Figure 1

Steps involved in a standard MIP Assay (Steps A – E). H1 & H2 – Two segments homologous to the target genome, P1 & P2 – Two universal primers common for all MIPs, R – Cleavage site. A) A Molecular inversion probe constructed using PathogenMIPer software, for each of the organism to be detected in the MIP assay. An oligo of user specified length is extracted from the target genome, and cut into two halves, reversed and tailored with the primer and other tags. B) Oligo hybridization with the samples. Done enzymatically – a mixture of the genomic DNA, MIP probes specific for all the targets, a thermostable polymerase and ligase, is heat denatured and brought to annealing temperature. i) Two sequences targeting each terminus of the probe hybridize to complementary sites in the genome creating a circular conformation with a single nucleotide gap between the termini of the probe. All the MIP probes are designed to have the same nucleotide at this position. ii) Unlabeled dATP/dGTP/dCTP/dTTP (any one selected by the user while designing the probe) is added to the reaction and the polymerase adds the nucleotide to the gap, and then iii) the ligase closes the gap to form a circular molecule that encircles the genomic strand, to which it's hybridized. C) Exonucleases are added to remove the excess unreacted linear probes and any linear genomic DNA. The reactions are then heated to inactivate the exonucleases. D) The probes are released from the genomic DNA by reacting with uracil-N-glycosylase. E) PCR reagents are added along with the common PCR primer pair. The reactions are subjected to thermal cycling, with the result that only circularized probes which bound to the specific target, are amplified. The probes are detected using a tag microarray or pyrosequencing.