Site-Directed Insertion of Long Single-Stranded DNA Fragments into Plasmid DNA

Alexander V. Mazin, Murat K. Saparbaev, Ludmila P. Ovchinnikova, Grigory L. Dianov, Rudolf I. Salganik

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

A new site-directed method for inserting long single-stranded DNA fragments into any region of a duplex vector is described. Its major advantage is independence of the location of the restriction sites. The method involves the assembly of single-stranded DNA fragments by ligation to both ends of the inserted fragments of two cohesive flanks that are complementary to the target region. Short oligonucleotide templates are used to direct the ligation. The resulting fragments, designated further as omega fragments with cohesive flanks, are hybridized with a gapped DNA vector. The heteroduplexes are transformed into Escherichia coli cells without enzymatic filling and sealing of gapped DNA. As a consequence of intracellular repair and heteroduplex resolution, insertion mutants are recovered. To demonstrate the method's efficiency, we inserted a 51-nucleotide synthetic DNA fragment containing a modified glucocorticoid receptor binding site into the region of pBR322, near the transcription starting point of the tet gene. The method we developed makes possible site-directed insertion of synthetic and genome-derived DNA fragments at least 200 nucleotides long.

Original languageEnglish (US)
Pages (from-to)63-69
Number of pages7
JournalDNA and Cell Biology
Volume9
Issue number1
DOIs
StatePublished - Jan 1990
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

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