Schematic illustration of PLA analysis with DTM readout.

<p><b>a</b>) The PLA probes, each carrying unique DNA sequences (A1, B1, …, N1, or A2, B2, …, N2) that code for their target antigens, are incubated with the samples to be examined. <b>b</b>) The oligonucleotides on pairs of PLA probes that have bound their targets in close proximity are ligated to give rise to reporter DNA strands. In the ligation step a sample barcode is introduced in the ligation product to allow for dual color comparisons of results for two samples in the same array spot. <b>c</b>) The ligation products are amplified by PCR and treated with DNA modifying enzymes to generate single stranded reporter molecules with barcodes identifying the targeted proteins at both ends. <b>d</b>) The reporter molecules are hybridized to oligonucleotides complementary to pairs of protein tags (e.g. N1N2), on a microarray, thereby allowing the reporter strands to be ligated into circles<b>. e</b>) The circularized reporter molecules finally template RCA, primed by the oligonucleotides on the array, and the RCA products are detected by hybridization with Cy3 or Cy5 labeled detection oligonucleotides. <b>f</b>) The Cy3 and Cy5 intensities are measured, and the ratios between the two colors are analyzed for each feature to detect differences in interaction patterns and protein abundances between the two samples.