SYBR Green chemistry

SYBR® Green is a fluorescent dye that binds only to double-stranded DNA. Fluorescence is emitted proportionally to the amount of double-stranded DNA. In a PCR reaction, the input DNA or cDNA is minimal and, therefore, the only double stranded DNA present in sufficient amounts to be detected is the PCR product itself. As for other real-time PCR chemistries, read-outs are given as the number of PCR cycles ('cycle threshold' Ct) necessary to achieve a given level of fluorescence. During the initial PCR cycles, the fluorescence signal emitted by SYBR® Green I bound to the PCR products is usually too weak to register above background. During the exponential phase of the PCR, the fluorescence doubles at each cycle. A precise fluorescence doubling at each cycle is an important indicator of a well optimized assay. After 30 to 35 cycles, the intensity of the fluorescent signal usually begins to plateau, indicating that the PCR has reached saturation. As Ct correlates to the initial amount of target in a sample, the relative concentration of one target with respect to another is reflected in the difference in cycle number (ACt = Ctsample - Ctreference) necessary to achieve the same level of fluorescence.

There are now many commercially available SYBR® Green chemistry kits (SYBR® Green core reagents master mix by ABI; QuantiTech SYBR® Green

PCR master mix by Qiagen; DNA master SYBR® Green by Roche; IQ™ SYBR® Green supermix by Biorad; Superarray by BioScience Corp.; DyNAsin™ II DNA polymerase with SYBR® Green by MJ Research and probably others). There is no clear indication for choosing one over the others, however, once an assay has been optimized with one chemistry, changing kit requires re-optimization. Prices differ and may guide towards one kit or another depending on amount of usage. To minimize variability, assays using master mixes may be recommended, although multi-user facilities may prefer individual reagents. It is also possible to use homemade SYBR® Green master mixes to reduce cost even further (Karsai et al., 2002).

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