The downside of the SYBR Green assay is that the dye binds to any double-stranded DNA sequence. This means that you could also detect fluorescence emitted from non-specific qPCR products, such as primer dimers. To eliminate this risk, check the reaction specificity by performing a melting curve...
c. During each round of PCR, the target and reference sequences are simultaneously amplified by AmpliTaq® Gold DNA Polymerase. This enzyme has a 5′ nuclease activity that cleaves probes that are hybridized to each amplicon sequence. When an oligonucleotide probe ...
However, it often leads to side reactions because of its short length, and it does not reflect the effect of flanking sequences that may interfere with PCR by wrapping onto the template [26]. Such interference can be significant in the initial cycles of the PCR, when the original template ...