Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule
First Claim
1. 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;
(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.
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Abstract
The invention relates to methods for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule. 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 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.
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Citations
12 Claims
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1. 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;
(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 (2, 3, 4, 5, 6, 7, 8, 9, 10, 12)
- -side of the mismatch by the mismatch-specific endonuclease;
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11. 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; and
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.
- -side of a mismatch; and
Specification