Auckland, March 16th 2021
Isothermal chemistry
Wairau River Lagoons, Marlborough
Isothermal DNA/RNA amplification
The discovery of thermostable DNA and RNA polymerases brought about the genomics revolution; thermostable enzymes made possible the enzymatic (polymerase chain reaction, PCR) copying, detection, cloning, and sequencing of DNA sequences. PCR has permeated all the life sciences.
In the past few years, new DNA amplifying methods have emerged, making it easy to perform DNA amplification at a single temperature, avoiding the need for temperature cycling and hence greatly reducing the complexity of instruments and the power required to run the instruments.
For traditional PCR, thermocycling to different temperatures is used to denature DNA, facilitate primer binding, and initiate amplification. Isothermal amplification, however, makes use of strand-displacing polymerases (or other enzymes) that effectively unzips (displace) the double-stranded DNA during amplification, removing the requirement to cycle rapidly through different temperatures.
This shift from a multi-temperature reaction to a single temperature makes the isothermal methods ideal for molecular in-field and point-of-care diagnostics as it significantly reduces the costs of materials and instruments.
As the DNAiTECH device was initially developed for LAMP, the overview below focuses on LAMP. However, with the second-generation DNAiTECH, all isothermal methods can be carried out, and standard techniques are summarised.
Loop-Mediated Isothermal Amplification
Loop-mediated isothermal amplification, or LAMP, is a standard isothermal amplification method with assays carried out between 60 and 65 °C. LAMP uses 4-6 primers and the strand-displacing polymerase Bst or Bsm. The amplification reaction produces long DNA products with hairpin and stem-loop structures. Using 6 primers (rather than the standard 2 for PCR), grants LAMP improved specificity and sensitivity for targets. Compared to DNA polymerases in PCR, LAMP is also highly tolerant of sample impurities and inhibitors and can be run on complex or poorly purified samples. LAMP assays are robust and fast, with products typically detected within 30 minutes.
There are a variety of methods that can be used to detect LAMP amplification. As a by-product, LAMP produces magnesium pyrophosphate, which precipitates and turns the sample turbid and can be read using an instrument or by eye. pH indicators such as neutral red or hydroxynaphthol blue will change colour as the pH of the solution changes during amplification. Fluorescent molecules can also be used. Intercalating dyes like EvaGreen or SYBR green are incorporated into amplified DNA and fluoresce when exposed to specific wavelengths of light. Lateral flow and gel electrophoresis are other options for determining amplification.
See the following links for some great videos covering the LAMP method:
Helicase-Dependent Amplification
Helicase-dependent amplification (HDA) is a technique that utilises helicase to unwind the double-stranded DNA, which facilitates primer binding and amplification using DNA polymerase. Like LAMP, HDA generally runs at 65 °C but only requires two primers. Detection within 50 – 100 minutes.
Link to the original publication.
Rolling Circle Amplification
Rolling circle amplification (RCA) is a technique to rapidly amplify circular DNA/RNA using a single primer at
37 °C. The DNA is nicked, allowing primer binding and amplification around the circular genetic material, hence the “rolling circle”. This amplification can produce circular or long linear DNA products from DNA or RNA.
Recombinase Polymerase Amplification
Recombinase polymerase amplification (RPA) is carried out between 37 and 42 °C using a recombinase to enable primer binding to double-stranded DNA, a single-stranded binding protein, and the strand displacing polymerase Bsu. The single-stranded binding proteins stabilise the primer-DNA complex, and Bsu initiates amplification from these primer sites. Like PCR, RPA uses two primers per site for amplification but can be multiplexed by including more than one pair in a single reaction. RPA can quickly identify pathogens, with results typically readable within 30 minutes rapidly.
Developed by TwistDx.
Strand Displacement Amplification
Strand displacement amplification (SDA) uses an endonuclease activity that nicks the DNA with a strand-displacing polymerase. It consists of two phases: target production and target amplification. While SDA is run at 37 °C, during target production, an initial 95 °C heating step is used to denature the double-stranded DNA, allowing modified primes containing an endonuclease site to bind and synthesise a modified strand of the target. This strand is displaced by a ‘bumper primer’. The substituted DNA forms double-stranded DNA containing the endonuclease site. During target amplification, these sites are ‘nicked’ by an endonuclease and DNA polymerase replicates the strand, displacing the DNA. This sequence repeats to amplify the target sequence. SDA returns results in 1-2 hours.