The QuantiFast SYBR® Green PCR Kit reduces total PCR run time by up to 60% in real-time two-step RT-PCR on standard cyclers (40 cycles without melting curve analysis; comparison with standard QIAGEN real-time PCR kits). L: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500; A3: ABI PRISM 7000.
The QuantiFast SYBR® Green PCR Kit was used to detect the Y-chromosome-specific single-copy gene SRY in genomic DNA from a male donor using the Mastercycler ep realplex. [A] Curves for 1000 down to 1.5 copies can be clearly distinguished from each other. [B] A plot of copy number (log) versus CT value demonstrates high linearity.
Expression of MYC (a proto-oncogene) in human leukocytes was analyzed by real-time two-step RT-PCR on the Applied Biosystems 7500 Fast System. Duplicate reactions were run using 10-fold cDNA dilutions (10 ng to 10 pg). [A] Fast cycling mode with the QuantiFast Kit gave similar CT values to [B] standard cycling mode with the QuantiTect Kit. In contrast, a kit for standard cycling from Supplier AII gave worse CT values not only in [C] fast cycling mode, but also in [D] recommended standard cycling mode.
Expression of MYC (a proto-oncogene) in human leukocytes was analyzed using the iCycler iQ and [A] the QuantiFast SYBR® Green PCR Kit or [B] a kit from Supplier Bv. Duplicate reactions were run using 10-fold cDNA dilutions (100 ng to 0.1 ng). The instrument-dedicated kit from Supplier BV was used according to the fast cycling protocol. The QuantiFast Kit gave lower CT values, indicating greater sensitivity.
A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers and probes to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH4+ destabilizes weak hydrogen bonds between mismatched bases.
[A] Q-Bond in QuantiFast Buffer increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.