Cooperative amplification of templates by cross-hybridization (CATCH).

Abstract

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