Isothermal Nucleic Acid Amplification

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

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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

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:

• 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 here. 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.