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Simplified in vitro synthesis of mutated RNA molecules
Author(s) -
Krupp Guido,
Söll Dieter
Publication year - 1987
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(87)81359-5
Subject(s) - klenow fragment , oligonucleotide , microbiology and biotechnology , coding strand , t7 rna polymerase , dna polymerase i , biology , in vitro recombination , dna , dna polymerase , dna clamp , polymerase , transcription (linguistics) , dna polymerase ii , rna , chemistry , bacteriophage , molecular cloning , reverse transcriptase , genetics , complementary dna , gene , exonuclease , escherichia coli , linguistics , philosophy
We describe a simplified method for the in vitro synthesis of mutated RNA molecules. The method makes use of an oligodeoxyribonucleotide (T 7 ‐oligo) which contains the T 7 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 T 7 ‐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 T 7 RNA polymerase; cloning of mutants is not required.