A short report on optimization of nucleic acid probes by DNA microarray synthesis and next generation sequencing

Initial single 2fold probe was designed at the Landegren lab in Uppsala University, and the following experiments were performed at the Broad Institute at the Mikkelsen lab. 12000 2fold probes were synthesized by a B3 Custom Array DNA synthesizer in their reverse complementary oligonuleotide-order. After stripped off from microarrays, synthesized products were pooled, emulsion-PCR amplified, lambda exonuclease and restriction enzymes treated and resulted in single stranded 2fold probes – briefly, the 9 nucleotides positions next to an nicking enzyme binding site at the stem structure within a probe were computationally designed to be variable thus a set of 4000 probe sequences, namely MlyI-Nb.BtsI in parallel with two matched control-sets: MlyI-Nb.BbvCI and MlyI-NoNick. A solid phase (magnetic beads) assay protocol was utilized and end-result products were subsequently analyzed in a HiSeq2000 sequencer whereas variable sequences were read and served as tags to generate a count file.