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US 2003/0154034 A1Published application
Methods for detecting polymorphisms in nucleic acids
AssigneePanomics, Inc.
InventorQinghong Yang
Life of this patent
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Abstract
The present invention provides methods for determining the genotype of a nucleic acid at the site of a polymorphism. The methods achieve sensitivities great enough to detect the presence of any difference between the nucleic acids, even single nucleotide polymorphisms. In the methods, the nucleic acid is compared to a reference nucleic acid having a known genotype. The nucleic acids can be of any length, even less than 100 base pairs. In methods, one or more extra mismatches are introduced into the nucleic acids at or near the site of the polymorphism. The nucleic acids are contacted under conditions in which they are capable of forming a stable four-way complex that can be detected to indicate that the nucleic acids differ in genotype.
Claims26
A method for determining the genotype at the site of a polymorphism in a target polynucleotide sequence comprising:
- a. contacting a first partial duplex comprising a target polynucleotide sequence with a second partial duplex comprising a mutated reference polynucleotide sequence under conditions in which said first and said second partial duplexes form a four-way complex, wherein said mutated reference polynucleotide is identical to a reference polynucleotide everywhere except at the site of a mutation that is not at the site of the polymorphism, and wherein said reference polynucleotide sequence corresponds to said target polynucleotide sequence and has a known genotype at the site of the polymorphism;
- b. subjecting said four-way complex to branch migration conditions, wherein branch migration in said four-way complex is impeded at or near the site of the polymorphism if the target polynucleotide sequence has a genotype at the site of the polymorphism that differs from that of the reference polynucleotide sequence, thereby forming a stabilized four-way complex, and wherein branch migration in the four-way complex is capable of continuing until complete strand exchange occurs if the target polynucleotide sequence and the reference polynucleotide sequence share the same genotype at the site of the polymorphism, thereby resulting in the resolution of said four-way complex into two duplex nucleic acids;
- and
- c. detecting said stabilized four-way complex as an indication that said target polynucleotide sequence has a genotype that is different from the known genotype of said reference polynucleotide sequence at said site of the polymorphism, or detecting resolution of said four-way complex as an indication that said target polynucleotide sequence has a genotype that is the same as the known genotype of said reference polynucleotide sequence at said site of the polymorphism.
1 more element
A method for determining the genotype at the site of a polymorphism in a target polynucleotide sequence comprising:
- a. Contacting a first partial duplex comprising a mutated target polynucleotide sequence with a second partial duplex comprising a reference polynucleotide sequence, wherein said mutated target polynucleotide sequence is identical to a target polynucleotide sequence everywhere except at the site of a mutation that is not at the site of the polymorphism, and wherein said reference polynucleotide sequence corresponds to said target polynucleotide sequence and has a known genotype at the site of the polymorphism;
- b. Subjecting said four-way complex to branch migration conditions, wherein branch migration in said four-way complex is impeded at or near the site of the polymorphism if the target polynucleotide sequence has a genotype at the site of the polymorphism that differs from that of the reference polynucleotide sequence, thereby forming a stabilized four-way complex, and wherein branch migration in the four-way complex is capable of continuing until complete strand exchange occurs if the target polynucleotide sequence and the reference polynucleotide sequence share the same genotype at the site of the polymorphism, thereby resulting in the resolution of said four-way complex into two duplex nucleic acids;
- and
- c. detecting said stabilized four-way complex as an indication that said target polynucleotide sequence has a genotype that is different from the known genotype of said reference polynucleotide sequence at said site of the polymorphism, or detecting resolution of said four-way complex as an indication that said target polynucleotide sequence has a genotype that is the same as the known genotype of said reference polynucleotide sequence at said site of the polymorphism.
1 more element
A method for determining the genotype at the site of a polymorphism in a target polynucleotide sequence comprising:
- a. contacting a first partial duplex comprising a mutated target polynucleotide sequence with a second partial duplex comprising a mutated reference polynucleotide sequence having a known genotype at the site of the polymorphism under conditions in which said first and said second partial duplexes form a four-way complex, wherein said mutated target polynucleotide sequence is identical to a target polynucleotide sequence everywhere except at the site of a mutation that is not at the site of the polymorphism, wherein said mutated reference polynucleotide is identical to a reference polynucleotide everywhere except at the site of a mutation that is not at the site of the polymorphism, and wherein said reference polynucleotide sequence corresponds to said target polynucleotide sequence and has a known genotype at the site of the polymorphism;
- b. subjecting said four-way complex to branch migration conditions, wherein branch migration in said four-way complex is impeded at or near the site of the polymorphism if the target polynucleotide sequence has a genotype at the site of the polymorphism that differs from that of the reference polynucleotide sequence, thereby forming a stabilized four-way complex, and wherein branch migration in the four-way complex is capable of continuing until complete strand exchange occurs if the target polynucleotide sequence and the reference polynucleotide sequence share the same genotype at the site of the polymorphism, thereby resulting in the resolution of said four-way complex into two duplex nucleic acids;
- and
- c. detecting said stabilized four-way complex as an indication that said target polynucleotide sequence has a genotype that is different from the known genotype of said reference polynucleotide sequence at said site of the polymorphism, or detecting resolution of said four-way complex as an indication that said target polynucleotide sequence has a genotype that is the same as the known genotype of said reference polynucleotide sequence at said site of the polymorphism.
1 more element
A method for determining the genotype at the site of a polymorphism in a target polynucleotide sequence comprising:
- a. contacting a first partial duplex comprising a target polynucleotide sequence with a second partial duplex comprising a reference polynucleotide sequence having a known genotype at the site of the polymorphism under conditions in which said first and said second partial duplexes form a four-way complex, and wherein said first and said second partial duplex each comprise a nucleic acid modification;
- b. subjecting said four-way complex to branch migration conditions, wherein branch migration in said four-way complex is impeded at or near the site of the polymorphism if the target polynucleotide sequence has a genotype at the site of the polymorphism that differs from that of the reference polynucleotide sequence, thereby forming a stabilized four-way complex, and wherein branch migration in the four-way complex is capable of continuing until complete strand exchange occurs if the target polynucleotide sequence and the reference polynucleotide sequence share the same genotype at the site of the polymorphism, thereby resulting in the resolution of said four-way complex into two duplex nucleic acids;
- and
- c. detecting said stabilized four-way complex as an indication that said target polynucleotide sequence has a genotype that is different from the known genotype of said reference polynucleotide sequence at said site of the polymorphism, or detecting resolution of said four-way complex as an indication that said target polynucleotide sequence has a genotype that is the same as the known genotype of said reference polynucleotide sequence at said site of the polymorphism.
1 more element
Dependencies are inferred from the claim text.