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    • 6. 发明授权
    • Method for detecting DNA polymorphism applying triple strand DNA formation technique
    • 检测应用三链DNA形成技术的DNA多态性的方法
    • US06849410B2
    • 2005-02-01
    • US09989526
    • 2001-11-21
    • Yasushi ShigemoriMichio OishiOsamu Ohara
    • Yasushi ShigemoriMichio OishiOsamu Ohara
    • C12N15/09C07H21/02C07H21/04C12P19/34C12Q1/68
    • C12Q1/6827C12Q1/6839C12Q2537/119C12Q2527/107C12Q2521/507
    • An objective of this invention is to provide a method for detecting DNA polymorphism that has high sensitivity and efficiency and does not need long DNA searching region.A homologous recombination protein RecA makes partial triple strand DNA from target double DNA and oligonucleotide probe complementary to the DNA. The triple strand DNA maintains stable triple strand DNA after RecA protein is removed. The present inventors found that the thermostability of triple strand DNA changes greatly when there is a mismatch between target DNA and oligonucleotide probe because of the existence of polymorphism in the target DNA. Utilizing this change of thermostability, efficient detection of polymorphism in labeled DNA is possible by examining whether oligonucleotide probe is released and the triple strand DNA is solved after heat treatment of triple strand DNA formed using homologous recombination protein.
    • 本发明的目的是提供一种检测DNA多态性的方法,其具有高灵敏度和高效性并且不需要长的DNA搜索区。同源重组蛋白RecA使得来自靶双重DNA的部分三链DNA和与DNA互补的寡核苷酸探针 。 在除去RecA蛋白后,三链DNA保持稳定的三链DNA。 本发明人发现,由于目标DNA中存在多态性,当靶DNA和寡核苷酸探针之间存在错配时,三链DNA的热稳定性变化很大。 利用这种热稳定性的变化,可以通过检查寡核苷酸探针是否被释放并且在使用同源重组蛋白形成的三链DNA热处理后求解三链DNA来有效地检测标记的DNA中的多态性。
    • 8. 发明授权
    • Partial homologous recombination of DNA chain
    • DNA链的部分同源重组
    • US07220548B2
    • 2007-05-22
    • US10798750
    • 2004-03-10
    • Kazuhiro KondoMichio OishiOsamu Ohara
    • Kazuhiro KondoMichio OishiOsamu Ohara
    • C12Q1/68
    • C12N15/1082C12N15/102C12Q1/6858C12Q2521/507C12Q2537/119C12Q2521/319
    • The present invention provides a method of constructing a circular DNA library having an increased content of a desired first dsDNA by removing a second dsDNA using RecA protein to introduce a target single strand nucleic acid by homologous recombination at the 3′ terminal portion of the second dsDNA, whereby the target DNA has a 3′ terminal portion that differs from the 3′ terminal portion of the second dsDNA to prevent circularization, thereby creating a triple stranded DNA portion at the 3′ terminal end of the second dsDNA, adding Exonuclease I to digest the displaced first strand of the second dsDNA, ligating the DNA fragments to circularize the desired first dsDNA, removing the linear second dsDNA, thereby constructing the circularized DNA library having an increased content of the desired first dsDNA.
    • 本发明提供了一种通过使用RecA蛋白去除第二个双链DNA来构建所需第一个dsDNA含量增加的环状DNA文库的方法,以通过在第二个双链DNA的3'端部分同源重组引入靶单链核酸 由此目标DNA具有与第二dsDNA的3'末端部分不同的3'末端部分以防止环化,从而在第二个dsDNA的3'末端产生三链DNA部分,加入外切核酸酶I进行消化 连接DNA片段以使所需的第一个dsDNA环化,除去线性第二个dsDNA,从而构建具有增加的所需第一个dsDNA含量的环化的DNA文库。