Solid-phase assembly of DNA duplexes from synthetic oligonucleotides.

A new method of rapid and efficient assembly of extended DNA duplexes in solid phase was developed. Subassemblies of separately annealed oligonucleotides were stepwise hybridized to each other on a solid support. Two types of supports with anchor oligonucleotide were tested: Fractosil-1000 with oligo-dT sequence and Sephacryl S-500 with an oligonucleotide bound via CNBr-activation procedure. Sephacryl S-500 turned out to be the support of choice since all enzymatic reactions of the assembly procedure (phosphorylation, ligation, restriction enzyme digestion) could be efficiently performed with DNA immobilized on Sephacryl S-500 particles.