FRET probes are SNP specific <i>in vitro</i> at 37°C.

Abstract

<p>Multicomponent plots indicating fluorescent signals from the anchor (blue line) and the allele specific probe (red line) as a course of temperature increase (37–75°C). <b>A</b> – Anchor/<b>A, T</b> allele specific probe or O – no probe/<b>A, U</b> – target RNA oligo complement to A and T probes respectively. Perfect match between the probe and target results in an energy transfer between the donor anchor and acceptor probe. When both the donor anchor and acceptor probe are bound to target at 37°C, their proximity causes energy transfer resulting in red fluorescence. As a course of temperature increase, the oligos denaturate from target (51°C), energy transfer ceases and only the donor fluorescence can be detected, causing a typical “8” shaped FRET plot to be generated (<b>A, B</b>). A single mismatch in target sequence results in low affinity of the oligos therefore no FRET response is detected (<b>C, D</b>). No target control (<b>E</b>), no donor control (<b>F</b>), no acceptor control (<b>G</b>).</p