Hybridization-based fluorescence assay
AssigneeHybridon, Inc.
InventorsEkambar Kandimalla, Sudhir Agrawal
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Abstract
The invention provides a method for detecting the presence or absence of an oligonucleotide in a sample. In the method according to the invention, a sample is obtained that may contain a target oligonucleotide. An oligonucleotide that is complementary to the target oligonucleotide and a fluorophore are added to the sample to form a mixture. The mixture is then incubated under hybridization conditions to allow binding of the complementary oligonucleotide to the target oligonucleotide to form a duplex. The fluorophore binds to the duplex; and fluorescence is then detected by standard procedures. The detection of fluorescence is indicative of the presence of the target oligonucleotide in the sample.
Claims12
A method for detecting the presence or absence of an oligonucleotide in a sample, comprising the steps of:
- a) obtaining a sample;
- b) adding to the sample an oligonucleotide that is complementary to a target oligonucleotide to form a mixture;
- c) adding a fluorophore to the mixture;
- d) incubating the mixture under conditions to allow binding of the complementary oligonucleotide to the target oligonucleotide to form a duplex;
- e) binding of the fluorophore to the duplex;
- and
- f) detecting fluorescence being indicative of the presence of the target oligonucleotide in the sample.
A method for determining the concentration of an oligonucleotide present in a sample, comprising the steps of:
- a) Obtaining a sample;
- b) Adding to the sample an oligonucleotide that is complementary to a target oligonucleotide to form a mixture;
- c) Adding a fluorophore to the mixture;
- d) Treating the mixture under conditions to allow binding of the complementary oligonucleotide to the target oligonucleotide to form a duplex;
- e) Binding of the fluorophore to the duplex;
- f) Detecting fluorescence being indicative of the presence of the target oligonucleotide in the sample;
- and
- g) Measuring the level of fluorescence
- wherein the level of fluorescence corresponds to the concentration of the target oligonucleotide in the sample.
A method for detecting the presence or absence of two or more oligonucleotides in a sample, comprising the steps of:
- a) Obtaining a sample;
- b) Conjugating oligonucleotides that are complementary to two or more target oligonucleotides to different fluorphores;
- c) Adding to the sample said complementary oligonucleotide-fluorophore conjugates to form a mixture;
- d) Treating the mixture under conditions to allow binding of the complementary oligonucleotide-fluorophore conjugates to the two or more target oligonucleotides to form a duplex;
- e) Detecting fluorescence of one of the complementary oligonucleotide-fluorophore conjugates being indicative of the presence of one of the target oligonucleotides;
- and
- f) Repeating step e) for the remaining complementary oligonucleotide-fluorophore conjugate(s).
A method for determining the concentration of two or more oligonucleotides in a sample, comprising the steps of:
- a) Obtaining a sample;
- b) Conjugating oligonucleotides that are complementary to two or more target oligonucleotides to different fluorphores;
- c) Adding to the sample said complementary oligonucleotide-fluorophore conjugates to form a mixture;
- d) Treating the mixture under conditions to allow binding of the complementary oligonucleotide-fluorophore conjugates to the two or more target oligonucleotides to form a duplex;
- e) Detecting fluorescence of one of the complementary oligonucleotide-fluorophore conjugates being indicative of the presence of one of the target oligonucleotides;
- f) Measuring the level of fluorescence;
- and
- g) Repeating steps e) and f) for the remaining complementary oligonucleotide-fluorophore conjugate(s);
- wherein the level of fluorescence corresponds to the concentration of the two or more oligonucleotides in the sample.
Dependencies are inferred from the claim text.