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26 条记录 (耗时 0.016 秒)

    Method for engineering strand-specific, sequence-specific, DNA-nicking enzymes
    1.
    发明授权
    Method for engineering strand-specific, sequence-specific, DNA-nicking enzymes 有权

    公开(公告)号：US07081358B2

    公开(公告)日：2006-07-25

    申请号：US10223074

    申请日：2002-08-16

    申请人： Daniel Heiter ,  Keith Lunnen ,  Geoffrey G. Wilson

    发明人： Daniel Heiter ,  Keith Lunnen ,  Geoffrey G. Wilson

    IPC分类号： C12N15/55 ,  C12N9/22

    CPC分类号： C12N9/22

    摘要： Methods are provided for converting into a sequence specific strand specific and location specific DNA nicking endonuclease, a restriction endonuclease that recognizes an asymmetric DNA sequence, the endonuclease having two catalytic sites and one or more single sequence specific DNA-binding domains. In one embodiment the method requires inactivating one of the catalytic sites of the restriction endonuclease. In another embodiment, the restriction endonuclease is a dimer having a first and second subunit each comprising a sequence specific DNA binding domain, a catalytic site and a dimerization domain. The nicking endonuclease is formed from combining one subunit having an inactivated catalytic site and a second subunit having an inactivated DNA binding domain. The nicking endonuclease may be converted into a restriction endonuclease by the addition of manganese cations in the digestion buffer.

    Cloning restriction and modification genes
    3.
    发明授权
    Cloning restriction and modification genes 失效
    克隆限制和修饰基因

    公开(公告)号：US5200333A

    公开(公告)日：1993-04-06

    申请号：US672158

    申请日：1991-03-19

    申请人： Geoffrey G. Wilson

    发明人： Geoffrey G. Wilson

    IPC分类号： C12N9/10 ,  C12N9/22 ,  C12N15/55

    CPC分类号： C12N9/1007 ,  C12N9/22

    摘要： Methods for cloning restriction enzymes and their corresponding modification enzymes by selecting clones resistant to digestion by the restriction enzyme and subsequent selection of clones containing the restriction gene.

    摘要翻译： 通过选择能够通过限制酶消化的克隆并随后选择含有限制性基因的克隆来克隆限制酶及其相应的修饰酶的方法。

    Cloning the Hae II restriction and modification genes
    4.
    发明授权
    Cloning the Hae II restriction and modification genes 失效
    克隆Hae II限制和修饰基因

    公开(公告)号：US5196332A

    公开(公告)日：1993-03-23

    申请号：US699146

    申请日：1991-05-13

    申请人： Geoffrey G. Wilson

    发明人： Geoffrey G. Wilson

    IPC分类号： C12N9/10 ,  C12N9/22 ,  C12N15/55

    CPC分类号： C12N9/22 ,  C12N9/1007

    摘要： Methods for cloning restriction enzymes and their corresponding modification enzymes by selecting clones resistant to in vitro digestion by the restriction enzyme and subsequent screening to identify those clones containing the restriction gene.

    摘要翻译： 通过选择通过限制酶对体外消化有抗性的克隆并随后进行筛选以鉴定含有限制性基因的克隆来克隆限制酶及其相应修饰酶的方法。

    Method for cloning and producing the RsaI restriction endonuclease in E. coli and purification of the recombinant RsaI restriction endonuclease
    7.
    发明授权
    Method for cloning and producing the RsaI restriction endonuclease in E. coli and purification of the recombinant RsaI restriction endonuclease 有权
    在大肠杆菌中克隆和产生RsaI限制性内切核酸酶的方法，并重组RsaI限制性内切核酸酶的纯化

    公开(公告)号：US06210945B1

    公开(公告)日：2001-04-03

    申请号：US09587066

    申请日：2000-06-02

    申请人： Keith D. Lunnen ,  Richard D. Morgan ,  Timothy Meixsell ,  Geoffrey G. Wilson

    发明人： Keith D. Lunnen ,  Richard D. Morgan ,  Timothy Meixsell ,  Geoffrey G. Wilson

    IPC分类号： C12N922

    CPC分类号： C12N9/22

    摘要： RsaI, a restriction enzyme from the bacterium Rhodopseudomonas sphaeroides, recognizes the DNA sequence 5′-GTAC-3′. Because RsaI is commercially valuable, we sought to overproduce it by cloning the genes for RsaI and its accompanying, modification, enzyme. The ‘methylase-selection’ method, the customary procedure for cloning restriction and modification genes, was applied to RsaI. The method yielded clones containing the methylase gene (rsaIM), but none containing both the methylase gene and the restriction gene (rsaIR). Inverse-PCR was then used to recover sections of the DNA downstream of rsaIM. These sections were sequenced, and the sequences were joined in silico to reveal the gene organization of the RsaI R-M system. By comparing the coding potential of the DNA with the N-terminal amino acid sequence of the purified RsaI restriction enzyme, we discovered that the RsaI R and M genes, rather than being adjacent-the situation that pertains in most R-M systems-are separated by an intervening gene of unknown function. Based on this information, the rsaIR gene was cloned by PCR instead of methylase-selection. These new clones proved to be highly unstable, however, even in the presence of the rsaIM gene. Various attempts were made to stabilize the gene, but most met with failure. Stability was finally achieved by introducing a second methylase gene, mjaVM, to augment the protection provided by rsaIM, and by tightly controlling the expression of rsaIR using a special two-promoter, anti-sense transcription, expression vector.

    摘要翻译： 来自细菌Rhodopseudomonas sphaeroides的限制酶RsaI识别DNA序列5'-GTAC-3'。 由于RsaI具有商业价值，我们试图通过克隆RsaI及其伴随的修饰酶的基因来过度生产。 将“甲基化酶选择”方法，克隆限制和修饰基因的常规方法应用于RsaI，该方法产生含有甲基化酶基因（rsaIM）的克隆，但不含有甲基化酶基因和限制性基因（rsaIR）。 然后使用反向PCR来回收rsaIM下游DNA的切片，对这些切片进行测序，并将序列与硅胶连接以揭示RsaI RM系统的基因组织，通过将DNA的编码潜力与N 纯化的RsaI限制酶的末端氨基酸序列，我们发现RsaI R和M基因而不是与大多数RM系统相关的情况相邻 - 由未知功能的介入基因分离，基于该信息 ，通过PCR克隆rsaIR基因而不是甲基化酶选择，这些新克隆被证明是高度不稳定的，然而，即使在rsaIM基因的存在下，也进行了各种尝试 lize基因，但大多数遇到失败。 通过引入第二个甲基化酶基因mjaVM来增加rsaIM提供的保护，并通过使用特异的双启动子，反义转录，表达载体来严格控制rsaIR的表达，最终达到稳定性。