Isothermal Nucleic Acid Amplification
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.
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 the 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.
There are many isothermal nucleic acid amplification techniques available. Please click on each accordion below to see a diagram for each technique.
Figure 1. 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.
Reference: Notomi T, Okayama H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63. doi:10.1093/nar/28.12.e63
Figure 2. Multiple displacement amplification (MDA). 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.
Reference: Zhang DY, Brandwein M, et al.Ramification amplification: a novel isothermal DNA amplification method. Mol Diagn. 2001;6(2):141-150. doi:10.1054/modi.2001.25323
Figure 3. 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.
Reference: Piepenburg O, Williams CH, et al.DNA detection using recombination proteins. PLoS Biol. 2006;4(7):e204. doi:10.1371/journal.pbio.0040204
Figure 4. Helicase-dependent amplification (HDA). HDA 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.
Reference: Vincent M, Xu Y, Kong H. Helicase-dependent isothermal DNA amplification. EMBO Rep. 2004;5(8):795-800. doi:10.1038/sj.embor.7400200
Figure 5. Nucleic acid sequence-based amplification (NASBA). NASBA 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.
Reference: Compton J. Nucleic acid sequence-based amplification. Nature. 1991;350(6313):91-92. doi:10.1038/350091a0
Figure 6. Transcription-mediated amplification (TMA). TMA 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.
Reference: Kacian DL, Fultz TJ. Nucleic acid sequence amplification methods. Biotechnol. Adv. 1995;3(13):569. doi: 10.1016/0734-9750(95)94160-8.
Figure 7. Strand displacement amplification (SDA). SDA is a technique that combines the nicking action of restriction endonuclease and the activity of strand displacing polymerase. Repeated cycles of nicking and extension results in exponential amplification of DNA target.
Reference: Walker GT, Little MC, et al. Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. Proc Natl Acad Sci U S A. 1992;89(1):392-396. doi:10.1073/pnas.89.1.392
Figure 8. Exponential amplification reaction (EXPAR). EXPAR 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.
Reference: Van Ness J, Van Ness LK, Galas DJ. Isothermal reactions for the amplification of oligonucleotides. Proc Natl Acad Sci U S A. 2003;100(8):4504-4509. doi:10.1073/pnas.0730811100
Figure 9. Rolling circle amplification (RCA). RCA 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.
Reference: Fire A, Xu SQ. Rolling replication of short DNA circles. Proc Natl Acad Sci U S A. 1995;92(10):4641-4645. doi:10.1073/pnas.92.10.4641
Table 1. Comparison between different INAATs
|Technology||Reaction temperature||Reaction time||Primers||Amplicon size||Detection method|
|60–65 °C||15–60 mins||4–6 primers||>20 kb||Fluorescence, colorimetric, turbidity, lateral flow|
Multiple displacement amplification
Whole genome amplification
|30–40 °C||60–180 mins||Random hexamers||Unlimited||Fluorescence, colorimetric|
Recombinase polymerase amplification
|37 °C||30–60 mins||2 primers||<1 kB||Fluorescence, lateral flow|
Helicase dependent amplification
|65 °C||~90 mins||2 primers||~150 nt||Fluorescence, colorimetric, lateral flow|
Nucleic acid sequence based amplification
|40–50 °C||~60 mins||2 primers||~150 nt||Fluorescence|
Transcription mediated amplification
|40–55 °C||30–90 mins||2 primers||~150 nt||Fluorescence, chemiluminescence|
Rolling circle amplification
|30–65 °C||60–90 mins||1 primer||~150 nt||Fluorescence, colorimetric, turbidity|
Exponential amplification reaction
|55–60 °C||<30 mins||DNA trigger||~120 nt||Fluorescence, colorimetric|
Strand displacement amplification
|30–55 °C||~120 mins||4 primers||~100 nt||Fluorescence|
Table 2. Enzymes for INAATs
|Lyo-ready Bst DNA Polymerase||LAMP, HDA, EXPAR, RCA|
|SuperScript IV RT-LAMP Master Mix||LAMP|
|SuperScript IV Reverse Transcriptase and other RTs||LAMP, NASBA, TMA|
|T7 RNA Polymerase||NASBA, TMA|
|RNase inhibitors (Ribolock, RNaseOUt, and others)||LAMP, NASBA, TMA|
|Phi29 DNA Polymerase||MDA, WGA, RCA|
|EquiPhi29™ DNA Polymerase||MDA, WGA, RCA|
|Klenow Fragment, exo–||SDA|
These enzymes 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.
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.
The SuperScript IV RT-LAMP Master Mix (Cat. A51801) 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:
For Research Use Only. Not for use in diagnostic procedures.