Figure 1
Schematic overview of the proposed assay. (A) OpenArray™ architecture. The OpenArray™ has 48 subarrays and each subarray contains 64 microscopic through-holes of 33 nl volume. The primers are pre-loaded into the holes. The sample combined with the reaction mix is auto-loaded due to the surface tension, provided by the hydrophilic coating of the holes and the hydrophobic surface of the array. (B) PRI-lock probe design. T1a and T1b indicate target complementary regions. Unique primer sites ensure specific amplification (forward: F1 and reverse: R1) and each PRI-lock contains a universal sequence (US) and a desthiobiotin moiety (dBio). (C) Multiple target specific PRI-lock probes are ligated on fragmented DNA samples. T1a and T1b bind to adjacent sequences of the target and in case of a perfect match, the probe is circularized by a ligase. The probes are captured via the desthiobiotin moiety using magnetic streptavidin-coated beads. The PRI-lock probes are washed and quantitatively eluted from the beads. Unreacted probes are removed by exonuclease treatment. (D) Circularized probes are loaded and independently amplified on the Biotrove OpenArray™ platform using PRI-lock probe specific primers. The amplification is monitored using SYBR-Green and the ligated PRI-lock probes are quantified based on the threshold cycle number (CT).