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The Construction of a New Budding Yeast Plasmid for Chromosome Integration of Exogenous DNA

Author: LiuKeKe
Tutor: MaPingSheng
School: Tianjin University
Course: Biochemistry and Molecular Biology
Keywords: Saccharomyces cerevisiae Gene targeting Homologous recombination Carrier
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 66
Quote: 0
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Abstract


This study investigated a yeast chromosome site-specific integration vector construction method and its chromosomal site-specific integration feasibility . Reference the SGD database of yeast genome sequence information, to select chromosome 4 the upstream gene HEM13 - 750 - 2150bp at the DNA sequence of the gene activity as Gene targeting target point . 1400bp DNA sequence selected no coincidence of two parts , the size of 744bp and 602bp , as two homologous arm hom1 of integration vector and System HOM2 . Vector pUC18 - INT4 is the purpose of this study need to build carrier , it builds on the vector pUC18 - RYUR our laboratory has been built . vector pUC18 -INT4 is the two-stage the homology arms hom1 and System HOM2 substituted the pUC18 - RYUR on two sections , respectively , in accordance with its position and orientation on the chromosome repeat sequence . This build into pUC18 - INT4 carrier . Used in this test yeast alcohol dehydrogenase 1 ( ADH1 ) gene as the authentication verification gene the pUC18 - INT4 carrier Gene targeting . ADH1 gene sequence amplified by PCR method using restriction endonuclease sites , and it is connected in the pUC18 - INT4 vector , substituted URA3 sequence to construct pUC18 -INT4 - A vector . the pUC18-INT4 carrier Gene targeting requires two steps , the first step : the the pUC18 - INT4 carrier missing yeast strains transformed URA3 sequence , coating to save uracil medium plate , the use of the selectable marker gene URA3 and PCR screening URA3 yeast strains correct sequence integrated ; second step : the the pUC18-INT4-A to achieve proper integration of the URA3 sequence of a yeast strain transformed with the vector in the first step , using the URA3 gene encoding the enzyme-catalyzed 5 - fluoro orotic acid ( 5 - FOA ) to generate cytotoxic characteristics of PCR to select yeast strains to achieve the correct integration of the ADH1 gene . From the results , the gene integration vector pUC18 - INT4 able to use this method to build the successful realization of the gene in the yeast chromosome site-specific integration .

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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