Strand displacement amplification (SDA) relies on a strand-displacing DNA polymerase, typically Bst DNA polymerase, Large Fragment (NEB #M0275) or Klenow Fragment (3'→5' exo-), to initiate replication at nicks created by a strand-limited restriction endonuclease or nicking enzyme at a site contained in a primer. The nicking site is regenerated with each polymerase displacement step, resulting in exponential amplification. SDA is typically used in clinical diagnostics.
Helicase-dependent amplification (HDN) employs the double-stranded DNA unwinding activity of a helicase to separate strands, enabling primer annealing and extension by a strand–displacing DNA polymerase. Like PCR, this system requires only two primers. HDA has been employed in several diagnostic devices and FDA-approved tests.
Nicking enzyme amplification reaction (NEAR) employs a strand-displacing DNA polymerase initiating replication at a nick created by a nicking enzyme, rapidly producing many short nucleic acids from the target sequence. This process is extremely rapid and sensitive, enabling the detection of small target amounts in minutes. NEAR is already being used commonly used for pathogen detection in clinical and biosafety applications.
Thermophilic Helicase-Dependent Amplification (tHDA) is a novel method for isothermal amplification of nucleic acids. Like PCR, the tHDA reaction selectively amplifies a target sequence defined by two primers. However, unlike PCR, tHDA uses a helicase enzyme, rather than heat, to separate dsDNA. This allows DNA amplification without the need for thermocycling. The tHDA reaction can also be coupled with reverse transcription for RNA analysis.
The IsoAmp® II Universal tHDA Kit (NEB #H0110) can be used to amplify and detect short DNA sequences (70 bp - 120 bp) at a constant temperature. The kit can be used with a variety of templates, including microbial genomic DNA, viral DNA, plasmid DNA, and cDNA. When optimized primers and buffer are used, even a single copy of target DNA can be amplified by tHDA and detected by agarose gel electrophoresis.
The IsoAmp II Universal tHDA Kit is based on a second-generation thermophilic Helicase-Dependent Amplification platform. The reactions supported by IsoAmp II Universal tHDA Kit include tHDA, reverse transcription HDA (RT-HDA), real-time quantitative HDA (qHDA) and real-time quantitative RT-HDA (qRT-HDA), from a single reaction buffer. The IsoAmp II Universal tHDA Kit contains an enzyme mix, dNTP solution and buffer, allowing flexibility in reaction setup. A control template and a set of specific primers are supplied for the positive control reaction.
Recommended storage conditions for the tHDA Kit are -20°C for storage shorter than 6 months and -80°C for storage greater than 6 months. Repeated freeze-thaw cycles should be avoided as this may decrease the performance of the kit.
IsoAmp® is a registered trademark of New England Biolabs, Inc.