Quantitative PCR by Continuous Fluorescence Monitoring of a Double Strand DNA Specific Binding Dye

Abstract

Introduction While there are many methods available for quantification of nucleic acids, quantitative PCR is becoming the method of choice (1). The high sensitivity and the wide dynamic range of the technique are advantages that outweigh many of the difficulties of the procedure. While extremely useful, quantitative PCR can be laborious to perform. Most of the difficulties arise because only a very small number of the cycles in a PCR reaction contain useful information. The early cycles have undetectable amounts of the DNA product, late cycles (the so-called plateau phase) are almost as uninformative. The quantitative information in a PCR reaction comes from those few cycles where the amount of DNA grows logarithmically from barely above background to the plateau. Often only 4 or 5 cycles out of 40 will fall in this “log-linear” portion of the curve. The position of these precious few cycles contains most of the quantitative information. The log-linear phase appears at higher cycle numbers as the number of template copies in the reaction decreases. How are these cycles identified? In traditional quantitative PCR, the sample is divided into aliquots and placed in multiple reaction tubes. One tube is removed for analysis after each cycle. The reactions are run out on a gel and the amount of DNA at each cycle is quantified, sometimes by staining with a double strand specific DNA binding dye such as ethidium bromide or SYBR Green I Dye. The brightness of the fluorescent band is measured and the fluorescence is plotted against cycle number. The cycle number where the fluorescence of the sample is first detected (the “threshold”) is compared to the threshold of samples of known concentration (2). Quantification with the LightCyclerTM greatly simplifies the process (3). The samples are continuously monitored during the PCR, so a single reaction takes the place of many reactions. The log-linear region is easily identified as the fluorescence data appears on the computer screen. The simplest way to monitor the progress of the reaction in the LightCycler is to include a dsDNA binding dye in the reaction (4). Ethidium bromide is the most common example of this kind of dye, but newer dyes like SYBR Green I Dye give stronger signals. This dye is thought to bind in the minor groove of dsDNA. In its unbound state SYBR Green I Dye has relatively low fluorescence, but when bound to DNA it fluoresces brightly. As the amount of DNA in the PCR increases, the amount of fluorescence from the dye increases proportionally. SYBR Green I Dye has the virtue of being easy to use. Because it has no sequence specificity it can be used to detect any PCR product. All you need is a primer pair and a template and you can do quantitative PCR.However, this virtue has a drawback. SYBR Green I Dye binds to any dsDNA molecule, whether it is the intended product or a non-specific product like a primer dimer. This non-specific binding of SYBR Green I Dye will particularly make the quantification of low copy numbers more difficult. ey Words LightCyclerTM, PCR, SYBR Green I Dye, quantification