De novo polynucleotide synthesis using rolling templates

摘要

Disclosed is a method for synthesizing polynucleotide molecules such as genes or gene segments. A primer having 5' and 3' ends is incubated with a relatively shorter template having a 5' region non-complementary to the primer, a 3' region complementary to the 3' end of the primer, and a non-reactive 3' terminus to allow the 3' region of the template to anneal to the primer. The annealed product is reacted with at least one nucleotide in the presence of a template-dependent polynucleotide polymerase to produce a primer extended at its 3' end by at least one nucleotide complementary to the 5' region of the template. The extended primer is then dissociated from the template. The extended primer is further extended by repeating this cycle for sufficient cycles, wherein the templates and enzymes may differ from cycle to cycle, to obtain the object polynucleotide. Also disclosed are template libraries and kits containing said libraries for use in conjunction with the polynucleotide synthesis method.