Methods and apparatus for detecting nucleic acid polymorphisms
First Claim
1. A method of detecting an allele at a polymorphic site in a nucleic acid molecule, the method comprising:
- locating a sample containing a nucleic acid molecule at an examination site;
contacting the sample with a primer and at least one photoluminescent dideoxy terminator, where the primer binds specifically to a nucleic acid sequence adjacent to a polymorphic site in the nucleic acid molecule;
modifying the primer so that the polarization of light emitted by the terminator in response to illumination with polarized light is larger upon incorporation of the terminator into the modified primer than upon incorporation of the terminator into an unmodified primer;
illuminating the sample with polarized light capable of stimulating photoluminescence emission from the terminator;
detecting polarized light emitted from the sample sequence; and
determining the identity of an allele at the polymorphic site based on the extent of polarization of the light transmitted from the sample.
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Abstract
Methods and apparatus for detecting polynucleotide hybridization in luminescence-based assays. The methods may include (1) allele-specific hybridization, using luminescence detection, (2) allele-specific oligonucleotide ligation, using dye-labeled oligonucleotide ligation with luminescence resonance energy transfer (FRET) detection, and (3) allele-specific nucleotide incorporation, using primer extension with luminescence polarization (FP) detection. More specifically, the methods may include (1) locating a sample containing a nucleic acid material at an examination site, (2) illuminating the sample, (3) detecting light transmitted from the sample, and (4) determining the presence, absence, and/or identity of a nucleic acid target in the sample using the light transmitted from the sample.
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Citations
52 Claims
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1. A method of detecting an allele at a polymorphic site in a nucleic acid molecule, the method comprising:
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locating a sample containing a nucleic acid molecule at an examination site;
contacting the sample with a primer and at least one photoluminescent dideoxy terminator, where the primer binds specifically to a nucleic acid sequence adjacent to a polymorphic site in the nucleic acid molecule;
modifying the primer so that the polarization of light emitted by the terminator in response to illumination with polarized light is larger upon incorporation of the terminator into the modified primer than upon incorporation of the terminator into an unmodified primer;
illuminating the sample with polarized light capable of stimulating photoluminescence emission from the terminator;
detecting polarized light emitted from the sample sequence; and
determining the identity of an allele at the polymorphic site based on the extent of polarization of the light transmitted from the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method of detecting an allele at a polymorphic site in a nucleic acid molecule, the method comprising:
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locating a sample containing a nucleic acid molecule at an examination site;
contacting the sample with a primer and first and second photoluminescent dideoxy terminators, where the primer binds specifically to a nucleic acid sequence adjacent the polymorphic site, and where the first and second terminators have different photoluminescence lifetimes;
illuminating the sample with light capable of simultaneously stimulating photoluminescence emission from the first and second terminators;
detecting light emitted from the sample;
converting the detected light to a signal;
processing the signal based on the difference in lifetimes to discriminate between a first portion of the signal attributable to light emitted by the first terminator and a second portion of the signal attributable to light emitted by the second terminator; and
determining the identity of the allele at the polymorphic site based on a difference in the first and second portions. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A method of detecting an allele at a polymorphic site in a nucleic acid sample comprising
packaging in stabilized form in a microplate well at least one reagent for performing a single base extension assay, where the reagent is selected from the group consisting of DNA polymerase, at least one ddNTP, and an extension primer capable of hybridizing to a known sequence in the nucleic acid sample adjacent the polymorphic site, hydrating the reagent in the microplate well, adding to the microplate well any additional reagents that are required to perform a single base extension assay, contacting the hydrated reagent with a nucleic acid sample, extending a primer that is hybridized to the nucleic acid sample by one terminating base corresponding to the polymorphic site, and determining the allele at the polymorphic site and the nucleic acid sample based on the identity of the terminating base.
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45. A method of detecting an allele at a polymorphic site in a nucleic acid molecule, the method comprising:
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locating a sample containing a nucleic acid molecule at an examination site;
contacting the sample with a primer and a photoluminescent dideoxy terminator, where the primer binds specifically to a nucleic acid sequence adjacent a polymorphic site in the nucleic acid molecule;
directing excitation light from a surface toward the sample, where the excitation light is capable of stimulating photoluminescence emission from the terminator, and where the surface conveys substantially more than half of the incident excitation light;
directing emission light from the surface toward a detector, where the surface conveys substantially more than half of the incident emission light; and
determining the identity of the allele at the polymorphic site based on the emission light conveyed to the detector. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52)
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Specification