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    Title: 合成致死對啤酒酵母菌JHD1功能相關基因之篩選與分析
    Other Titles: Synthetic lethal analysis of JHD1 from saccharomyces cerevisiae
    Authors: 林俊良;Lin, Chun-liang
    Contributors: 淡江大學生命科學研究所碩士班
    陳銘凱;Chern, Ming-kai
    Keywords: 合成致死;突變;Synthetic Lethal;JHD1;Mutagenesis
    Date: 2008
    Issue Date: 2010-01-11 02:26:54 (UTC+8)
    Abstract: 合成致死篩選是藉由兩個基因間交互作用的一種很有效的基因篩選法。原則上,經此種方式可篩選出合成致死性基因。合成致死即在篩選需仰賴興趣基因方可存活的菌株,當中使用菌落顏色分析的技術以視覺確認符合需求的突變株。

    合成致死篩選必須遵照以下四個步驟 :
    一.感興趣的基因功能需要在實驗菌株中被阻斷,此實驗菌株的ade2 ade3/ade8需無法作用,菌株最終呈現白色。
    二.將含有野生型的感興趣基因與ADE3/ADE8的重組質體,導入步驟一的實驗菌株中。導入後的菌株會有紅白相間的扇形菌落產生,而白色菌落表示所導入的重組質體已遺失。
    三.將步驟二的菌株執行隨機性突變,所要的突變株需仰賴重組質體上才得以存活,並呈現純紅色的菌落。
    四.篩選出的合成致死菌株將會導入基因庫質體,如果突變株上的基因庫質體含有與原本的重組質體互補的DNA,則細胞將不需要在長期的依賴重組質體上的感興趣基因,就會再次呈現出紅白的扇形菌落。

    最後,可從篩選出的互補性質體定序出其上所帶的合成致死基因。
    The synthetic lethal screen is a powerful genetic screen that relies on finding the secondary molecular targets. In principle, this screen can identify any mutant gene which causes cell death with a nonlethal “primary” mutation. The synthetic lethal screen is a method of isolating novel mutants whose survival is dependent on the gene of interest. Combining the colony-color assay, this method will offer a means to visually detect a mutant that depends on a plasmid for survival.

    The synthetic lethal screen must be carry out in the following four processes :
    (i) The gene function of interest should be disrupted in a strain containing ade2 ade3/ade8 mutations, which result in a white phenotype.
    (ii) Combining the wild-type gene of interest and ADE3/ADE8 in a plasmid which is then transformed into the strain of step (i). The cells will produce red-white sector, and the white colonies have lost the plasmid.
    (iii) A mutagenesis will be performed to introduce random mutations into the strain genome. The mutant must dependents on the gene of interest from plasmid of step (ii) for survival and produce red solid colonies.
    (iv) The synthetic lethal(SL) colonies are transformed with a library. Then the mutants containing complementing DNA are no longer dependent on the gene of interest, and the cells with red-white sectoring are identified.

    Finally,the synthetic lethal gene can be sequencd from the complement plasmid.
    Appears in Collections:[生命科學研究所] 學位論文

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