Self-sustained sequence replication (3SR): an isothermal transcription-based amplification system alternative to PCR.

T h e processes of DNA replication, DNA ligation, and RNA transcription have provided the basis of distinct and separate in vitro nucleic acid amplification strategies. The first described and the most widely utilized amplification protocol is the polymerase chain reaction (PCR).O,2) The PCR method consists of multiple cycles of oligonucleotide primer-directed DNA replication that are punctuated by short periods of elevated temperature (92-97~ to permit strand separation of newly synthesized DNA. The modifications, adaptations, and enhancements of the basic PCR protocol now number in the hundreds (reviewed partially in refs. 3 and 4) and have arisen primarily to meet the challenges of the numerous biological questions to which this valuable technology has been applied. More recently, the description of an amplification method based on cycles of oligonucleotide-targeted ligation, called the ligase chain reaction (LCR) (see review by Barany in this issue), has offered users the ability to amplify short (oligonucleotide-length) segments of DNA and simultaneously to monitor these amplified segments for the presence of mutations. (s-7) Similar to the PCR protocol, the LCR method includes thermocycling steps to permit the denaturing of newly ligated oligonucleotide duplexes so that these products can serve as templates for subsequent cycles of amplification. The requirement for multiple heatdenaturation steps by both the PCR and LCR amplification methods has prompted the rapid development of programmable thermocycling instruments and has hastened the isolation and purification of thermostable forms of DNA polymerase (8,9) and ligase. (7,10,11) These thermostable enzymes allow the amplification reactions to be carried out over many cycles of heating and cooling without debilitating loss of catalytic activities. The necessity of employing thermocycling as an essential part of an in vitro amplification strategy has been obviated by changing the product of the amplification process from double-stranded DNA to single-stranded RNA, thereby eliminating the heat denaturation step from the procedure. RNA transcription is a process employed by all cellular and viral systems to copy discrete segments of nucleotide sequences into multiple single-stranded RNA molecules. In exploring modifications of the previously described in vitro transcription-based amplification system (TAS), (12,13) it was discovered that isothermal replication of a targeted nucleic acid is possible using a concerted three-enzyme, in vitro reaction. This amplification strategy has been termed a selfsustained sequence replication (3SR) reaction, and was modeled after the general scheme employed during retroviral replication. (~4) In this reaction, activities of avian myeloblastosis virus (AMV) reverse transcriptase (RT), Escherichia coli RNase H, and T7 RNA polymerase produce an average 10-fold amplification every 2.5 min (for the first 15 min), leading to 107-fold amplification in 60 min. (14) Figure 1 provides a description of the steps in the 3SR amplification reaction. This report discusses additional developments in the originally described 3SR reaction. (14) Such developments include: (1) optimization of substrate concentrations, (2) the effect of temperature, pH, and ionic strength on amplification productivity, and (3) the reaction conditions required that enable the 3SR reaction to be carried out with only AMV RT and T7 RNA polymerase (two-enzyme 3SR reaction).