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

TLDR

Isothermal in vitro amplification of nucleic acids can be achieved by a multienzyme reaction that mimics retroviral replication, employing reverse transcriptase, RNase H, and a DNA‑dependent RNA polymerase. The reaction mimics retroviral RNA replication via cDNA intermediates, accumulating cDNA and RNA copies of the target sequence. The system achieves exponential amplification, reaching ten‑million‑fold increases within 1–2 h (≈10‑fold every 2.5 min), and its self‑sustained replication makes it valuable for detecting and sequencing rare RNAs and DNAs.

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 DNA-dependent RNA polymerase. By mimicking 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.