What are the different methods of isothermal amplification?

The different methods of isothermal amplification include nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), and multiple displacement amplification (MDA).  

• LAMP involves using two sets of unique primers (known as inner and outer primers) along with a DNA polymerase that can displace DNA strands. LAMP is highly efficient and can produce a significant amount of amplified DNA within just one hour. 
• RCA relies on a circular DNA template and a strand-displacing DNA polymerase. The polymerase continuously extends the DNA strand, creating a long, single-stranded DNA molecule. This process occurs at a constant temperature and results in the creation of a DNA strand with repeated sequences from the circular template. 
• HDA utilizes the action of a DNA helicase to unwind the double-stranded DNA (dsDNA), eliminating the need for temperature cycling to generate single-stranded templates. This process mirrors the natural mechanism of DNA replication in living organisms, where DNA helicase unwinds the DNA strands to facilitate DNA polymerase activity. 
• MDA is a method used for whole genome amplification, which can generate numerous amplification products from a small number of DNA molecules. MDA utilizes random exonuclease-resistant primers and the strand displacement activity of the φ29 DNA polymerase to create DNA strands up to 70 kb in length. 
• NASBA is designed specifically for amplifying single-stranded RNA or DNA sequences. It uses two RNA target-specific primers and three enzymes: avian myeloblastosis virus reverse transcriptase, RNase H, and T7 DNA-dependent RNA polymerase (DdRp). NASBA is not capable of amplifying double-stranded DNAs unless they undergo a denaturation step. 
• RPA is performed at a low temperature of around 37°C. It combines recombinase-driven primer targeting with strand-displacement DNA synthesis. In this technique, nucleoprotein complexes formed by oligonucleotide primers and recombinase proteins facilitate primer binding to the template DNA.