Method for identifying the sequence of one or more variant nucleotides in a nucleic acid molecule
View Patent ↗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.
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.
2. The method of claim 1 , wherein the 5′-end of at least one strand of the double-stranded nucleic acid molecule in step (a) is bound to a solid support.
3. The method of claim 1 , wherein the 3′-ends of the double-stranded nucleic acid molecule in (a) are blocked.
4. The method of claim 1 , wherein the strand with the one or more variant nucleotides is labeled.
5. The method of claim 1 , wherein step (c) is carried out by sequencing-by-synthesis from the predetermined linker sequence.
6. The method of claim 1 , wherein step (c) is carried out by
(i) denaturing the double-stranded portion of the linker;
(ii) hybridizing a complementary primer to the predetermined linker sequence in the presence of an intercalating fluorescence resonance energy transfer (FRET) donor;
(iii) contacting the product of step (ii) with DNA Polymerase and at least one ddNTP labeled with a FRET acceptor to extend the complementary primer; and
(iv) detecting FRET, wherein the presence of FRET is indicative of the sequence of the variant nucleotide.
7. The method of claim 1 , wherein the double-stranded linker is detectably labeled.
8. The method of claim 1 , wherein the double-stranded nucleic acid molecule of step (a) is produced by a polymerase chain reaction.
9. The method of claim 8 , wherein the polymerase chain reaction is carried out with a universal primer.
10. The method of claim 9 , wherein the 5′-end of the universal primer is bound to a solid support.
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.
12. The kit of claim 11 , further comprising
a primer complementary to the predetermined sequence of the linker;
an intercalating FRET donor; and
ddCTP, ddATP, ddTTP, and ddGTP each labeled with a different FRET acceptor.