"World's first" RPA kits promise more accessible DNA sequencing

JULY 17, 2009--TwistDx (Cambridge, England), developer of point-of-use nucleic acid diagnostics, announced today the launch of its TwistAmp kits, which it calls "the world's first DNA amplification products based on recombinase polymerase amplification (RPA)." Concurrently, TwistDx launched Twista, an "easy-to-use," portable, real-time fluorescence-based RPA reader.

RPA promises to make genetic sequencing faster and more accessible. Each of these first four TwistAmp kits promises to detect DNA molecules in a mixed sample within 10-15 minutes, less than half the time of most polymerase chain reaction PCR systems. RPA operates at low, constant temperatures and does not require initial melting of sample DNA, opening the way to broader access to end-users without reliance on expensive thermocyclers. RPA has also been shown to be highly resistant to crude samples compared to PCR, suggesting applications in on-the-spot field testing requiring no nucleic acid extraction.

The Twista fluorometer is a customized platform containing proprietary fluorescent probe systems developed by the company to accurately monitor RPA reactions. Although seamlessly integrated with the Twista device, TwistAmp kits can be used in combination with various, widely used assay platforms, such as conventional analysis tools (e.g. on agarose gels), dipsticks, as well as broadly available fluorescence plate readers and real-time PCR machines.

"Our TwistAmp kit offers researchers a flexible and cost-effective solution to achieve DNA amplification in significantly less time. With laboratory analysis a critical but often time-consuming aspect of biomedical research, we believe these new research tools provide our customers with a viable alternative to PCR, which although very effective is costly to access and time consuming," said Dr. Niall Armes, CEO of TwistDx. "In particular, real-time PCR devices are very expensive; a laboratory could purchase multiple Twista devices for the same price. Field researchers may also be able to easily perform their measurements in a mobile setting as TwistAmp enzyme and nucleotide mixes are provided in a freeze-dried form that demonstrates good stability at ambient temperatures. The Twista device will be available with portable battery pack and standalone software."

RPA is a proprietary technique, employing recombinase enzymes, used to amplify nucleic acids in any biological sample, with high analytical sensitivity and specificity.

TwistDx's initial product range includes:

TwistAmp Basic
The TwistAmp Basic kit contains all enzymes and reagents necessary for the amplification of DNA. Primers and the template must be supplied by the user. Amplification success is typically assessed by an end-point method, such as gel electrophoresis. Amplified material can also be purified and used for down-stream applications (e.g. subcloning).

TwistAmp exo
The TwistAmp exo kit is recommended for users who want to combine the TwistDx amplification technology with the company's proprietary fluorescent exo GapProbe in a homogenous format. In addition to the basic components, it contains a powerful nuclease (Exonuclease III) which processes exo GapProbe during the amplification reaction and generates a real-time read-out. The presence of Exonuclease III reduces the final overall yield of amplified material at endpoint and so is not suitable for analysis on gels, however it is the preferred system for generating strong fluorescence signal kinetics in the RPA system.

TwistAmp nfo
The TwistAmp nfo kit is designed for users who want to detect the success of their amplification reaction by means of end-point 'sandwich-assays', such as lateral-flow (LF) strips. Besides the basic amplification reagents, it includes a nuclease (nfo) that can generate new polymerase extension substrates within suitable LF GapProbe. The amplified material can then be used in instrument–free detection formats. The TwistAmp nfo kit can also be used for fluorescence monitoring using fluorescent exo GapProbe as an alternative to the TwistAmp exo kit as nfo processes these probes (often with slightly slower kinetics) and will in addition permit product to be analysed by gels at endpoint.

TwistAmp fpg
The TwistAmp fpg kit is tailored for users who want to combine the TwistDx amplification technology with an alternative TwistDx reporter probe system, fluorescent fpg GapProbe, in a homogenous format. These probes have less design constraints than exo GapProbe, although kinetics of fluorescence accumulation may be slower. In addition to the basic components it contains a powerful nuclease (fpg) that processes fpg GapProbe during the amplification reaction itself and generate a real-time read-out. The presence of fpg will not reduce the final overall yield of amplified material, in contrast to the use of Exonuclease III in the TwistAmp exo kit, allowing end point gel analysis, but the kinetics of probe cutting can be slower than that with the exo system.

TwistAmp RT
The TwistAmp RT kit will contain all enzymes and reagents necessary for the amplification of RNA – all that has to be supplied by the user are the primers and the template.

Twista portable real-time fluorometer
The Twista is a customized device developed for monitoring RPA reactions containing TwistDx's fluorescent probe systems, exo GapProbe, fpg GapProbe, or intercalating dyes should they be employed. It contains a heated incubation chamber for a strip of 8 tubes perfectly designed for the RPA reagents supplied in the TwistAmp kits, and can monitor fluorescence in real-time in two colour channels with exquisite sensitivity. The Twista has a small footprint and can be rendered entirely portable with the addition of a rechargeable battery pack. Data can be analysed by PC-link, or alternatively the device can function as a standalone unit capable of storing and running user-defined methods to call samples positive and negative for any characterised assay.

Learn more about TwistDx at the company's website. Also, find out more about RPA at the Public Library of Science (PLoS) site.

Posted by Barbara G. Goode, [email protected], for BioOptics World.