( Increasing the [dNTP] will re...)Real-Time PCR: General Considerations, Rev A

Increasing the [dNTP] will require an increase in [MgCl₂]
Source and concentration (1.254.5 U/50 l reaction) of Taq DNA polymerase
Primer concentration (100500 nM)
An asymmetric primer concentration may be helpful
Fluorescent probe or intercalation dye concentration

Conditions
Optimization of the following amplification conditions will be required to obtain the maximum efficiency and specificity:
Annealing temperature (50 to 65C) and time (dependent on primer T_m and chemistry)
Extension time (dependent on chemistry and product length)
Denaturation temperature and time (dependent on target sequence)
2-step v. 3-step PCR

Experimental Design and Interpretation of Results
Primer Selection
This section demonstrates the importance of primer optimization using the human cyclophilin 40 gene. Two sets of primers, differing in location, were designed to amplify the same region of the human cyclophilin 40 gene (IMAGE Consortium clone 71154, ATCC). Figure 1 illustrates the location of the primer sets (primer sets A and B use the same forward primer).

Five replicates for a 10x dilution series (10⁷ to 10³ copies) using identical primer concentrations (300 nM/reaction) were performed on the iCycler iQ system. The reaction mixture consisted of custom-made Life Technologies Supermix (Platinum Taq polymerase, 1.25 U, 20 mM Tris, pH 8.4, 3 mM MgCl₂, 0.2 mM of each dNTP, 50 mM KCl). Real-time amplification was detected using the intercalating dye SYBR* Green I (Molecular Probes; 1:75,000 dilution of the 10,000x stock solution).

Optimizing primer location to reduce template seco
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