Isothermal Nucleic Acid 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.

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.

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.

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.

Our RPA products leverage a unique combination of proteins and enzymes, allowing for fast, efficient target detection:

• Invitrogen Lyo-ready Bst DNA Polymerase (engineered, strand-displacing)
• Invitrogen Lyo-ready T4 UvsX and UvsY Proteins (recombinase complex)
• Invitrogen Lyo-ready T4 Gene 32 Protein (single-strand DNA binding)

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.

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 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 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 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.

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.