One-Step qHDA (Real-time quantitative tHDA)

Overview

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.

Protocol

1. Set up a 50 μl reaction in a MicroAmp optical tube (Applied BioSystems) in a sterile hood or a PCR Workstation.
   

   H2O	X μl
   10X Annealing buffer II	5 μl
   MgSO4 (100 mM)*	2 μl
   NaCl (500 mM)*	4 μl
   IsoAmp® dNTP Solution	3.5 μl
   DNA template	X μl
   Forward Primer (5 μM)*	0.75 μl
   Reverse Primer (5 μM)*	0.75 μl
   IsoAmp® Enzyme Mix	3.5 μl
   EvaGreen (20X, Biotium)	0.5 μl
   ROX Reference Dye (50X, Invitrogen)	1 μl
   Total volume	50 μl

2. Mix the reaction by brief vortexing or by pipetting followed by brief centrifugation. Overlay the reaction mixture with 50 μl min eral oil. Place the tubes on ice.
3. 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