Two-Step qHDA (Real-time quantitative tHDA)


The following protocol is intended for real-time detection using the Applied Biosystems 7300 Real-Time PCR System. 

The kit also can be coupled with real-time detection methods to conduct realtime quantitative tHDA (qHDA) and RT-HDA (qRT-HDA) to monitor amplification as it progresses. For optimal performance, use EvaGreen as a reporter dye and ROX as a passive reference dye. Sequence-specific probes can also be designed for qHDA experiments.


  1. To set up a 50 μl qHDA reaction, prepare a 25 μl Mix A in a 0.2 ml MicroAmp optical tube (ABI) and a 25 μl Mix B in a 0.5-ml micro centrifuge tube in a sterile hood or a PCR Workstation.
    H2O X μl
    10X Annealing buffer II 2.5 μl
    DNA template X μl
    Forward Primer (5 μM)* 0.75 μl
    Reverse Primer (5 μM)* 0.75 μl
    Total volume of Mix A 25 μl

    H2O 8 μl
    10X Annealing buffer II 2.5 μl
    MgSO4 (100 mM)* 2 μl
    NaCl (500 mM)* 4 μl
    IsoAmp® dNTP Solution 3.5 μl
    IsoAmp® Enzyme Mix 3.5 μl
    EvaGreen (20X, Biotium) 0.5 μl
    ROX Reference Dye (50X, Invitrogen) 1 μl
    Total volume of Mix B 25 μl
  2. Gently mix each of the mixes by brief vortexing or by pipetting followed by brief centrifugation. Overlay the reaction mixture with 50 μl mineral oil. Place the tubes on ice.
  3. Incubate Mix A at 95ºC for 2 minutes and place promptly on ice. Add 25 μl of Mix B into Mix A underneath the oil layer and gently mix the reaction by pipetting. Place the tubes on ice.
  4. Real-time detection is carried out on a 7300 Real-Time PCR System (ABI) with the following well inspector setting: reporter dye: SYBR; quencher: none; passive reference dye: ROX. Use the following program:
    Stage 1: (60 X)
      Step1: 66ºC for 0:05
      Step2: 65ºC for 1:55
      Data collection and real-time analysis enabled
    Stage 2: (1 X)
      Dissociation Stage (default settings of the machine)
      Melt curve data collection and analysis enabled

    * The condition of tHDA reactions can be further optimized by titering thefollowing components:
    Components Recommended concentration Recommended concentration for titering
    MgSO4 3.5 to 4 mM 3 to 4.5 mM
    NaCl 30 to 40 mM 20 to 50 mM
    Primer 75 to 100 nM 50 to 200 nM
    ProtoScript II RT (NEB M0368S) 1 to 2 units 0.5 to 10.5 units