Patent ID: 7247428
Filing Date: 2007-07-24
Classification: C12Q

Abstract:
1. A method of identifying at least two nucleic acid alterations in a single target nucleic acid, which comprises: (a) preparing the target nucleic acid and a control nucleic acid; (b) hybridizing the target nucleic acid with the control nucleic acid to create a heteroduplex, wherein the control nucleic acid is the wild type nucleic acid corresponding to the target nucleic acid, and wherein a mismatch is formed between the control and the target nucleic acid at each location where a difference is present; (c) removing any 5′ phosphate groups or 3′ hydroxyl groups from the heteroduplex nucleic acid ends by circularizing said heteroduplex nucleic acid and selectively degrading any non-circularized heteroduplex nucleic acids; (d) cleaving said circularized heteroduplex nucleic acid at the site of any mismatch using any agent that generates a single or double strand break at the site of the mismatch, thereby creating free 5′P or 3′OH ends at the site of said any mismatch; (e) ligating a linker at the 5′ phosphate group at any of the newly generated 5′ phosphate groups, or ligating a linker at any of the newly generated 3′ hydroxyl groups; (f) selectively amplifying the cleaved, heteroduplex nucleic acid fragments which have a linker ligated thereto using a polymerase chain reaction; and (g) determining if at least two different nucleic acid fragments are present to identify the at least two nucleic acid alterations present in the target nucleic acid.