Methods and systems for identifying nucleotides by primer extension
First Claim
1. A method of identifying a nucleotide in at least a first position in a polynucleotide sequence, comprising:
- providing a polynucleotide target sequence;
hybridizing the target sequence with a first oligonucleotide probe, wherein;
the probe comprises a first subsequence of nucleotides, a first 3′
-terminal nucleotide, and a first florescent label coupled to the 3′
-terminal nucleotide;
the subsequence is complementary to a portion of the target sequence that is immediately adjacent to the first position; and
the 3′
-terminal nucleotide is complementary to one possible nucleotide in the first position;
contacting the hybridized probe and target sequence with polymerase extension reagents in a first extension reaction mixture;
measuring a level of polarized fluorescence emitted from the first extension reaction mixture, a decrease in polarized fluorescence indicating the presence of polymerase extension of the probe, the presence of polymerase extension of the probe indicating that the 3′
-terminal nucleotide is complementary to the nucleotide in the first position; and
identifying the nucleotide in the first position.
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Abstract
The methods and systems for identifying nucleotides monitor changes in fluorescent signal that occur during the polymerase-mediated extension of dye labeled primer molecules. In certain aspects, the methods monitor changes in a fluorescent signal, e.g., relative polarization, emitted from a reaction mixture that contains a dye labeled primer that hybridizes to a target sequence such that the primer ends at the position of interest. Perfect hybridization at the position results in extension of the primer while imperfect hybridization, e.g., a mismatch at the position of interest results in no extension reaction. The occurrence of extension reaction is monitored by a change in the fluorescent signal of the mixture.
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Citations
9 Claims
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1. A method of identifying a nucleotide in at least a first position in a polynucleotide sequence, comprising:
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providing a polynucleotide target sequence;
hybridizing the target sequence with a first oligonucleotide probe, wherein;
the probe comprises a first subsequence of nucleotides, a first 3′
-terminal nucleotide, and a first florescent label coupled to the 3′
-terminal nucleotide;
the subsequence is complementary to a portion of the target sequence that is immediately adjacent to the first position; and
the 3′
-terminal nucleotide is complementary to one possible nucleotide in the first position;
contacting the hybridized probe and target sequence with polymerase extension reagents in a first extension reaction mixture;
measuring a level of polarized fluorescence emitted from the first extension reaction mixture, a decrease in polarized fluorescence indicating the presence of polymerase extension of the probe, the presence of polymerase extension of the probe indicating that the 3′
-terminal nucleotide is complementary to the nucleotide in the first position; and
identifying the nucleotide in the first position. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
hybridizing the target sequence with a second oligonucleotide probe that comprises;
the first subsequence of nucleotides, a second 3′
-terminal nucleotide, and a second florescent label coupled to the second 3′
-terminal nucleotide, the second fluorescent label being distinguishable from the first fluorescent label; and
the second 3′
-terminal nucleotide is different from the first 3′
-terminal nucleotide and is complementary to one possible nucleotide in the first position; and
wherein the measuring step comprises measuring a level of polarized fluorescence emitted from each of the first and second fluorescent labels, a decrease in the amount of polarized fluorescence from one of the first and second fluorescent labels being indicative of polymerase extension of the first or second oligonucleotide probe, respectively.
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9. A method for identifying a nucleotide in a first position in a target nucleic acid sequence, comprising:
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amplifying the target nucleic acid sequence in a first reaction mixture that includes effective amounts of polymerase enzyme and four dNTPs;
introducing into the first reaction mixture a first primer sequence to produce a second reaction mixture under conditions conducive to a polymerase mediated primer extension, wherein the first primer sequence comprises a first subsequence of nucleotides, a first 3′
-terminal nucleotide, and a first florescent label coupled to the 3′
-terminal nucleotide, wherein the subsequence is complementary to a portion of the target sequence that is immediately adjacent to the first position, and the first 3′
-terminal nucleotide is complementary to one possible nucleotide in the first position;
measuring a level of polarized fluorescence emitted from the second reaction mixture, a decrease in amount of polarized fluorescence being indicative of the presence extension of the first primer sequence, the presence of polymerase extension of the first primer sequence indicating that the 3′
-terminal nucleotide is complementary to the nucleotide in the first position; and
identifying the nucleotide in the first position.
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Specification