Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication.

Abstract

A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNAdependent RNA polymerase. By mimickisg the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1-to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.The transfer of genetic information from RNA to DNA and then back to RNA is a scheme characteristic of retroviruses. Such a scheme provides a mechanism for the replication of RNA genomes (reviewed in refs. 1-3). In exploring variations of an in vitro transcription-based amplification system (TAS) (4), it was discovered that it was possible to devise a concerted, three-enzyme, in vitro reaction to carry out an isothermal replication of target nucleic acid sequences, analogous to the strategy used in retroviral replication. This reaction is a self-sustained sequence replication (3SR) system involving the collective activities of avian myeloblastosis virus (AMV) reverse transcriptase, Escherichia coli RNase H, and T7 RNA polymerase. The accumulation ofboth target nucleic acid-specific RNA Approximately 25 mg (wet weight) of Trisacryl beads containing oligonucleotide 86-273 (5'-AGTCTAGCAGAA-GAAGAGGTAGTAATTAGA-3') was prehybridized in 250 pA of hybridization solution (5 x standard saline phosphate/ EDTA/10% dextran sulfate/0.1% SDS) in a 2-ml microcolumn (Isolab) for 30 min at 370C. The prehybridization solution was removed, and 40 p.1 of fresh hybridization solution was added to the beads, together with the 60 p.1 of solution from the solution hybridization step. The beads were then incubated at 370C for 1 hr with occasional mixing and then washed six times with 1 ml of 2x standard saline citrate (6) at 370C. The radioactivity of the beads and the combined washes was measured by Cerenkov counting for 1 min. The amount of target detected was determined by calculating the percentage of total radioactivity captured on the beads and Abbreviations: 3SR, self-sustained sequence replication; TAS, transcription-based amplification system; HIV-1, human immunodeficiency virus type 1; AMV, avian myeloblastosis virus.§To whom reprint requests should be addressed. 1874The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Proc. Natl. Acad. Sci...

Signal amplification through nucleotide extension and excision on a dendritic DNA platform

“…In isothermal target amplification methods, such as the strand displacement assay and transcription-mediated amplification, the magnitude of amplification is dependent on the incubation time of the reaction. The isothermal methods have produced 10 6 -to 10 8 -fold target amplification after 1-2 h incubation (3,4). The magnitude of signal amplification obtained by the NEESA approach employing DNA dendrimer substrates falls well within the range of amplification obtained with exponential target amplification methods.…”

“…Detection of extremely low viral loads became feasible with the development of nucleic acid amplification technologies. Due to their ability to be amplified using different enzymatic approaches (1)(2)(3)(4)(5), nucleic acid molecules have been detected at extremely low concentrations, as low as a single molecule (6). Nucleic acid sequences at sparingly low concentrations are pre-amplified to enrich their concentrations, allowing them to be detected by less sensitive methods.…”

Analysis of Specific Gene by Integration of Isothermal Amplification and Electrophoresis on Poly(methyl methacrylate) Microchips

“…10-13 However, polymer microchips, which are expected to be very promising in the future, 14 can hardly endure the high temperature during PCR. Accordingly, amplification at a relatively low temperature should be especially developed for them.Up until now, several isothermal amplification methods under mild conditions have been proposed, including nucleic acid sequence-based amplification, 15 self-sustained sequence replication, 16 and strand displacement amplification. 17,18 Each of them has its own innovation to reinitiate new rounds of DNA synthesis, but there are still drawbacks to overcome.…”

“…Up until now, several isothermal amplification methods under mild conditions have been proposed, including nucleic acid sequence-based amplification, 15 self-sustained sequence replication, 16 and strand displacement amplification. 17,18 Each of them has its own innovation to reinitiate new rounds of DNA synthesis, but there are still drawbacks to overcome.…”

Extremely sensitive, background-free gene detection using binary probes and beta replicase.

“…Although a very large number of copies are synthesized in target-sequence amplification assays (1)(2)(3)(4)(5), such as those that use the polymerase chain reaction, their design creates practical problems that restrict their use to specialized laboratories. These assays are usually carried out in crude cellular extracts, where they can be inhibited by cellular components and where the presence of unrelated nucleic acids can lead to falsepositive signals; different sample preparation protocols are needed for different tissues and for different infectious agents; relatively expensive equipment is often required to alternately raise and lower the temperature; and additional steps are needed to detect the amplified nucleic acid, increasing the risk of contaminating other samples.…”