Nucleic acid detector and method of detecting targets within a sample
First Claim
1. A method of detecting the presence of a nucleic acid target within a biological sample comprising the steps of:
- supplying a substrate-bound, hairpin-shaped nucleic acid captor;
contacting the captor with a suspected target under conditions favorable for denaturing of the captor;
placing the captor under conditions favorable for renaturing;
contacting the captor with a labeled detector probe;
washing the captor; and
determining if the labeled detector probe remains adhered to the captor, thereby detecting the presence of the nucleic acid target.
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Abstract
A nucleic acid detector and a method of using the detector are disclosed for detecting the presence of target nucleic acid sequences within a sample. The nucleic acid detector comprises a substrate-bound, hairpin-shaped nucleic acid captor in conjunction with a labeled universal nucleic acid detector probe. The captor has a segment that is complementary to at least a portion of the target nucleic acid and is denatured and hybridized to the target under test conditions. Hybridization of the captor to the target maintains the captor in an open conformation which exposes an end portion of the captor to the universal detector probe. The detector probe is able to hybridize with the exposed end portion of the captor if the captor has hybridized with a target. The labeled detector probe is detectable by external detection methods. Detector probes having identical universal detector probe sequences may be used to identify the presence of multiple targets having various target sequences within a sample. An assay embodying the detector and method is also contemplated.
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Citations
28 Claims
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1. A method of detecting the presence of a nucleic acid target within a biological sample comprising the steps of:
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supplying a substrate-bound, hairpin-shaped nucleic acid captor;
contacting the captor with a suspected target under conditions favorable for denaturing of the captor;
placing the captor under conditions favorable for renaturing;
contacting the captor with a labeled detector probe;
washing the captor; and
determining if the labeled detector probe remains adhered to the captor, thereby detecting the presence of the nucleic acid target. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13)
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2. The method of claim 2 wherein the captor is bound to the substrate at its 5′
- terminus, and wherein the captor sequence serially comprises, from the 5′
terminus to the 3′
terminus,a first tail sequence, an optional first spacer sequence, a loop sequence that is complementary to a sequence within the target, an optional second spacer sequence, and a second tail sequence that is complementary to the first tail sequence. - View Dependent Claims (3, 4, 5)
- terminus, and wherein the captor sequence serially comprises, from the 5′
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14. A method of detecting the presence of a nucleic acid target within a biological sample comprising the steps of:
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supplying a single-stranded nucleic acid captor sequence that is bound to a substrate at its 5′
terminus, wherein the captor sequence serially comprises, from the 5′
terminus to the 3′
terminus,a first tail sequence, an optional first spacer sequence, a loop sequence that is complementary to a sequence within the target, an optional second spacer sequence, and a second tail sequence that is complementary to the first tail sequence;
denaturing the captor sequence;
contacting the captor sequence with the biological sample, whereby targets within the sample tend to hybridize with the loop sequence of the captor;
placing the captor sequence under conditions favorable for renaturing the captor, whereby the first and second tail sequences tend to hybridize to one another if not hindered by a target sequence;
contacting the captor with a labeled detector probe that is complementary to the second tail sequence of the captor;
washing the captor; and
detecting the presence of labeled detector probes remaining upon the captor after the washing step, whereby the presence of the labeled probes indicates the presence of the target within the sample.
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15. A method of detecting the presence or absence of multiple nucleic acid targets within a biological sample comprising the steps of:
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supplying multiple single-stranded nucleic acid captors, wherein each captor comprises, from the 5′
terminus to the 3′
terminus,a first tail sequence, an optional first spacer sequence, a loop sequence that is complementary to a sequence within a target, an optional second spacer sequence, and a second tail sequence that is complementary to the first tail sequence;
grouping the multiple captors into clusters such that all captors of a common cluster have a loop sequence complementary to a common target;
disposing the captor clusters upon a substrate;
contacting the captors with the biological sample under conditions favorable for denaturing of the captors, whereby targets within the sample tend to hybridize captors having loop sequences that are complementary to sequences within the targets;
subjecting the captors under conditions favorable for renaturing, whereby the first and second tail sequences tend to hybridize to one another if not hindered by a target sequence;
contacting the captors with labeled universal detector probes that are complementary to the second tail sequences of each captor;
washing the captors; and
detecting the presence of labeled detector probes remaining upon the captors after the washing step, whereby the presence of the labeled probes in a particular cluster of captors indicates the presence of the target having a sequence complementary to the loop portion of the captors of that cluster. - View Dependent Claims (16)
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17. A multi-component nucleic acid detector for indicating the presence of a nucleic acid target within a biological sample, comprising
a nucleic acid captor sequence that is bound to a substrate at its 5′ - terminus,
wherein the captor sequence serially comprises, from the 5′
terminus to the 3′
terminus,a first tail sequence, an optional first spacer sequence, a loop sequence that is complementary to a sequence within the target, an optional second spacer sequence, and a second tail sequence that is complementary to the first tail sequence; and
a nucleic acid detector probe having a sequence complementary to the second tail sequence of the captor and having at least one label attached thereto. - View Dependent Claims (18, 19, 20, 21, 22)
- terminus,
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23. An array of nucleic acid captors capable of simultaneously and specifically detecting the presence of multiple nucleic acid targets from a heterogeneous sample, comprising
a substrate having at least two captor clusters adhered thereto; -
wherein each of the captor clusters corresponds to a respective pre-selected target, and wherein each captor comprises a nucleic acid sequence that is bound to a substrate at its 5′
terminus, wherein the captor sequence serially comprises, from the 5′
terminus to the 3′
terminus,a first tail sequence, an optional first spacer sequence, a loop sequence that is complementary to a sequence within the target, an optional second spacer sequence, and a second tail sequence that is complementary to the first tail sequence and suitable for hybridization with a detector probe. - View Dependent Claims (24, 25, 26, 27, 28)
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Specification