One-Pot Making of Sequence-Restricted DNA Dumbbells

A highly efficient and simple method to build DNA dumbbells was developed. 5'-exonuclease as used in Gibson Assembly converts end sequences of linear DNA targets into sticky ends. Self-looping oligonucleotides with complementary 3'-overhangs are ligated to form dumbbells by DNA polymerase and ligase in a sequence-restricted manner. These reactions proceed in one pot and depend on end nucleotides of input DNA. We demonstrated one use of this method to 'tunnel' sequencing libraries into dumbbells for the Pacific Biosciences (PacBio) platform. Readouts of an Illumina P5/P7-ended 16S library from a standard microbial community confirmed successful tunneling. Twelve fecal samples additionally showed significant correlations between standard and tunneled 16S sequence variants on the PacBio platform. We further extended the method to build a genome-scale dumbbell of nearly half million base pairs in the Human Leukocyte Antigen II region on chromosome 6. Sequences inside the dumbbells were successfully protected from a cocktail of exonucleases. Roughly 11-fold enrichment was achieved for the dumbbell-guarded region compared to the vicinity.