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Real-Time Quantification of Genomic DNA Using DyNAzyme II DNA Polymerase and SYBR Green I Dye

uction of the fluorescence signal. Addition of DMSO will also lower the Tm for both the primers and the amplicon. Incremental addition of DMSO is recommended.

Selection of Read Temperatures Enhances Specificity
Melting curve analysis indicates this protocol can be used for analysis of amplified template without complications from side reactions, such as primer-dimer formation. To avoid primer-dimer interference with C(t) value determination, the temperature at which the fluorescence was read during each cycle was adjusted to 78C, a temperature above the melting point of the primer-dimers. At this temperature, the double stranded primer-dimers should be denatured, releasing bound SGI and diminishing their contribution to the fluorescence signal. At this same temperature, the longer PCR product remains annealed, and is directly correlated to the fluorescence detected. Typically, the read temperature is set three degrees below the Tm of the amplicon. Melting temperatures for both the primer-dimers and the PCR product are determined by analyzing melting curve data.

The results discussed here highlight DyNAzyme II as a robust polymerase that can be used with SYBR Green I dye in real-time PCR applications. The protocol listed here can be used to provide accurate, reproducible quantitation data for both lambda and human genomic DNA templates over a broad concentration range. The discussion of read temperatures, as well as the guidelines for DMSO use and denaturing and annealing temperatures, provides the user with some basic tools for optimizing real-time fluorescence detection of amplification reactions using SYBR Green I.


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