METHODS FOR GENERATING LIBRARIES WITH CO-VARYING REGIONS OF POLYNULEOTIDES FOR GENOME MODIFICATION

摘要

The invention provides methods for introducing co-varying paired nucleic acids into a vector such under separate transcriptional control. An oligonucleotide is synthesized including the paired nucleic acids. The oligonucleotide is then assembled with a spacer nucleic acid encoding a promoter. After assembly the spacer nucleic acid is to one side of the oligonucleotide encoding the paired segments. However, on circularization of the assembled nucleic acid and cleavage between the DNA segments the spacer oligonucleotide and its components now occur between the paired segments. The resulting nucleic acid can now be cloned into a vector with a single step, such that each of the paired nucleic acid segments is linked to its own promoter. The present method can readily be extended to library screening without proportionately increasing the effort.

主权项

1. A method of inserting a pair of nucleic acid segments into a vector, comprising
(a) synthesizing an oligonucleotide comprising the paired segments; (b) performing an assembly reaction between the oligonucleotide and a spacer oligonucleotide to form a nucleic acid in which the spacer oligonucleotide is to one side of the oligonucleotide comprising the paired segments; (c) circularizing the nucleic acid; (d) cleaving the circularized nucleic acid at a site that generates a linear nucleic acid in which the spacer oligonucleotide is flanked by the paired segments; and (e) inserting the linear nucleic acid into a vector.