The RNA primer region of the regenerated double-stranded cDNA is digested by RNaseH so that another second chimeric primer can anneal by partially displacing the strand that covers the DNA region of the primer (red line). Elongation by a strand-displacing polymerase displaces the first cDNA strand and regenerates the cDNA without the RNA primer region of the original template.

By addition of RNaseH, the RNA region of the first chimeric is removed so that a complementary second hybrid primer comprising DNA (dark red arrow) and RNA region (wavy blue line) can anneal. By heating, parts of the RNA template is fragmented and removed from the cDNA, leaving a small portion that can act as a primer (gray wavy arrow) for second strand synthesis. The resulting cDNA (straight black line) represents a full-length copy of the template. The mRNA template (gray wavy line with a polyA tail in green) is first contacted with a first chimeric primer comprising DNA (dark green arrow) and RNA regions (wavy red and blue line) for reverse transcription. This review will serve as an overall outlook on the journey and development of isothermal amplification methods as a whole. We would also compare the efficiencies and usefulness of each method while highlighting the potential applications and detection methods involved. This review pays special attention to the various isothermal amplification methods by classifying them based on the mechanistic characteristics which include reaction formats, amplification information, promoter, strand break, and refolding mechanisms. With all the advantages isothermal amplification brings, the issues or complications surrounding each method are heterogeneous making it difficult to identify the best approach for an end-user.

The ease with which isothermal amplification methods have allowed nucleic acid amplification to be carried out has had a profound impact on the way molecular diagnostics are being designed after the turn of the millennium. Isothermal amplification methods have since been introduced utilizing different mechanisms, enzymes, and conditions. Since the introduction of PCR, a deeper understanding of molecular mechanisms and enzymes involved in DNA/RNA replication has spurred the development of novel methods devoid of temperature cycling. The advent of PCR catalyzed the increasing application of DNA, not just for molecular cloning but also for molecular based diagnostics. The introduction of nucleic acid amplification techniques has revolutionized the field of medical diagnostics in the last decade.

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