Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications

Xiaofeng Zheng, Philip C. Bevilacqua

Research output: Contribution to journalArticlepeer-review


We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson- Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert (~20 bp) containing the modification or non- Watson-Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields (~60%overall yield) and ease of use. Thismethod has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of nonsequence- specific nucleic acid-protein interactions.

Original languageEnglish (US)
Article numbere6
JournalNucleic acids research
Issue number2
StatePublished - Jan 1 2021

All Science Journal Classification (ASJC) codes

  • Genetics


Dive into the research topics of 'Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications'. Together they form a unique fingerprint.

Cite this