Patent ID: 6475731
Filing Date: 2002-11-05
Classification: C12N,C12Q

Abstract:
A method of obtaining sequence information from two portions of a nucleic acid molecule separated by at least 10 kb, the method comprising the steps of:a) isolating a large nucleic acid molecule, wherein said large nucleic acid molecule is at least 20 kb in length; b) ligating said large nucleic acid molecule into a plasmid vector molecule to anchor the ends of the large nucleic acid molecule to the plasmid vector molecule, thereby forming a closed circular plasmid containing the large nucleic acid molecule; c) cutting said closed circular plasmid with a restriction endonuclease, wherein said restriction endonuclease cuts said large nucleic acid molecule in at least two sites separated by at least 10 kb but does not cut said plasmid vector molecule, thereby forming a pair of cut ends separated by an internal deletion of at least 10 kb; d) ligating said cut ends to form a closed circular plasmid containing a shortened nucleic acid molecule, wherein the shortened nucleic acid molecule is at least 10 kb shorter than the large nucleic acid molecule; e) transforming a host cell with said closed circular plasmid containing a shortened nucleic acid molecule to propagate said shortened nucleic acid molecule; and f) determining the sequence of the shortened nucleic acid molecule, wherein said steps (b) and (c) are performed consecutively without transforming a host cell with the product of step (b).