Figure 6

Description of the CLPA protocol. CLPA uses chemical ligation chemistry to generate target specific reaction products which contain a 5′-biotin group to allow for easy capture and purification of the ligated product, universal primer sites to allow for multiplex amplification by PCR that is specific for only the ligated product, and identifier sequences that allow for easy readout using existing instrumentation. Two probes (S and L) are designed for each target RNA species. The S-probe contains a 5′ biotin moiety, a universal upstream primer sequence (black), a target hybridizing domain (red), and a target identifier domain (blue) to enable separation for subsequent measurement. The L-probe contains a 5′-leaving group (L), a hybridizing domain (red), and a sequence complementary to a universal downstream primer (black). Upon hybridization to adjacent sequences in the target RNA, the nucleophilic group on the S-probe reacts with the L-probe to displace the leaving group and ligate the two oligonucleotides. The ligation products from many probe pairs are produced in proportion to the concentrations of their corresponding targets, and are then isolated by capture on streptavidin-coated magnetic particles, and amplified by PCR. The PCR products are then analyzed on a capillary electrophoresis system.