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Description:
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 an enzyme called a helicase to separate DNA, rather than heat. This allows DNA amplification without the need for thermocycling. The tHDA reaction can also be coupled with reverse transcription for RNA analysis.
IsoAmp® II Universal tHDA kit 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. A single copy of target DNA can be amplified by tHDA and detected by agarose gel electrophoresis when optimized primers and buffer are used.
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. 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.
Reommended storage conditions for the tHDA kit are -20°C for storage shorter than 6 months and -80°C for storage greater than 6 months. Please avoid repeated freeze-thaw cycles as this may decrease performance of the kit.
Positive Control Assays:
The Control Template and Control Primers provided in this kit can be used as a positive control in tHDA (Fig. 2A) and qHDA (Fig. 2B) reactions. This control reaction amplifies a 85 bp target sequence. The expected melting temperature (Tm) of the amplicon in the melt-curve analysis is 79°C ± 1°C. To set up a positive control, follow the one-step or two-step tHDA or qHDA protocols using 1 μl of Control Template (Plasmid: 1 ng/μl), 0.75 μl of Control Forward Primer (5 μM) and 0.75 μl of Control Reverse Primer (5 μM).
General Recommendations:
As tHDA, RT-HDA, qHDA and qRT-HDA are exponential amplification reactions, extra attention should be paid to avoid sample cross-over and carryover contamination. Reaction assembly, amplification, and gel electrophoresis-based detection steps should be carried out in physically separated locations. Always wear gloves. Open the reaction tubes only when adding reagents into them during reaction setup and keep them closed at any other time. Never open the tubes after qHDA or qRT-HDA reactions are done.
Performance of tHDA, RT-HDA, qHDA and qRT-HDA is extremely sensitive to changes in the magnesium and salt concentrations of the reaction. Avoid introducing any substances that may affect these concentrations.
Every freshly thawed reagent should be gently vortexed and spun down before being added to the reaction. Autoclaved Milli-Q water should be used for tHDA and qHDA reactions. DEPC-treated water should be used for RT-HDA and qRT-HDA reactions. Use sterilized filter tips only and change tips after each pipetting step.
Figure 1. HDA Technology.
Figure 2. Positive control assays with varying amount of Control Template. A, two-step tHDA positive control with 10 pg - 1 ng Control Template. B, two-step qHDA positive control using 500 fg - 5 ng, in triplicate.
Kit Components:
Annealing Buffer II (10X)
Control Forward Primer (5 μM)
Control Reverse Primer (5 μM)
Control Template (Plasmid: 1 ng/�l)
IsoAmp® dNTP Solution (50 reactions)
IsoAmp® Enzyme Mix (50 reactions)
MgSO4 (100 mM)
NaCl (500 mM)
Storage Conditions
Storage Temperature:
-20°C
FAQs
Protocols
Protocols for IsoAmp® II Universal tHDA Kit
Legal
Research Use Assurance:
Developed by BioHelix Corporation, a New England Biolabs, Inc. - affiliated company.
Licenses/Patents/Disclaimers:
This product is covered by pending U.S. and foreign patents. The purchase price of this product includes a limited, non-transferable license for research use only under the patents owned by BioHelix. No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of the product. The Product is not to be used for diagnostic purposes nor is it intended for human use. This product may not be resold, modified for resale, or used to manufacture commercial products without written approval of BioHelix Corp.