One-pot making of sequence-restricted DNA dumbbells

Ya-Hui Chang; Yu-Cheng Lin; Hong-Hsing Liu

doi:10.1016/j.heliyon.2023.e16035

One-pot making of sequence-restricted DNA dumbbells

Heliyon. 2023 May 9;9(5):e16035. doi: 10.1016/j.heliyon.2023.e16035. eCollection 2023 May.

Authors

Ya-Hui Chang¹, Yu-Cheng Lin^{2

3}, Hong-Hsing Liu^{1

4}

Affiliations

¹ Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Miaoli County, 35053, Taiwan.
² Pediatrics, Far Eastern Memorial Hospital, Pan-Chiao District, New Taipei City, 220216, Taiwan.
³ Electronic Engineering, Oriental Institute of Technology, Pan-Chiao District, New Taipei City, 220216, Taiwan.
⁴ Pediatrics, En Chu Kong Hospital, Sanxia District, New Taipei City, 237414, Taiwan.

Abstract

A method to build sequence-restricted DNA dumbbells was developed. 5'-exonuclease converts end sequences of 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 at one temperature. 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 at a genomic scale to build a ∼0.45 Mbp giant dumbbell 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.

Keywords: Dumbbell DNA; Genome enrichment; Next-generation sequencing.