Nucleic acid detector and method of detecting targets within a sample

• US 20040110141A1
• Filed: 12/10/2002
• Published: 06/10/2004
• Est. Priority Date: 12/10/2002
• Status: Active Grant

• Current Assignee: University of Alabama (University of Alabama System)
• Original Assignee: University of Alabama (University of Alabama System)
• Inventors: Chittur, Krishnan K., Ng, Joseph D., Pusey, Marc L., Dowell, Jeffrey J.
• Granted Patent: US 7,291,459 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

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.

Claims

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.

6. The method of claim 1 wherein denaturing comprises raising the temperature of the captor above a critical temperature.

7. The method of claim 1 wherein the suspected target is presented within a heterogeneous biological sample.

8. The method of claim 1 wherein the step of placing the captor under conditions favorable for renaturing comprises lowering the temperature of the captor below a critical temperature.

9. The method of claim 1 wherein the captor is denatured and placed under conditions for renaturing while maintained in a buffer solution.

10. The method of claim 1 wherein the suspected target is maintained in a medium prior to contact with the captor, and wherein the medium is treated with nuclease inhibitors prior to contact of the suspected target with the captor.

11. The method of claim 10 wherein the nuclease inhibitor is selected from the group consisting of sodium dodecyl sulfate (SDS), ethylene diamine tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), and combinations thereof.

12. The method of claim 1 wherein the detector probe is labeled with a molecule selected from the group consisting of fluorophores and radioisotopes.

13. The method of claim 12 wherein the detector probe is labeled with a fluorophore and the presence of the probe upon the captor is determined with a fluoroscope.

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.

3. The method of claim 2 wherein the tail sequences comprise a series of four repeat tetramers.

4. The method of claim 2 wherein the tail sequences are between 8 and 20 residues in length, the loop sequence is between about 10 and 40 residues in length, and the optional spacer sequences are from 0 to 10 nucleotides in length.

5. The method of claim 2 wherein the captor and detector probe comprise DNA, RNA, PNA, or a combination thereof.

14. A method of detecting the presence of a nucleic acid target within a biological sample comprising the steps of:

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.

15. A method of detecting the presence or absence of multiple nucleic acid targets within a biological sample comprising the steps of:

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.

16. The method of claim 15 wherein all captors have a common second tail sequence and wherein the universal detector probes all contain a common nucleic acid sequence.

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.

18. The detector of claim 17 wherein the tail sequences comprise a series of four repeat tetramers.

19. The detector of claim 17 wherein the tail sequences are between 8 and 20 residues in length, the loop sequence is between about 10 and 40 residues in length, and the optional spacer sequences are from 0 to 10 nucleotides in length.

20. The detector of claim 17 wherein the captor and detector probe comprise DNA, RNA, PNA, or a combination thereof.

21. The detector of claim 17 wherein the detector probe is labeled with a molecule selected from the group consisting of fluorophores and radioisotopes.

22. The detector of claim 18 wherein the label molecule is selected from group consisting of fluorescein, Lucifer Yellow, erythrosine, tetramethylrhodamine, and coumarin.

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.

24. The array of claim 23 wherein the tail sequences are between 8 and 20 residues in length, the loop sequence is between about 10 and 40 residues in length, and the optional spacer sequences are from 0 to 10 nucleotides in length.

25. The array of claim 23 wherein the captors comprise DNA, RNA, PNA, or a combination thereof.

26. The array of claim 23 further comprising universal detector probes.

27. The array of claim 26 wherein the universal detector probes have common nucleic acid sequences that are complementary to the second tail sequence of the captors.

28. The array of claim 27 wherein the universal detector probe is labeled with a molecule selected from the group consisting of fluorophores and radioisotopes.