Isothermal Amplification | Thermo Fisher Scientific

Isothermal amplification techniques offer a fast, cycling-free alternative to PCR, enabling exponential amplification of nucleic acids at constant temperatures. Each technique leverages specific enzymes and reaction conditions, but all require polymerases with strand-displacement activity. DNA polymerases like Bst DNA Polymerase, Klenow exo-, and Phi29 (EquiPhi29) exhibit rapid and strong strand displacement activity, making them an excellent choice for isothermal nucleic acid amplification. This method has garnered significant attention and is becoming increasingly important in fields such as infectious disease diagnostics, genomic research, environmental monitoring, and food safety due to its speed, simplicity, and robustness.

Why choose isothermal amplification techniques?

Speed and efficiency: Helps provide rapid results without the need for thermal cycling
Versatility: Suitable for a variety of applications, from point-of-care (POC) to laboratory and field-based assays
Minimal sample processing: Designed to simplify the workflow, making it accessible for various testing environments

Applications of isothermal amplification

The widespread adoption of isothermal amplification technology has revolutionized molecular assay developments in these areas:

Point-of-Care Testing (POCT): An excellent choice for near-patient environments, offering quick and reliable results
Infectious and genetic disease detection: Essential tools for developing assays to detect a wide range of diseases
Next-generation testing devices: Many modern testing devices now incorporate these technologies for enhanced performance

Isothermal amplification techniques

Loop-mediated isothermal amplification (LAMP)

LAMP is a technique for the amplification of DNA or RNA (when reverse transcriptase is incorporated) based on a strand displacement reaction and the formation of stem-loop structures under isothermal conditions. It uses the Bacillus stearothermophilus DNA polymerase (Bst DNA polymerase) and a set of four to six specifically designed primers that hybridize to six or eight different parts of the target DNA sequence.

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Figure 1. Loop-mediated isothermal amplification (LAMP). [1]

Recombinase polymerase amplification (RPA)

RPA is an isothermal amplification mechanism that operates at low temperature and is based on strand invasion that is accomplished by a cocktail of recombinase enzymes, single-stranded binding proteins, and strand displacing DNA polymerases.

These products leverage a unique combination of proteins and enzymes, allowing for fast, efficient target detection.

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Figure explaining Isothermal RPA reaction

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Figure 2. Recombinase Polymerase Amplification.

Multiple displacement amplification/whole genome amplification (MDA-WGA)

Multiple displacement amplification (MDA) is the most widely used whole-genome amplification (WGA) technique that utilizes a strand-displacing DNA polymerase, such as Phi29 polymerase and random hexamers to amplify the genome under isothermal conditions.

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Diagram showing multiple displacement amplification (MDA)

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Figure 3. Multiple displacement amplification (MDA). [2]

Rolling circle amplification (RCA)

Rolling circle amplification is an isothermal amplification technique where short DNA or RNA primer is amplified into a long single stranded DNA or RNA using a circular DNA template and strand-displacing DNA polymerase, such as Phi29 DNA polymerase. RCA generates a concatemer that contains numerous tandem repeats that are complementary to the circular template.

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Diagram showing rolling circle amplification

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Figure 4. Rolling circle amplification (RCA). [3]

Why choose our enzymes?

Customizable solutions: Tailor enzyme formulations to your specific assay requirements.
Enhanced performance: Our enzymes are optimized for a wide range of isothermal amplification techniques, designed for reliable and efficient amplification.
Comprehensive support: Access detailed product information and expert support for your assay development.

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Help enhance your isothermal amplification assays with our high-quality, customizable enzyme solutions. Visit www.thermofisher.com/mdx or contact us to learn more and place your order.

Explore other isothermal amplification techniques

Helicase-dependent amplification (HDA)

Helicase-dependent amplification is an isothermal amplification method that utilizes helicase to unwind double-stranded DNA, enabling primer annealing and extension with strand displacing DNA polymerase, such as Bst DNA polymerase.

Figure explaining Isothermal HDA reaction

Figure 5. Helicase-dependent amplification.

Nucleic acid sequence-based amplification (NASBA)

Nucleic acid sequence-based amplification is a technique for amplifying RNA, based on initial target extension by reverse transcriptase and subsequent transcript generation by RNA polymerase, such as T7 RNA polymerase. NASBA reactions require isothermal conditions and additional RNase H enzyme, that is used to degrade the RNA strand in an RNA/DNA hybrid.

Figure explaining Isothermal NASBA reaction

Figure 6. Nucleic acid sequence-based amplification (NASBA).

Transcription-mediated amplification (TMA)

Transcription-mediated amplification is a technique that involves isothermal amplification of RNA by utilizing two enzymes: reverse transcriptase (RT) and T7 RNA polymerase. The main difference from NASBA is the intrinsic RNase H activity of RT enzyme that hydrolyzes the RNA strand in an RNA/DNA hybrid.

Figure explaining Isothermal TMA reaction

Figure 7. Transcription-mediated amplification (TMA).

Strand displacement amplification (SDA)

Strand displacement amplification is a technique that combines the nicking action of restriction endonuclease and the strand-displacing activity of polymerase. Repeated cycles of nicking and extension results in exponential amplification of target DNA.

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Figure 8. Strand displacement amplification (SDA). Reference: Walker GT, Little MC, Nadeau JG, et al. (1992) Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. Proc Natl Acad Sci U S A 89(1):392–396.

Exponential amplification reaction (EXPAR)

Exponential amplification reaction is a technique for the amplification of short oligonucleotides at isothermal conditions. The reaction is initiated by a DNA trigger and further amplification occurs repeatedly and exponentially by utilizing a nicking enzyme and a strand-displacing polymerase.

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Figure 9. Exponential amplification reaction (EXPAR).
Reference: Van Ness J, Van Ness LK, Galas DJ. (2003) Isothermal reactions for the amplification of oligonucleotides. Proc Natl Acad Sci U S A 100(8):4504–4509.

Comparing isothermal amplification techniques

Isothermal amplification methods offer important alternatives to lab-based methods that depend on expensive equipment and protocols for sequential cycling to amplify target of interest.

Table 1. Comparison between different isothermal amplification techniques

Technology	Featured products	Reaction temperature	Reaction time	Primers	Amplicon size	Detection method
LAMP Loop-mediated amplification	SuperScript IV Reverse Transcriptase and other RTs Lyo-ready Bst DNA Polymerase SuperScript IV RT-LAMP Master Mix RNase inhibitors	60–65 °C	15–60 mins	4–6 primers	>20 kb	Fluorescence, colorimetric, turbidity, lateral flow
MDA Multiple displacement amplification WGA Whole genome amplification	Phi29 DNA Polymerase EquiPhi29 DNA Polymerase EquiPhi29 DNA Amplification Kit	30–40 °C	60–180 mins	Random hexamers	Unlimited	Fluorescence, colorimetric
RPA Recombinase polymerase amplification	Lyo-ready Bst DNA Polymerase Lyo-ready RPA Kit Lyo-ready T4 UvsX Lyo-ready T4 UvsY Lyo-ready T4 Gene 32	37 °C	30–60 mins	2 primers	<1 kb	Fluorescence, lateral flow
HDA Helicase dependent amplification		65 °C	~90 mins	2 primers	~150 nt	Fluorescence, colorimetric, lateral flow
NASBA Nucleic acid sequence based amplification	SuperScript IV Reverse Transcriptase and other RTs T7 RNA Polymerase RNase inhibitors RNase H	40–50 °C	~60 mins	2 primers	~150 nt	Fluorescence
TMA Transcription mediated amplification	SuperScript IV Reverse Transcriptase and other RTs T7 RNA Polymerase RNase inhibitors	40–55 °C	30–90 mins	2 primers	~150 nt	Fluorescence, chemiluminescence
RCA Rolling circle amplification	Phi29 DNA Polymerase EquiPhi29 DNA Polymerase EquiPhi29 DNA Amplification Kit	30–65 °C	60–90 mins	1 primer	~150 nt	Fluorescence, colorimetric, turbidity
EXPAR Exponential amplification reaction		55–60 °C	<30 mins	DNA trigger	~120 nt	Fluorescence, colorimetric
SDA Strand displacement amplification	Klenow Fragment, exo–	30–55 °C	~120 mins	4 primers	~100 nt	Fluorescence

FAQs about isothermal amplification

What are the differences between conventional vs lyo-ready enzyme? Do you offer enzymes/assays in lyophilized/dry format?

We focus on supplying the raw materials for your assay development. All our enzymes come in liquid form; conventional enzymes contain glycerol in the storage buffer formulation. We do not offer dried-down enzymes or lyophilization services.

Enzymes in lyo-ready format are beneficial when portable, room-temperature stable assays are being developed. Lyo-ready enzymes retain all conventional enzyme characteristics like reproducibility, sensitivity, and specificity required for these assays.

Do you have a Lyo-ready version of the Invitrogen SuperScript IV RT-LAMP Master Mix? Is there an OEM offering for this product?

SuperScript IV RT-LAMP Master Mix is available for commercial use and optimized for the best results in LAMP and RT-LAMP. SuperScript IV RT-LAMP Master Mix is provided in glycerol format only.

Nevertheless, we offer lyo-ready components of the Master Mix—the SSIV Reverse Transcriptase, Bst DNA Polymerase, and RNaseOUT RNase inhibitor, as well as the user manual for LAMP reaction setup. These components are available in a glycerol-free format and can be customized further upon request.

How can I eliminate non-specific amplification?

Isothermal amplification methods are fast and robust, but they often result in non-specific amplification, leading to false positive results. To help ensure a reliable test result and control non-specific amplification:

Make sure the work environment and reagents are clean. It is recommended to clean your workspace regularly to prevent contamination.
Set up the reactions on ice to avoid non-specific amplification products.
To enhance specificity in methods such as LAMP, primer design and optimization is required. We recommend following the primer design guidelines. Ensure the primer melting temperature is neither too high nor too low. Low temperature may result in non-specific primer binding, and high temperature may inhibit primer binding to the template.
Other reaction parameters such as reaction time or Bst DNA polymerase concentration can also be optimized. Depending on primer design and template concentration, Bst DNA polymerase amount per reaction can be decreased down to 1 U. If end-point detection is used, time can be an important factor for non-specific amplification. Reducing the incubation time by several minutes may help distinguish the NTC from the sample.