Solid phase ligation of synthetic DNA fragments
Filippov, S.A.; Kalinichenko, S.V.; Esipov, D.S.; Shibanova, E.V.; Shingarova, L.N.; Korobko, V.G.; Dobrynin, V.N.
Bioorganicheskaia Khimiia 16(8): 1045-1051
1990
ISSN/ISBN: 0132-3423 PMID: 2285422 Document Number: 350404
A synthetic oligodeoxyribonucleotide (oligo) covalently bound by an internucleotide linkage to the succinylated Sephacryl S-500 support through 1,9-diaminononane spacer was used as starting compound to assemble the Escherichia coli rec A promoter DNA fragment from synthetic oligos by means of T4 DNA ligase. The solid-phase assembly of the designed DNA was performed by two ways: stepwise ligation of two pairs of oligos (2 diads) or simultaneous ligation of four oligos (tetrad). Both ways gave equal results with some preference in the tetrad case. The reliability of E. coli promoter DNA fragment assembly was demonstrated by cloning it in a plasmid vector and sequencing the cloned DNA by the solid-phase Maxam-Gilbert technique.