| Summary: | 碩士 === 淡江大學 === 生命科學研究所碩士班 === 96 === 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.
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