Patent ID: 7781190
Filing Date: 2010-08-24
Classification: C12N

Abstract:
1. A method of red mediated bacterial artificial chromosomal construction of DNA sequences in excess of about 20 kb, comprising the steps of: a.) providing a first DNA sequence or segment in excess of about 20 kB; b.) adding a first selectable marker cassette flanked by a recombination sequence on a first end of the first DNA segment or sequence; c.) adding an Sp6 element containing a NotI cutting site at the first end to form a first insert; d.) providing a linearized bacterial artificial chromosome retrieval vector that has an origin of replication and a second selectable marker different from the first insert, and that has homologous arms longer than about 200 base pairs that correspond to the first insert on either end thereof; e.) inserting the first insert and retrieval vector into a DY380 cell under conditions that enable the DY380 cell to effect homologous recombination between inserted DNA molecules; f.) selecting the recombined insert-vector molecules using first and second selectable markers; g.) linearizing the complex of recombined molecules using the NotI site in Sp6 fragment; h.) identifying and isolating a second DNA segment for combination with the first DNA segment that has an overlapping homology region of at least 200 base pairs with the first DNA segment on one end; i.) cloning the second DNA segment into a second retrieval vector that has a selectable marker different from both the cassette used in the first DNA insert and the selectable marker in the first retrieval vector, and an Sp6 fragment with a NotI cutting site; j.) inserting the linearized complex from step (g) with the second DNA segment-vector complex into a DY380 cell, and allowing the DY380 cell to effect a homologous recombination event between the two inserted molecules; l.) selecting the recombined insert-vector molecules utilizing the selectable marker from the first insert and the selectable marker from the second retrieval vector; and m.) transferring the isolated DNA from step (j) into a cell line that mediates the site-specific recombination mechanism indicated by flanking sites selected in step (b), to remove the selection cassette from the first insert, thereby forming a vector-insert construct containing a contiguous segment of DNA equal to or larger than about 40 kb.