Patent ID: 7579155

Claim:
A method for producing a library of genomic DNA molecules containing genetic variations comprising (a) fragmenting one or more samples of genomic DNA; (b) denaturing and annealing the fragmented genomic DNA to generate double-stranded genomic DNA molecules, wherein one strand of said double-stranded genomic DNA molecules has one or more variant nucleotides which create at least one mismatch in the double-stranded genomic DNA molecules; (c) blunt ending, dephosphorylating 3′ ends, and phosphorylating 5′ ends of the double-stranded genomic DNA molecules; (d) ligating a Double-Stranded Adaptor onto ends of the double-stranded genomic DNA molecules, wherein said Double-Stranded Adaptor contains a restriction enzyme cut site and a functional group at one 5′ end; (e) size fractionating the double-stranded genomic DNA molecules of step (d) to remove the Double-Stranded Adaptor; (f) immobilizing the size fractionated, double-stranded genomic DNA molecules via binding of the functional group of the Double-Stranded Adaptor to reactive groups of a first solid support; (g) removing double-stranded genomic DNA molecules which are not immobilized by the first solid support; (h) contacting the immobilized double-stranded genomic DNA molecules with a mismatch-specific endonuclease so that the double-stranded genomic DNA molecules are cleaved at the 3′-side of the mismatches therein; (i) ligating a Double-Stranded Linker to the 3′-end of the immobilized genomic DNA molecules, wherein one strand of the Double-Stranded Linker contains a nick, a functional group at the 5′ end and a degenerate sequence at the 3′ end, wherein the degenerate sequence is a 3′-overhang, and the double-stranded portion of the linker is of a predetermined sequence; (j) repairing nicked DNA of the product of step (i); (k) immobilizing the Double-Stranded Linker ligated to the immobilized double-stranded genomic DNA molecules via binding of the functional group of the Double-Stranded Linker to reactive groups of a second solid support; and (l) contacting the product of step (k) with a restriction enzyme which cleaves the Double-Stranded Adaptor at the restriction enzyme cut site thereby releasing the double-stranded genomic DNA molecules from the first solid support thereby creating a library of genomic DNA molecules containing genetic variations.