Method for synthesizing DNA sequentially

Abstract

Abstract A method and a DNA linker are described for synthesizing relatively long double-stranded deoxyribonucleic acid sequences of defined composition. Short complementary single strand segments of oligonucleotides comprising part of the full sequence desired are synthesized using known procedures. Overlapping single strand segments are annealed forming double-stranded fragments which are inserted in cloning vectors and cloned in an appropriate host, both purifying the DNA fragments and amplifying the amount thereof. An adjacent fragment is then similarly synthesized in quantity and such fragments are inserted adjacent to the first synthetic introduced fragments in the cloning vectors, followed by cloning in an appropriate host. The procedure continues until the entire desired sequence has been formed, at which time it may be excised or cloned directly in the vectors upon which it was made. The described DNA linker contains a restriction enzyme recognition sequence for a restriction enzyme which cuts offset from the recognition sequence and exactly at the end of the linker, enabling synthesized DNA to be inserted at the end of the linker without disturbing the recognition sequence.

Inventors

• 👤 Stanley N. Cohen

Bibliographic Details