3′ Branch Ligation: A Novel Method to Ligate Non-Complementary DNA to Recessed or Internal 3′ OH Ends in DNA or RNA
Brock A. Peters,
Posted 29 Jun 2018
bioRxiv DOI: 10.1101/357863 (published DOI: 10.1093/dnares/dsy037)
Posted 29 Jun 2018
Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various molecular tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to join 5′ phosphorylated blunt-end double-stranded DNA to DNA breaks at 3′ recessive ends, gaps, or nicks to form a 3′ branch structure. Therefore, this base pairing-independent ligation is termed 3′ branch ligation (3′BL). In an extensive study of optimal ligation conditions, similar to blunt-end ligation, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency. A low level of nucleotide preference was observed at the junction sites using different synthetic DNAs. Furthermore, we discovered that T4 DNA ligase efficiently ligated DNA to the 3′ recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, whereas RNA ligases are less efficient in this reaction. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important applications. We performed a proof-of-concept study of a new directional tagmentation protocol for next generation sequencing (NGS) library construction that eliminates inverted adapters and allows sample barcode insertion adjacent to genomic DNA. 3′BL after single transposon tagmentation can theoretically achieve 100% usable template, and our empirical data demonstrate that the new approach produced higher yield compared with traditional double transposon or Y transposon tagmentation. We further explore the potential use of 3′BL for preparing targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.
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