LAMP Fast Mastermixes with SybrGreen and Isothermal Amplification Products

List of isothermal amplification techniques

Isothermal amplification is not a new technique. It has been around since the 1990s and has continued to evolve. As of now, there are several different approaches depending on amplification goals, some which will be discussed a little bit in this article. Among the techniques available are:

• Helicase-Dependent Amplification (HDA): Helicase-dependent amplification is an amplification approach that based on the in vivo DNA replication process by using helicase to unwind DNA, allowing primers to bind. To prevent single stranded DNA from associating with its complimentary strand or destabilizing, two accessory proteins: MutL and single-stranded DNA-binding protein (SSB) are also used. This technique is a simple approach to isothermal amplification that has been used to develop sensitive viral and bacterial detection (Barreda-García, Miranda-Castro, de-Los-Santos-Álvarez, Miranda-Ordieres, & Lobo-Castañón, 2018).
• Isothermal Multiple Displacement Amplification (IMDA): Isothermal multiple displacement amplification amplifies nucleic acid sequences using a strand-displacing DNA polymerase and multiple primer sets (Gill, & Ghaemi, 2008). Variations of this technique have been used for isothermal whole genome sequencing. One advantage of IMDA over PCR is its sensitivity and specificity. This is especially helpful when the amount of DNA in the sample is very low (Mairinger et al., 2014).
• Loop-Mediated Isothermal Amplification (LAMP): Loop-mediated isothermal amplification is a very common isothermal technique that uses four to six primers and a strand displacing DNA polymerase. The LAMP technique is a very fast approach to synthesizing a lot of DNA in a very short period of time. Researchers have used this technique to develop rapid molecular tests or detect microorganisms (Gill, & Ghaemi, 2008).
• Recombinase Polymerase Amplification (RPA): Recombinase polymerase amplification amplifies DNA at a constant temperature (37–42 °C) using a recombinase, primers, a single-stranded DNA binding protein (SSB), and a strand displacing DNA polymerase. In this technique, the recombinase is complexed with the primer. The complex is able to bind with double-stranded DNA at homologous sequences through a strand exchange. After the exchange, a single-stranded binding protein, T4 gp32, stabilizes the displaced strand. Finally, Bsu polymerase extends the primers, creating a new complete copy of the template (Piepenburg, Williams, Stemple, & Armes, 2006).
• Rolling Circle Amplification (RCA): Rolling circle amplification synthesizes long single-stranded DNA using a short, circular single-stranded DNA template and a single primer. Like many other isothermal techniques, RCA enzymatically synthesizes DNA using a strand displacing DNA polymerase called phi 29 (ɸ29) DNA polymerase. This technique has been used particularly in nanotechnology, but has also been used for sensitive DNA detection in genomics and diagnostics (Zhao, Ali, Brook, & Li, 2008)
• Single Primer Isothermal Amplification (SPIA): Single primer isothermal amplification is an approach using only one DNA-RNA chimeric primer along with RNase H and a DNA polymerase with strand displacement activity. This approach is capable of amplifying more DNA than PCR when primer concentrations are increased. Applications using this technique include on-site diagnosis and DNA detection (Mukai et al., 2007).
• Strand Displacement Amplification (SDA): Strand displacement amplification is an isothermal technique that relies on a restriction enzyme (HincII) and an exonuclease-deficient DNA polymerase. The restriction endonuclease will nick the target DNA, allowing DNA polymerase to extend the 3’ end. This technique does have some limitations including lower primer specificity (Walker et al., 1992).

source: GoldBio Corporation