NEBUILDER® HIFI DNA ASSEMBLY® : Bridging dsDNA with a ssDNA Oligo

Learn how NEBuilder® HiFi DNA Assembly bridges dsDNA with a ssDNA oligo.


Bridge two double-stranded fragments with a synthetic single-stranded DNA oligo, for simple and fast construction. The bridge is designed as a single-stranded 60-basepair oligo that contains 25 to 30 basepairs of homology with an end of each DNA fragment. The bridge can be customized to include additional sequences, if desired.

During assembly, the T5 exonuclease activity chews back the 5-prime end of the DNA fragment, generating a 3-prime single-stranded overhang, which allows the oligo to anneal with it. The high-fidelity DNA polymerase activity fills the gap, and DNA ligase seals the nicks. The T5 exonuclease activity then cleaves again to allow it to anneal with the overhang on the other DNA fragment. The gaps are once again filled, and nicks are sealed.

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