Figure 3.

A sample RT–PCR using MultiPriDe-developed primers. (A) Demonstrates an amplification curve for these primers, which is a plot of fluorescence intensity on the ordinate axis and the number of cycles on the abscissa axis. The increase in fluorescence is the result of free SYBR Green binding to double-stranded DNA (initially at a very low concentration), which increases roughly 2-fold per amplification cycle until primers and other reagents begin to run out. The fluorescence reaches a maximum at high cycle numbers. A smooth exponential increase in the initial stages of the PCR suggests the amplification of only one product, consistent with the primers being specific. (B) Shows a representative dissociation curve for this reaction, plotting change in fluorescence against change in temperature. As double-stranded DNA product is melted with increasing temperature, it dissociates into two single strands and releases complexed SYBR green. The release of SYBR green results in diminished fluorescence. The central sharp peak seen indicates the homogenous melting behavior of the double-stranded DNA produced by the qRT–PCR, strongly suggesting the production of a single DNA species.