucleotides, and conducting a polymerase chain reaction using at least one primer to amplify the recombinant polynucleotides; and a method for constructing a recombinant DNA library comprising the steps of inserting the recombinant polynucleotide prepared by the above method into a vector and transforming an expression cell with said vector containing the recombinant polynucleotide to obtain a plurality of mutant clones. The method of the present invention can be used for in vitro recombination of homologous polynucleotides and the directed molecular evolution.
    • 本发明涉及一种生产重组多核苷酸的方法,包括以下步骤:从两个或多个同源双链多核苷酸产生单向单链多核苷酸片段池,使用单向单链多核苷酸进行包含多循环延伸反应的聚合方法 标记的多核苷酸片段作为模板和特异性寡核苷酸作为引物以获得重组多核苷酸,并使用至少一个引物进行聚合酶链反应以扩增重组多核苷酸; 以及构建重组DNA文库的方法,包括以下步骤:将通过上述方法制备的重组多核苷酸插入载体中,并用含有重组多核苷酸的载体转化表达细胞以获得多个突变克隆。 本发明的方法可用于同源多核苷酸的体外重组和定向分子进化。