Self-sustained sequence replication

Fahey, E., Kwoh, D. Y. and Gingeras, T. R. Self-sustained sequence replication (3SR) an isothermal transcription-based amplification system alternative to PCR , PCR Methods Appl., 1, 25-33 (1991). 

Figure 6. Amplification of RNA molecules by assays that are sequence- insensitive. The first assay (upper part) combines the polymerase chain reaction (PCR) of DNA templates with reverse transcription and transcription. Commonly used enzymes are TAQ-polym-erase, HIV reverse transcriptase and bacteriophage T7 RNA polymerase. The assay requires a temperature program applying higher temperatures for double strand dissociation. The second assay (lower part) shows the self-sustained sequence replication reaction (3SR) which can be carried out isothermally because double strand dissociation is replaced by enzymatic digestion of the RNA strand in the RNA-DNA duplex. The enzymes used are HIV reverse transcriptase, RNase H and T7 RNA polymerase.

The second assay makes use of the isothermal self-sustained sequence replication reaction of RNA (3SR Fahy et al., 1991). Instead of double strand melting to yield single strands the RNA DNA hybrid obtained through reverse transcription is converted into single stranded DNA by RNA digestion making use of RNase H. DNA double strand synthesis and transcription complete the cycle. Here, transcription by T7 polymerase represents the amplification step. 

Transcription-based amplification methods are modeled after the replication of retroviruses. These methods are known by various names including nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA)/ and self-sustained sequence repfi-cation (3SR) assays. Isothermal target amplification, using the collective activities of reverse transcriptase, RNase H, and RNA polymerase, is common to these methods. As illustrated in Figure 37-6, the method may be applied to... 

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