DAPE cloning with modified primers for producing designated lengths of 3’ single-stranded ends in PCR products

• DOI: 10.1371/journal.pone.0318015
• Publication Date: 2025-02-13T18:44:38Z
• Authors: Seoee Lee; Hyunyoung Kim; Aqsa; Kwangjin Jeung; Minho Won; Hyunju Ro
• Keywords: Cloning (programming); Exonuclease III; In vitro recombination; Sequencing by ligation

For in vitro DNA assembly, enzymes with exonuclease activities have been utilized to generate relatively long recessed ends on fragments, which can anneal other fragments if they complementary nucleotide sequences. The combined construct be directly delivered competent cells, where the gaps and nicks between are completely rectified. We introduce a versatile sequence- ligation-independent cloning (SLIC) method called ’DNA Assembly Phosphorothioate (PT) T5 Exonuclease’ (DAPE), generates precise lengths of 3’ overhangs at both linearized DNA. In contrast conventional SLIC techniques, not suitable for smaller than 50 base pairs (bp) due overzealous activity, such as gRNA epitope tags, DAPE efficiently precisely assemble several single reaction regardless size Thus, DAPE, an advanced toolkit synthetic biology, may further expedite construction more elaborate multi-gene circuitry.