MICRO-HOMOLOGY MEDIATED PCR TARGETING IN SACCHAROMYCES-CEREVISIAE
MICRO-HOMOLOGY MEDIATED PCR TARGETING IN SACCHAROMYCES-CEREVISIAE
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DOI：
10.1093/nar/23.14.2799
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发表时间：
1995-07-25
影响因子：
14.9
通讯作者：
SCHIESTL, RH
中科院分区：
文献类型：
--
作者：
MANIVASAKAM, P;WEBER, SC;SCHIESTL, RH
在本研究中，我们确定了DNA片段靶向整合到酵母基因组中所需的同源性的量。这里描述的程序有利于酵母基因组的操作，并消除了克隆与靶位点同源的序列的需要。此外，该方法对于其中仅可获得靶的有限序列信息的应用是有用的。该方法包括：（i）产生PCR引物以扩增含有与所选靶的侧翼同源性的选择性标记;（ii）转化酵母细胞和（iii）选择整合体。然而，靶向整合所需的最小同源性长度和作为同源性长度的函数的靶向的确切效率迄今尚未确定。
In the present study, we determined the amount of homology required for targeted integration of DNA fragments into the yeast genome. The procedure described here facilitates the manipulation of the yeast genome and eliminates the need to clone sequences homologous to a target site. In addition, this method is useful for applications in which only limited sequence information of the target is available. The procedure comprises of:(i) production of PCR primers to amplify a selectable marker containing flanking homology to the target of choice;(ii) transformation of yeast cells and (iii) selection of integrants.Since the first development of this technique (1) different groups reported versions of this technique (2-4). However, the minimum length of homology required for targeted integration and the exact efficiencies of targeting as a function of homology length has not to date been determined.