	US 7,579,155 B2
	Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule
Paul D. Taylor, Gilroy, Calif. (US); Reyes Candau, Olney, Md. (US); and Gary F. Gerard, Frederick, Md. (US)
Assigned to Transgenomic, Inc., Omaha, Nebr. (US)
Filed on Apr. 11, 2008, as Appl. No. 12/101,255.
Application 12/101255 is a continuation in part of application No. 11/854181, filed on Sep. 12, 2007.
Prior Publication US 2009/0068659 A1, Mar. 12, 2009
Int. Cl. C12Q 1/68 (2006.01); C12P 19/34 (2006.01); C07H 21/00 (2006.01); C07H 21/02 (2006.01); C07H 21/04 (2006.01)

U.S. Cl. 435—6 [435/91.2; 536/22.1; 536/23.1; 536/24.3]

15 Claims

1. 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;

(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.