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

Mimi Amutan and David Batey, Ph.D.


Abstract
Real-time quantification was performed for lambda and human genomic DNA on the DNA Engine Opticon system (MJ Research). DyNAzyme II DNA polymerase* was used for amplification, and SYBR Green I fluorescent dye was used for detection. The sensitivity and linear dynamic range of the assay was tested for ranges of 100 to 1x108 initial copies of lambda DNA and 75 to 7.5x104 initial copies of human DNA. Precision of the assay was measured with lambda template and standard deviations of 0.3 cycles or less were obtained for quadruplicate samples. This assay has been shown to be both reliable and precise for the quantification of genomic DNA templates.


Introduction
Quantification of DNA and RNA from biological samples can be effectively accomplished using real-time amplification methods1. The predominant chemistries are based on either the binding of fluorescent dyes to the double stranded structure of DNA or the hybridization of specific probes, such as TaqMan probes2 and molecular beacons3. The simplicity and directness of using binding dyes such as SYBRGreen I (SGI) make this a desirable alternative for many applications4.

SGI is a dsDNA binding dye that has proven exceptionally useful for assays requiring sensitive nucleic acid detection. The fluorescence of SGI is enhanced approximately 1000-fold upon binding dsDNA5, making it ideal for detection of amplification products. Because SGI binds to all dsDNA, it does not have to be customized for individual templates. Detection formats utilizing hybridization probes require careful design of template-specific primer sets. SYBR Gre
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