An Introduction to Isothermal Amplification

What is Isothermal Amplification

In molecular biology, when it comes to copying nucleic acid sequences, PCR gets all the glory. But there is another, less common term floating around: isothermal amplification, and maybe it’s got you wondering, “well, what is that exactly, and how is it different from PCR?” Isothermal amplification is the continuous, exponential amplification of nucleic acid sequences at a constant temperature using enzymes, usually strand displacing polymerases, rather than temperature changes.

That might seem pretty technical, so to break it down, isothermal amplification is a way of copying nucleic acids without temperature changing cycles. Instead, it uses specific DNA polymerases (usually), and specially designed primer sets to exponentially amplify a target sequence. And this technology has helped overcome certain limitations of PCR.

In this article, we’ll talk more about Isothermal amplification, the techniques used, how it differs from PCR, and more.

Figure 1. Simplistic overview of DNA polymerase with strand displacement activity. DNA polymerase will replicate a new strand of DNA while displacing one of the original strands. This process will continue repeatedly. And in some techniques, the displaced strand serves as a template for further amplification. Note, this figure is meant for conceptual understanding and does not represent all types of strand displacing behavior in isothermal amplification.

It is important for us to point out that when we talk about strand displacement, it’s in a very general context. Different variations employ different techniques and events, such as an initial nicking event, branching and the use of specialized primer sets.

There are also other isothermal amplification techniques that depend on other types of enzymes to unzip the target strand of DNA. For instance, helicase-mediated isothermal amplification uses T7 helicase to unwind DNA, allowing primer binding (Xu, Kim, Kays, Rice, & Kong, 2006).

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).

The difference between isothermal amplification and PCR

The key difference between isothermal amplification and PCR is isothermal amplification is carried out at a constant temperature using amplification machinery, whereas PCR requires temperature-changing cycles for amplification. What this also means is that isothermal amplification does not require a thermal cycler (though many machines can also perform isothermal amplification) for the reaction.

Another important difference between isothermal amplification and PCR is that DNA polymerase will extend primers on a single strand of DNA during PCR, while certain isothermal amplification techniques have special stand displacing DNA polymerases that extend primers on double-stranded DNA.

Illustration of the difference between PCR and Isothermal Amplification. PCR depends on temperature changing cycles whereas isothermal amplification depends on special polymerases or enzymes and primer sets.

Figure 2. Comparing the basic differences between PCR and isothermal amplification. PCR requires repeated cycles of programmed temperature changes to exponentially amplify sequences. Isothermal amplification relies on enzymatic characteristics to carry out amplification. This example shows the very basic concept of isothermal amplification via strand displacement. In this comparison, DNA polymerase can extend a primer while displacing a strand of double-stranded DNA. However, for PCR, DNA polymerase extends a primer using a single DNA strand as a template.

Advantages of isothermal amplification

One of the biggest advantages of isothermal amplification is that it doesn’t require thermal cycling, and therefore requires less power consumption, making it a compatible technique for hand-held equipment in the field.

Being able to carry out amplification on-site with handheld equipment opens the door for molecular biologists. They can perform point-of-care diagnostics or examine onsite environmental samples (Zanoli & Spoto, 2013).

The second advantage to isothermal amplification is its speed and sensitivity. Because isothermal provides continuous, exponential amplification independent of thermal cycling. Some methods can be performed in as little as 10 minutes (Zou, Mason, & Botella, 2020).

Depending on the technique used, primer design for isothermal amplification also enables greater target specificity (Zou, Mason, & Botella, 2020), which can be extremely useful, especially for whole genome amplification.

Why isothermal amplification is not as popularly used as PCR

If isothermal amplification doesn’t require thermal cycling, can be carried out on-site, is fast and sensitive, why isn’t it as commonly used as PCR?

We’ve listed a few reasons isothermal amplification is not as popular as PCR:

PCR was first in the industry.
Primer design can be more complicated.
More components are needed depending on technique.
There are still intellectual property barriers to isothermal amplification.
Advantages of time in high-throughput labs are not actually an advantage.

First-to-market advantage of PCR

PCR came just a little bit before isothermal amplification, and the science behind it was relatively easy to understand. At programed temperature intervals, the behavior of DNA, primers and polymerase could be manipulated into exponential amplification. Because it was first and easy to setup, it was quickly adopted by laboratories. From there, PCR continued to evolve. New techniques spun out fulfilling other research needs (Howard, 2019).

Primer design

The LAMP method, as an example, requires six primers rather than the two needed for PCR. Primer design can be a little frustrating, especially when you’re new at it, reducing that frustration is going to be extremely attractive. If your lab is already set up for PCR, there would have to be a case for spending extra time and money developing and purchasing special primers (Howard, 2019).

Keep in mind, however, if you’re considering isothermal amplification for a regular diagnostic test, you may only have to worry about primer design once. Which means you would only encounter this frustration one time. Furthermore, kits can ease a lot of frustration since there is no need for primer design.

Components

A general PCR master mix contains dNTPs, template, water, primers, Taq DNA polymerase, MgCl2 and reaction buffer. However, different types of isothermal amplification techniques require other enzymes, probes, proteins, etc. (Howard, 2019). Kits also succeed in overcoming this issue. Amplification kits will come with all the components needed for your reaction so you’re not stuck with extra reagents, or having to spend time shopping and price comparing.

Intellectual property

With basic PCR technology having been available for a few decades now, there are fewer intellectual property barriers.

Older isothermal amplification methods have fewer barriers, but the new, innovative techniques that are more advantageous do (Howard, 2019).

Speed is not always an advantage.

Isothermal amplification happens quickly. Depending on the setup, results could be achieved in as little as 30 – 60 minutes. In some cases, results are produced in as little as 10 minutes. Yet speed is not always an advantage. For example, high-throughput batch production using PCR saves money and time with a simple process. And since production is scaled, these advantages far outweigh the speed obtained from isothermal amplification.

When is isothermal amplification appropriate to use?

Thinking about some of the setbacks of isothermal amplification, you might wonder when is it right for your lab. When thinking about whether isothermal amplification is right for you, consider how important speed is to you, whether you need an extremely sensitive test, and if you need to perform the test on-site.

Deciding whether isothermal amplification would be a better approach for your lab involves a balance of the factors listed above. For instance, if you need a rapid, on-site test using the same diagnostic test every time, the advantages of isothermal amplification will become more critically important than the ease of PCR.

To help you better weight out these factors, we have provided a table for you to use when considering isothermal amplification.

Is Isothermal Amplification is Right for Your Lab Checklist
Do you need results in under an hour?	Yes / No
Do you need to perform on-site testing?	Yes / No
Do you have a smaller lab (not doing large-batch amplification)?	Yes / No
Are you doing a regular diagnostic test that would use the same primers?	Yes / No
Are you working with small sample amounts needing high sensitivity?	Yes / No
Do you have equipment that will carry out isothermal amplification? (Many thermal cyclers can carry out the reaction.)	Yes / No
Do you have the budget for the components listed your intended protocol?	Yes / No

The more YESes checked off on this list (with the exception of the last two questions, whose NOs may in fact be deal breakers), the more likely isothermal amplification would be appropriate for your lab.

The advantages of isothermal amplification kits

If you discover isothermal amplification is critical for your laboratory, you might not want to deal with the frustration of selecting which type of amplification method to use, designing primers, shopping for components and optimizing the test.

If you’re running a standard diagnostic, this is where kits provide an excellent benefit. With a kit, you can be assured your setup is optimized and you have all the components you need. And if you’re nervous about ease of use, kits typically make reaction setup extremely user-friendly.