Double-Stranded DNA Binding Dyes
Small molecules that bind to double-stranded DNA can be divided into two classes: intercalators and minor groovebinders.8 Higuchi et al. used the intercalator ethidium bromide for their real-time detection of PCR. Hoeschst 33258is an example of a minor groove binding dye whose fluorescence increases when bound to double-stranded DNA.9Regardless of binding mechanism, there are two requirements for a DNA binding dye for real-time detection of PCR:1) increased fluorescence when bound to double-stranded DNA; 2) no inhibition of PCR. PE Biosystems has developedconditions that permit the use of the SYBR® Green I dye10 in PCR without inhibition and with increased sensitivitycompared to ethidium bromide. The mechanism of SYBR® Green I dye’s interaction with DNA (intercalator vs. groove binding) is not known.
Both the advantage and disadvantage of using a DNA binding dye for real-time detection of PCR are that thedye allows detection of any double-stranded DNA generated during PCR. On the plus side, this means versatilitybecause the same dye can be used to detect any amplified product. Thus, any PCR amplification can be monitoredsimply by including the generic DNA binding dye with the other PCR reagents. On the negative side, both specificand non-specific products generate signal. Thus, any mis-priming events that lead to spurious bands observed onelectrophoretic gels will generate false positive signal when a generic DNA binding dye is used for real-time detection.
Another aspect of using DNA binding dyes is that multiple dyes bind to a single amplified molecule. Thisincreases the sensitivity for detecting amplification products. A consequence of multiple dye binding is that theamount of signal is dependent on the mass of double-stranded DNA produced in the reaction. Thus, if the amplificationefficiencies are the same, amplification of a longer product will generate more signal than a shorter one.This is in contrast to the use of a fluorogenic probe, in which a single fluorophore is released from quenching foreach amplified molecule synthesized, regardless of its length.
Instrumentation
PE Biosystems has two instruments designed to detect fluorescence during the thermal cycling of PCR. Thesimpler system is the GeneAmp® 5700 Sequence Detection System. This complete system consists of an OpticalDetector and a GeneAmp® PCR System 9600, coordinately controlled by software running on a Windows®-basedcomputer. The system has been designed for efficient detection of PCR product accumulation using either SYBR®Green I double stranded DNA binding dye or TaqMan® fluorogenic probes. The 96 reaction tubes are irradiatedwith a white light source and the resulting fluorescence is detected using a CCD array to capture an image of all96 wells. The software collects the images throughout the thermal cycling of PCR and analyzes the data to generatean amplification plot for each reaction. Fluorogenic probes labeled with fluoroscein can be detected on the 5700system, but the instrument does not have the capability of distinguishing two or more fluorophores. Despite thissingle-color detection limitation, however, the 5700 system is still able to use an internal reference dye (ROX) to normalize for non-PCR-related, well-to-well fluctuations in fluorescence. This ability to normalize is achieved byusing fluorescence readings taken at 95 ºC in the baseline region and is essential for reproducible results.
The ABI PRISM® 7700 Sequence Detection System is a more flexible system designed to take full advantage of thebenefits of fluorogenic probe detection. The 7700 system has a built-in thermal cycler and a laser directed via fiberoptic cables to each of the 96 sample wells. The fluorescence emission travels back through the cables to a CCDcamera detector. Because each well is irradiated sequentially, the dimensions of the CCD array can be used forspectral resolution of the fluorescent light. This contrasts with the 5700 system, in which the CCD is used for spatialresolution of the 96 wells. Because the 7700 instrument detects an entire fluorescence spectrum, the system iscapable of distinguishing and quantitating multiple fluorophores in each sample well. The software analyzes thedata by first calculating the contribution of each component dye to the experimental spectrum. Each reporter signalis then divided by the fluorescence of an internal reference dye (ROX) in order to normalize for non-PCR relatedfluorescence fluctuations occurring well-to-well or over time. The use of this internal reference dye, enabled by theability to distinguish fluorophores, increases the precision of the data obtained with the 7700 system. The fluorescenceemissions of SYBR® Green I dye and ROX dye are well resolved, so the benefit of using an internal referencedye is obtained for SYBR® Green I dye detection of PCR on the 7700 system. The other advantage of distinguishingfluorophores is that probes labeled with different reporter dyes can be used so that more than one PCR target can bedetected in a single tube.
