We compared Stratagenes Brilliant Plus quantitative PCR core reagent kit with another commercially available core reagent kit to amplify and detect a 294-bp region in exon 3 of the human b-actin gene using a TaqMan probe (Figure 1). Three concentrations (100, 1, and 0.01 ng) of human genomic DNA were amplified (Methods), and both tests included dUTP and UNG (Methods), identical primers and linear hydrolysis probe, and a passive reference dye. Our data show that for each concentration of target DNA, the threshold cycle (Ct; the cycle at which fluorescence is first detected above background) was comparable or better for the Brilliant Plus kit. For 100, 1, or 0.01 ng of human genomic DNA, the Ct values generated with Brilliant Plus reagents were 18.0, 25.6, or 33.0, respectively; the corresponding Ct values for reactions with the other commercially available kit were 18.3, 26.0, or 34.3 (Figure 1).
To demonstrate the sensitivity and absolute quantification capabilities
of Stratagenes Brilliant Plus QPCR core reagent kit, we used molecular
beacon chemistry to amplify and detect a 148-bp fragment from the gene
encoding the mouse muscle nicotinic acetylcholine receptor g-subunit.
In this experiment, the passive reference dye was not included. We achieved
a detection level of 101 to 108 copies of plasmid
DNA (Figure 2) with our standard protocol (Methods). When the Ct was plotted
versus the input amount of target, the resulting standard curve was linear
over eight orders of magnitude, with an R2 value of 0.998<