Claims
- 1. A method of determining the identity of a polymorphic nucleotide, said method comprising:
contacting under hybridizing conditions, a target nucleic acid comprising a polymorphic site, and a solid substrate comprising one or more bound locus-specific primer pairs; amplifying said target nucleic acid with said locus-specific primer pair, wherein said amplifying results in an amplification product bound to the solid support at each end; contacting said amplification product with a labeled probe comprising at least one detecting nucleotide that will specifically base pair with said polymorphic nucleotide, in the presence of an enzyme that catalyzes the formation of a covalent bond between said detecting nucleotide and said amplification product; and detecting said label; wherein the identity of the label on said detecting nucleotide indicates the complement of the polymorphic nucleotide.
- 2. The method according to claim 1, wherein said enzyme is DNA polymerase.
- 3. The method of claim 2, wherein said amplification product is denatured and contacted with an extension primer that hybridizes to a site immediately adjacent to said polymorphic nucleotide, prior to contacting with said labeled probe, wherein DNA polymerase extends from said extension primer to covalently attach a labeled probe to its 3′ end.
- 4. The method of claim 2, wherein said amplification product is cleaved with an endonuclease to generate a free end; and
cleaving with a distance-cleaving endonuclease, resulting in a cleavage product having an overhang strand and a recessed strand comprising a 3′ terminus, wherein the polymorphic nucleotide is on the single-stranded overhang of the cleavage product, wherein said recessed strand provides an extension primer for said DNA polymerase.
- 5. The method of claim 2, wherein said amplification product is contacted with a plurality of labeled probes selected from the group consisting of at least two differentially labeled dideoxynucleotides.
- 6. The method of claim 3, wherein a plurality of mutually distinguishable extension primers are used.
- 7. The method of claim 1, wherein said enzyme is ligase.
- 8. The method of claim 7, wherein said amplification product is cleaved with an endonuclease to generate a free end; and
cleaving said amplification product with a distance-cleaving endonuclease, resulting in a cleavage product having a single-stranded overhang strand and a recessed strand, wherein the recessed strand has a 3′ terminus, wherein the polymorphic nucleotide is on the single-stranded overhang of the cleavage product, wherein said recessed strand; contacting with ligase and at least one nucleotide complementary to said polymorphic nucleotide under conditions that permit covalent linkage.
- 9. The method of claim 8, wherein said amplification product is contacted with a plurality of differentially labeled oligonucleotide probes selected from the group consisting of all possible sequences of said single-stranded overhang.
- 10. The method of claim 10, wherein at least two different labels are used.
- 11. The method of claim 8, wherein the polymorphic nucleotide is the first nucleotide on the single-stranded overhang of the cleavage product.
- 12. The method of claim 1, wherein said amplification product comprising said detecting nucleotide is released from said substrate for detection.
- 13. The method of claim 1, wherein said amplification product comprising said detecting nucleotide is detected in situ.
- 14. The method according to claim 1, wherein said solid substrate comprises a capture primer.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to, and the benefit of, U.S. Provisional Application Serial No. 60/289,606, filed May 7, 2001, which status is pending and the entirety of which is incorporated herein by this reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60289606 |
May 2001 |
US |