Patent ID: 8053188
Filing Date: 2011-11-08
Classification: C12Q

Abstract:
1. A method of enriching a preselected nucleic acid from a mixture of nucleic acids, the preselected nucleic acid encompassing a sequence variant at a given position, the method comprising the steps of: (a) providing a mixture of nucleic acids which includes the preselected nucleic acid to be enriched; (b) cleaving the nucleic acids in the mixture to provide a nucleic acid fragment comprising the preselected nucleic acid, wherein the sequence variant is located 3′ of the 5′ end of the fragment, and denaturing the fragment if the fragment is at least partially double stranded; (c) providing a template oligonucleotide, wherein one end of the template oligonucleotide hybridizes to a downstream sequence beginning at the 3′ end of the fragment and the other end of the template oligonucleotide hybridizes to an upstream sequence of the fragment which is 5′ of the downstream sequence and which is 3′ of the 5′ end of the fragment, and wherein the sequence variant is located in the upstream sequence; (d) hybridizing the template oligonucleotide to said downstream sequence and to said upstream sequence such that said 3′ end of the fragment and the upstream sequence are brought into juxtaposition with each other, optionally with a gap therebetween filled by a complementary oligonucleotide or by extension of the upstream sequence or the downstream sequence, and such that a protruding sequence at the 5′ end of the fragment that is a substrate for a structure specific enzyme is generated; (e) cleaving the substrate with a structure specific enzyme, wherein only the protruding sequence is removed; (f) after cleavage by the structure specific enzyme, joining said 3′ end of the fragment to the 5′ end of the fragment, optionally with the complementary oligonucleotide or extension therebetween, to form a circularized ligated-product containing the preselected nucleic acid, which circularized ligated-product is protected from degradation; and (g) enriching for the ligated product by degrading unligated product and/or amplifying the circularized ligated-product.