Isothermal nucleic acid amplification techniques (INAATs) are fast and cycling-free alternatives to PCR, which enable exponential amplification of nucleic acids at constant temperatures. Each INAAT utilizes specific enzymes and reaction conditions, but all require polymerases with strand-displacement activity. DNA polymerases, such as Bst DNA Polymerase, Klenow exo-, and Phi29 (EquiPhi29) exhibit rapid and strong strand displacement activity, and are suitable for isothermal nucleic acid amplification.

Applications of isothermal nucleic acid amplification technology

The broader use of isothermal amplification technology has stimulated new developments in the molecular assay development field. Assays based on INAATs play a significant role in point-of-care (POC), laboratory-based, and field-based assay development. Valued for fast turnaround and minimal sample processing, INAATs became common assay development tools for infectious and genetic disease detection. Many next-generation testing devices employ INAAT technology for point-of-care testing (POCT) in near-patient environments. During the COVID-19 pandemic, one of the isothermal amplification techniques—loop-mediated isothermal amplification (LAMP)—became a routine method for fast-track SARS-CoV-2 detection.

Isothermal amplification techniques

There are many isothermal nucleic acid amplification techniques available. Click on each accordion below to see a diagram and description for each technique.

Loop-mediated isothermal amplification

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.

Multiple displacement amplification/whole genome amplification

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.

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.

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.

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.

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.

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.

Comparing isothermal nucleic acid 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 compares the different technologies made possible with isothermal nucleic acid amplification below.

Table 1. Comparison between different INAATs

TechnologyReaction temperatureReaction timePrimersAmplicon sizeDetection method
LAMP
Loop-mediated amplification
60–65 °C15–60 mins4–6 primers >20 kbFluorescence, colorimetric, turbidity, lateral flow
MDA
Multiple displacement amplification

WGA
Whole genome amplification
30–40 °C60–180 minsRandom hexamersUnlimitedFluorescence, colorimetric
RPA
Recombinase polymerase amplification
37 °C30–60 mins2 primers  <1 kbFluorescence, lateral flow
HDA
Helicase dependent amplification
65 °C~90 mins2 primers ~150 ntFluorescence, colorimetric, lateral flow
NASBA
Nucleic acid sequence based amplification
40–50 °C~60 mins2 primers ~150 ntFluorescence
TMA
Transcription mediated amplification
40–55 °C30–90 mins2 primers ~150 ntFluorescence, chemiluminescence
RCA
Rolling circle amplification
30–65 °C60–90 mins1 primer ~150 ntFluorescence, colorimetric, turbidity
EXPAR
Exponential amplification reaction
55–60 °C<30 minsDNA trigger ~120 ntFluorescence, colorimetric
SDA
Strand displacement amplification
30–55 °C~120 mins4 primers ~100 ntFluorescence

Enzymes for isothermal nucleic acid amplification

Enzymes for isothermal nucleic acid amplification can be customized by volume, concentration, glycerol content, and other components in the formulation and reaction buffers. For the information about our custom commercial supply of products, please visit www.thermofisher.com/mdx or contact us.

Table 2. Enzymes for INAATs

FAQs

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.

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.

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.
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