Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule
6 Assignments
0 Petitions
Accused Products
Abstract
The invention relates to methods for identifying the sequence of one or more variant nucleotides in nucleic acid molecules. The method involves cleaving a double-stranded nucleic acid molecule containing a mismatch with a mismatch-specific endonuclease which cleaves on the 3′ side of the mismatch, and preserving the integrity of the variant nucleotide by ligating a Double-Stranded Linker with a degenerate 3′-overhang to said variant nucleotide. Because the variant nucleotide is immediately adjacent to the linker, PCR and/or sequence-by-synthesis analysis can be readily carried out.
-
Citations
30 Claims
-
1-12. -12. (canceled)
-
13. 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. - View Dependent Claims (14, 24)
-
-
15-17. -17. (canceled)
-
18. A method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule comprising
(a) contacting a double-stranded nucleic acid molecule with a mismatch-specific endonuclease, wherein one strand of said double-stranded nucleic acid molecule has one or more variant nucleotides which create at least one mismatch in the double-stranded nucleic acid molecule so that the double-stranded nucleic acid molecule is cleaved at the 3′ - -side of the mismatch by the mismatch-specific endonuclease, wherein the double-stranded nucleic acid molecule is double-stranded genomic DNA produced by;
(i) fragmenting one or more samples of genomic DNA; (ii) 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; (iii) blunt ending, dephosphorylating 3′
ends, and phosphorylating 5′
ends of the double-stranded genomic DNA molecules;(iv) 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;(v) size fractionating the double-stranded genomic DNA molecules of step (iv) to remove the Double-Stranded Adaptor; (vi) 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; and (vii) removing double-stranded genomic DNA molecules which are not immobilized by the first solid support; (b) ligating a Double-Stranded Linker, containing a 3′
-overhang, to the 3′
-end of the strand with the one or more variant nucleotides, wherein the 3′
-overhang of the linker is degenerate and the double-stranded portion of the linker is of a predetermined sequence; and(c) determining the sequence of the one or more variant nucleotides via the predetermined linker sequence thereby identifying the sequence of the one or more variant nucleotides. - View Dependent Claims (19, 20, 21, 22, 23, 25, 26, 27, 28)
- -side of the mismatch by the mismatch-specific endonuclease, wherein the double-stranded nucleic acid molecule is double-stranded genomic DNA produced by;
-
29. A kit for identifying the sequence of one or more variants in a nucleic acid molecule comprising
a mismatch-specific endonuclease that cleaves a double stranded nucleic acid molecule at the 3′ - -side of a mismatch;
a Double-Stranded Linker, containing a 3′
-overhang, wherein the 3′
-overhang of the linker is degenerate and the double-stranded portion of the linker is of a predetermined sequence; anda Double-Stranded Adaptor containing a restriction enzyme cut site and a functional group at one 5′
end. - View Dependent Claims (30)
- -side of a mismatch;
Specification