In vitro genetic analysis of the Tetrahymena self-splicing intron.
The availability of methods for the amplification of nucleic acid sequences allows the genetic analysis in vitro of the structural and functional properties of many nucleic acids. We have now developed an in vitro selection and amplification system, and used it to analyse the self-splicing Tetrahymena ribozyme. A much wider range of selective conditions can be used in vitro than is possible with standard in vivo methods, and many more variants can be handled in vitro than in vivo. This method can be used to isolate the wild-type ribozyme, and structural variants that are as active as the wild type, from a pool of over 250,000 variants in only three cycles of selection and amplification.
Green
R
R
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Ellington
A D
AD
Szostak
J W
JW
D000818:Animals; D001426:Bacterial Proteins; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015246:Deoxyribonuclease EcoRI; D015252:Deoxyribonucleases, Type II Site-Specific; D014644:Genetic Variation; D007438:Introns; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000032:analysis*; D013768:Tetrahymena / Q000235:genetics*
D001426:Bacterial Proteins; D016337:RNA, Catalytic; D004247:DNA; D015246:Deoxyribonuclease EcoRI; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific