Real-time PCR measurement by fluorescence anisotropy

We have developed an instrument for monitoring real-time PCR using fluorescence anisotropy, enabling an assay chemistry in which the fluorescence from a labeled primer elucidates amplification. The instrument holds the sample temperature constant to within [+ or -] 0.03[degrees]C during measurement in the extension phase of each PCR cycle and achieves 0.116 mP FA resolution. Primer conjugation with Alexa-Fluor 488, when compared with other fluorophores, is shown to provide the greatest FA range between primer and product. Comparable reproducibility and linearity of the crossing point for a range of target copy numbers is observed between the FA-based assay run in our instrument and the SYBR green assay run in commercial instrumentation. Reproducibility is also consistent with Poisson-distributed experimental error in aliquoting starting copies, a theoretical limit to instrument/ assay performance.