Cooperative Amplification of Templates by Cross‐Hybridization (CATCH)

摘要: In vitro amplification systems not only serve as a tool for the processing of DNA, but have also provided important model investigation fundamental issues in evolutionary optimization. this work we present coupled system based on self-sustained sequence replication (3SR), known nucleic acid sequence-based (NASBA), which allows experimental evolving molecular cooperation. The 3SR reaction is an isothermal method and alternative to PCR. A target can be amplified exponentially using two enzymes: reverse transcriptase (RT) DNA-dependent RNA poly-merase (RNAP). has been constructed species cooperatively coupled. These are single-stranded (ss)DNA templates (D1 D2) lengths 58 68 nucleotides, respectively. Coupling occurs when D1 D2 anneal each other via complementary region (DB DB') situated at 3′ end template. RT elongates hybridized producing double-stranded (ds)DNA 106 base pairs (bp). This double strand contains promoters, either side of, directly adjacent DB, oriented towards other. promoters specify transcripts encompassing, respectively, portion dsDNA. After hybridization primers (P1 P2) (R1 R2) transcription, ss regenerated. Amplification cycles common dsDNA intermediate. Under optimized batch conditions shows expected growth phases: exponential, linear saturation phase. enzymes cycle tend misincorporate nucleotides produce abortive products. future experiments, intend use studies processes spatially distributed where new strategies optimization level possible.