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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 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. Click on each accordion below to see a diagram and description for each technique.
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.
Figure 1. Loop-mediated isothermal amplification (LAMP). Reference: Notomi T, Okayama H, Masubuchi H, et al. (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):E63.
Learn more: Loop-mediated isothermal amplification (LAMP)
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.
Figure 2. Rolling circle amplification (RCA). Reference: Fire A, Xu SQ. (1995) Rolling replication of short DNA circles. Proc Natl Acad Sci U S A 92(10):4641–4645.
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.
Figure 3. Multiple displacement amplification (MDA). Reference: Zhang DY, Brandwein M, Hsuih T, et al. (2000) Ramification amplification: a novel isothermal DNA amplification method. Mol Diagn 6(2):141–150.
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.
Figure 4. Recombinase polymerase amplification (RPA). Reference: Piepenburg O, Williams CH, Stemple DL, et al. (2006) DNA detection using recombination proteins. PLoS Biol 4(7):e204.
Our RPA products leverage a unique combination of proteins and enzymes, allowing for fast, efficient target detection:
The RPA reaction efficiency is fueled by the ATP regenerating buffer system, which is also supplied and optimized under our protocol.
For more information on the RPA protein characteristics please see this iconographic or contact our teams for samples, protocol, and support.
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 5. Helicase-dependent amplification (HDA). Reference: Vincent M, Xu Y, Kong H. (2004) Helicase-dependent isothermal DNA amplification. EMBO Rep 5(8):795–800.
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 6. Nucleic acid sequence-based amplification (NASBA). Reference: Compton J. (1991) Nucleic acid sequence-based amplification. Nature 350(6313):91–92.
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 7. Transcription-mediated amplification (TMA). Reference: Kacian DL, Fultz TJ. (1995) Nucleic acid sequence amplification methods. Biotechnol Adv 3(13):569.
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.
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 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.
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.
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.
Technology | Reaction temperature | Reaction time | Primers | Amplicon size | Detection method |
---|---|---|---|---|---|
LAMP Loop-mediated amplification | 60–65 °C | 15–60 mins | 4–6 primers | >20 kb | Fluorescence, colorimetric, turbidity, lateral flow |
MDA Multiple displacement amplification WGA Whole genome amplification | 30–40 °C | 60–180 mins | Random hexamers | Unlimited | Fluorescence, colorimetric |
RPA Recombinase polymerase amplification | 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 | 40–50 °C | ~60 mins | 2 primers | ~150 nt | Fluorescence |
TMA Transcription mediated amplification | 40–55 °C | 30–90 mins | 2 primers | ~150 nt | Fluorescence, chemiluminescence |
RCA Rolling circle amplification | 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 | 30–55 °C | ~120 mins | 4 primers | ~100 nt | Fluorescence |
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.
Product name | INAATs |
---|---|
Lyo-ready Bst DNA Polymerase | LAMP, HDA, EXPAR, RCA, RPA |
SuperScript IV RT-LAMP Master Mix | LAMP |
SuperScript IV Reverse Transcriptase and other RTs | LAMP, NASBA, TMA |
T7 RNA Polymerase | NASBA, TMA |
RNase inhibitors | LAMP, NASBA, TMA |
RNase H | NASBA |
Phi29 DNA Polymerase | MDA, WGA, RCA |
EquiPhi29™ DNA Polymerase | MDA, WGA, RCA |
Klenow Fragment, exo– | SDA |
Lyo-ready T4 UvsX * | RPA |
Lyo-ready T4 UvsY * | RPA |
Lyo-ready T4 Gene 32 * | RPA |
*Connect with us for more information or request samples.
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:
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