Simplified in vitro synthesis of mutated RNA molecules

FEBS Letters

Krupp, Guido¹  Söll, Dieter¹ 

[1] Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 6666, New Haven, CT 06511, USA

关键词: T7 RNA polymerase;    Transcription;    Oligonucleotide promoter;    Klenow enzyme;    Klenow fragment of DNA polymerase I;    ddA;    2';    3'-dideoxyadenosine;    ss;    single-stranded;    ds;    double-stranded;   

DOI  :  10.1016/0014-5793(87)81359-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We describe a simplified method for the in vitro synthesis of mutated RNA molecules. The method makes use of an oligodeoxyribonucleotide (T₇-oligo) which contains the T₇ RNA polymerase promoter sequence. In combination with a second oligonucleotide, a series of transcripts initiating and terminating at any chosen position on a cloned ss DNA (e.g. M 13 phage DNA) can be generated. The phage DNA represents the non-coding DNA strand for the desired transcript; the T₇-oligo determines the transcription start site, whereas the second oligonucleotide permits the choice of the transcription termination site. The synthesis of the required template DNA is achieved by hybridizing the two oligonucleotides to the phage DNA and subsequently synthesizing the coding DNA strand by a fill-in reaction with Klenow enzyme. The reaction product is used directly as a template for T₇ RNA polymerase; cloning of mutants is not required.

【 授权许可】

Unknown