Figure 1

A schematic of the asMIP methodology. (A) The asMIP design. Unreacted probes terminate with 3'- and 5'-interrogation sequences (no. 1) abutted by two DraI cleavage sites (no. 2). Each probe contains two unique sequence tags (no. 3) and two common primer binding sites (no. 4). (B) The asMIP assay. Step 1, sample preparation: immobilized cDNA was reverse transcribed (arrow) from polyadenylated RNA (curved lines followed by AAAAAA) using oligo-dT primers (TTTTT), which were covalently attached to magnetic beads (gray circles). Following cDNA synthesis, RNA is digested and washed away. Step 2, asMIP hybridization: unreacted asMIP probes (flattened nicked circles) terminate with 3'- and 5'-interrogation sequences (gray and colored lines), which are homologous to the exon sequences that flank splice junctions on the cDNA. Probes quantitatively anneal to the appropriate exon-exon junction (colored probes). Probes that do not hybridize (gray probes) are washed away. Step 3, asMIP circularization: bound asMIPs are ligated into circles (small arrows). Step 4, asMIP amplification and quantitation: only the successfully ligated probes (contiguous circles) can be exponentially amplified using the common PCR primers (thin black lines). Each probe contains two unique sequence tags, which are amplified by PCR for multiplexed detection via array hybridization or high-throughput sequencing (bar graph).