Rapid Construction of Three-Fragment Recombinant DNAs by Polymerase Chain Reaction: Application for Gene-Targeting inSaccharomyces cerevisiae
✍ Scribed by Mingfu Ling; Brian H. Robinson
- Publisher
- Elsevier Science
- Year
- 1996
- Tongue
- English
- Weight
- 137 KB
- Volume
- 242
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
son with previous TLC systems for the isolation of the phosphoinositides. (1) It is the first protocol for the TLC separation of all known isomers of phospho-Conventionally, the preparation of a three-fragment inositides. ( 2) It provides excellent separation of the construct to be used for gene targeting (gene knockout) phosphoinositides without tending to streak from involves at least two consecutive cloning experiments. the origin, as often occurs in chloroform-containing Each cloning experiment involves first ligating two solvent systems. (3) It avoids the use of chloroform, DNA fragments together and then transforming the which has been suspected of being carcinogenic. (4) ligation into competent bacteria, followed by prepara-There are neither visible secondary solvent fronts tion of plasmid DNAs to screen recombinants. Finally, nor migration of boric acid, which can result in circular plasmid DNAs must be digested with restricpoorly resolved lipids. (5) The ''activation'' of the tion enzymes to release the linear DNA construct to plates and the use of chelating agents is not necesbe used in the gene-targeting experiment. The whole sary.
process of cloning for constructing such three-fragment DNA recombinants usually takes a minimum of 10 to
Acknowledgments. I thank Dr. Kazimierz Jaroszewicz, Medical 14 working days. In reality, because of difficulties (1-School, Bialystok, Poland, for introducing me into TLC methodology.
4) in many steps of cloning, this process may take much
This study was supported by the Deutsche Forschungsgemeinschaft, longer.
through Grant SFB 197/A4.
This DNA construct has been successfully used for disrupting the pyruvate dehydrogenase (pdh) E 1 b subunit 7.