SYBR Green® is the DNA binding dye most commonly used in real-time PCR applications. Chapters 1, 7, 8, and 9 all contain reviews and comparisons of this technology to others available and again, the reader is directed to these chapters for detailed information.
DNA binding dyes are molecules which bind non-specifically to double-stranded DNA molecules. This includes specific products, misprimed nonspecific products and primer-dimer molecules. Due to this non-specific binding, amplicon selection and primer design are of utmost importance with this technology. Self-self primer binding, dimer formation and loop structures can all introduce non-specific signal. DNA binding dye technology allows the investigator to view the number of products contributing to the observed fluorescent signal. This is achieved by the gradual increase of the temperature, post-amplification, causing the amplicon to dissociate to become single-stranded. As this occurs, the DNA binding dye is released and the fluorescence signal decreases. Based on the DNA sequence of the amplicon there is a specific temperature at which it will be completely dissociated. However, as different regions of the amplified product will dissociate at different temperatures before the whole product is single-stranded, this means there will be a graded decrease in fluorescence. Software packages translate this data into a 'melting curve', represented as a single peak per product in the negative first-derivative curve of the actual melting curve. As informative as this is, when first setting up a method the melting curve data should not be relied on alone for determining whether you have a single product or not. The nature of the melting curve means that the product peaks are broad. It is very possible that this could mask the presence of secondary products not that dissimilar in length or thermal profile. The degree to which this problem presents itself is dependent on the melting curve parameters. All primer pairs should be checked for specificity by separating the amplification products through a high percentage agarose gel.
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