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One-pot making of sequence-restricted DNA dumbbells.

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.

SUBMITTER: Chang YH

PROVIDER: S-EPMC10196794 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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One-pot making of sequence-restricted DNA dumbbells.

Chang Ya-Hui YH Lin Yu-Cheng YC Liu Hong-Hsing HH

Heliyon 20230509 5

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 Illu ...[more]

PMID: 37215900

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