Engineering DNA Capture Probes for Massively Multiplexed Cloning of Kilobase-Sized Genome Regions. Escherichia coli K-12

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.