title: Comparison of self-sustained sequence-replication reaction systems.
abstract: The 3SR (self-sustained sequence-replication) reaction is a very efficient method for isothermal amplification of target DNA or RNA sequences in vitro. This method requires three enzymatic activities: reverse transcriptase, DNA-dependent RNA polymerase and Escherichia coli ribonuclease H. We have modified the original protocol by using human immunodeficiency virus (HIV)-1 reverse transcriptase instead of avian myeloblastosis virus (AMV) reverse transcriptase to allow amplification with T7 RNA polymerase but without E. coli ribonuclease H. Comparison of the incorporation kinetics between the conventional three-enzyme 3SR and our two-enzyme 3SR shows differences in the kinetic behaviour. Furthermore, by the new two-enzyme 3SR, the amplified RNA is obtained in a purer form compared with the experiments with three-enzyme 3SR. The aim of our research is to adapt 3SR as a useful tool for darwinian evolutionary experiments.
authors.affiliation: Institute for Molecular Biotechnology, Jena, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Gebinoga
authors.forename: F
authors.initials: F
authors.lastname: Oehlenschläger
mesh_terms: D001483:Base Sequence; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D012321:DNA-Directed RNA Polymerases; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes; D054303:HIV Reverse Transcriptase; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009690:Nucleic Acid Conformation; D011804:Quinolines; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D013844:Thiazoles; D013942:Thymine Nucleotides / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D005456:Fluorescent Dyes; D011804:Quinolines; D013844:Thiazoles; D013942:Thymine Nucleotides; C051000:thiazole orange; D012313:RNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases; D016914:Ribonuclease H; C024157:thymidine 5'-triphosphate; D014544:Uridine Triphosphate