Engineering DNA Capture Probes for Massively Multiplexed Cloning of Kilobase-Sized Genome Regions

The ability to isolate or enrich specific genomic loci for downstream analyses has transformed ourunderstanding of molecular and cellular biology1. Molecular inversion probes (MIPs) are short single stranded DNA molecules (~150 bp) that become circularized by gap filling after annealing to target sequences that flank a desired DNA fragment. MIPs have proven to be a useful tool for target capture, since they exhibit high specificity and can be massively multiplexed2. However, traditional MIPs are ineffective at capturing large target sequences (>400 bp) because of constraints on physical bending of a short MIP-target DNA complex. Herein, we describe a new long adapter single strand oligonucleotide (LASSO) probe that can made in library formats and used to capture and clone thousands of kilobase-sized DNA fragments in a single reaction. As a proof-of-principle, we simultaneously cloned >3000 bacterial open reading frames from genomic DNA (spanning 400-5,000 bp sized targets) in just 2 hours. This technology will enable long-read sequencing library preparation and massively parallel cloning.