Methods for detecting and assaying nucleic acid sequences
First Claim
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1. A method for determining the presence of a specific nucleotide sequence in an RNA reagent of a target sample, said method comprising the steps of:
- a) incubating a mixture comprising;
(i) a first component including an RNA reagent extracted directly from a target sample, said RNA reagent having a target nucleotide sequence and a capture sequence, and (ii) a second component comprising a capture reagent having at least one first arm containing a label capable of emitting a detectable signal and at least one second arm having a nucleotide sequence complementary to the capture sequence of the RNA reagent of the first component, at a first temperature and for a time sufficient to induce the capture sequence of the RNA reagent of the first component to bind to the complementary nucleotide sequence of the capture reagent of the second component, and thereby form a pre-hybridized RNA-capture reagent complex comprising the target nucleotide sequence;
b) contacting the pre-hybridized RNA-capture reagent complex with a microarray having thereon a plurality of features each containing a particular probe nucleotide sequence; and
c) incubating the pre-hybridized RNA-capture reagent complex on the microarray at a second temperature and for a time sufficient to hybridize the target nucleotide sequence of the pre-hybridized RNA-capture reagent complex to the complementary probe nucleotide sequence contained within the feature, wherein the presence of such hybridization results in the emission of the detectable signal from the corresponding feature, and the absence thereof results in no emission of the detectable signal from the corresponding feature, thus generating a detectable hybridization pattern for subsequent analysis.
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Abstract
The present invention is directed to a method for determining the presence of a specific nucleotide sequence in an RNA reagent of a target sample utilizing a capture reagent having at least one first arm containing a label capable of emitting a detectable signal and at least one second arm having a nucleotide sequence complementary to a capture sequence attached to the RNA reagent on a microarray.
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Citations
38 Claims
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1. A method for determining the presence of a specific nucleotide sequence in an RNA reagent of a target sample, said method comprising the steps of:
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a) incubating a mixture comprising;
(i) a first component including an RNA reagent extracted directly from a target sample, said RNA reagent having a target nucleotide sequence and a capture sequence, and (ii) a second component comprising a capture reagent having at least one first arm containing a label capable of emitting a detectable signal and at least one second arm having a nucleotide sequence complementary to the capture sequence of the RNA reagent of the first component, at a first temperature and for a time sufficient to induce the capture sequence of the RNA reagent of the first component to bind to the complementary nucleotide sequence of the capture reagent of the second component, and thereby form a pre-hybridized RNA-capture reagent complex comprising the target nucleotide sequence;
b) contacting the pre-hybridized RNA-capture reagent complex with a microarray having thereon a plurality of features each containing a particular probe nucleotide sequence; and
c) incubating the pre-hybridized RNA-capture reagent complex on the microarray at a second temperature and for a time sufficient to hybridize the target nucleotide sequence of the pre-hybridized RNA-capture reagent complex to the complementary probe nucleotide sequence contained within the feature, wherein the presence of such hybridization results in the emission of the detectable signal from the corresponding feature, and the absence thereof results in no emission of the detectable signal from the corresponding feature, thus generating a detectable hybridization pattern for subsequent analysis. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)
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19. A method for determining the presence of a specific nucleotide sequence in an RNA reagent of a target sample, said method comprising the steps of:
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a) contacting a first component including an RNA reagent extracted directly from a target sample, said RNA reagent having a target nucleotide sequence and a capture sequence with a microarray having thereon a plurality of features each containing a particular probe nucleotide sequence;
b) incubating the RNA reagent and the complementary probe nucleotide sequences on the microarray at a first temperature and for a time sufficient to hybridize the target nucleotide sequence of the RNA reagent to the complementary probe nucleotide sequence contained within the feature;
c) contacting a second component comprising a capture reagent having at least one first arm containing a label capable of emitting a detectable signal and at least one second arm having a nucleotide sequence complementary to the capture sequence of the RNA reagent of the first component; and
d) incubating the capture reagent and the capture sequence of the RNA reagent at a second temperature and for a time sufficient to induce the capture sequence of the RNA reagent of the first component to hybridize to the complementary nucleotide sequence of the capture reagent of the second component, wherein the presence of the hybridization results in the emission of the detectable signal from the corresponding feature, and the absence thereof results in no emission of the detectable signal from the corresponding feature, thus generating a detectable hybridization pattern for subsequent analysis.
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Specification