Great Basin’s isothermal amplification method—helicase-dependent amplification (HDA)—is an innovative, proprietary approach that simplifies and speeds up the DNA amplification process. Our method utilizes helicase (a key component of in vivo DNA replication) instead of heat—as performed in PCR—to unwind double-stranded DNA. The single-stranded DNA can then be bound by DNA primers and amplified with DNA polymerase (as in PCR).
To prevent the formation of primer artifacts during amplification (a limitation inherent in competing isothermal amplification systems), Great Basin utilizes a novel Hot Start approach—RMA (ribonuclease-mediated amplification). In the RMA approach, we utilize DNA primers that contain a single RNA base linkage near a blocked 3’-end. By blocking the 3’-end, no amplification may occur. At elevated temperatures (>50C) a thermostable version of the enzyme RNase H2 is activated. If the blocked primer is hybridized to a target DNA sequence, then RNase H2 will cut the primer at the RNA base, removing the block and permitting DNA amplification. Great Basin’s RN2 approach allows us to drive the amplification system to permit detection of 10 molecules of DNA from a clinical specimen in less than 25 minutes.
Further, incorporation of RMA permits us to amplify multiple target sequences simultaneously or multiplex. Competing isothermal amplification methods are unable to multiplex due to the formation of primer artifact, which are created at low temperatures by DNA polymerase.