Nucleic acid hairpin probes and uses thereof

• US 6,596,490 B2
• Filed: 03/30/2001
• Issued: 07/22/2003
• Est. Priority Date: 07/14/2000
• Status: Expired due to Fees

• Current Assignee: Applied Gene Technologies Incorporated
• Original Assignee: Applied Gene Technologies Incorporated
• Inventors: Dattagupta, Nanibhushan
• Primary Examiner(s): Whisenant, Ethan C.
• Assistant Examiner(s): Lu, Frank Wei Min
• Application Number:
  US09/823,647
• Publication Number:
  US 20020142309A1
• Time in Patent Office: 844 Days
• Field of Search: 435/6, 435/91.1, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/6.15
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates generally to nucleic acid hybridization analysis. More specifically, an oligonucleotide probe for hybridization analysis is provided, which probe comprises a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be hybridized with. Arrays comprising the hairpin probes immobilized on a solid support and methods for nucleic acid hybridization analysis using the probes or array of immobilized probes are also provided. Methods for transcribing and/or amplifying a probe DNA sequence using a hairpin probe are further provided.

Claims

1. A method for detecting a target nucleotide sequence in a sample, which method comprises the steps of:

a) providing an oligonucleotide probe comprising a nucleotide sequence that forms a hairpin structure having a stein region comprising a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions, and wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region tat is complementary to a target nucleotide sequence to be detected;

b) contacting said probe provided in step a) with a sample containing or suspected of containing said target nucleotide sequence under conditions that favor intermolecular hybridization between said probe and said target nucleotide sequence over intramolecular hybridization of said probe itself, whereby said contacting breaks said intramolecular hybridization of said probe itself by dissociating said double stranded segment of said probe and forms a first intermolecular hybrid between said target and a region consisting of at least a portion of one stand of said stem and at least a portion of said loop within said probe that is complementary to said target nucleotide sequence; and

c) assessing said intermolecular hybrid formed in step b), wherein said assessing is effected by addition of a secondary probe tat specifically hybridizes with at least a portion of said intermolecular hybrid, thereby forming a secondary intermolecular hybrid formed between said secondary probe and said first intermolecular hybrid wherein said detection of said secondary intermolecular hybrid indicates the presence and/or amount of said target nucleotide sequence in said sample.

2. The method of claim 1, wherein the portion of the nucleotide sequences located within the double stranded segment and the single stranded loop is substantially complementary to its corresponding nucleotide sequence in the target nucleotide sequence.

3. The method of claim 1, wherein the portion of the nucleotide sequences located within the double stranded segment and the single stranded loop is a perfect match to its corresponding nucleotide sequence in the target nucleotide sequence.

4. The method of claim 1, wherein the single stranded loop of the probe comprises at least 3, 4, 5, 6, 7, 8, 9, 10 or 15 nucleotides.

5. The method of claim 1, wherein the conditions that favor intermolecular hybridization between the probe and the target nucleotide sequence over intramolecular hybridization of the probe itself is achieved by controlling compositions of the probe and the target nucleotide sequence so that the Tm of the intermolecular hybrid is higher than the Tm of the intramolecular hybrid.

6. The method of claim 5, wherein the Tm of the intermolecular hybrid is at least 2°

C. higher than the Tm of the intramolecular hybrid.

7. The method of claim 5, wherein the intermolecular hybrid is a RNA:

DNA, RNA;

RNA or a DNA;

DNA hybrid or a derivative thereof and the intramolecular hybrid is a RNA;

DNA, RNA;

RNA or a DNA;

DNA hybrid or a derivative thereof.

8. The method of claim 5, wherein the intermolecular hybrid is a RNA:

DNA hybrid whereas the intramolecular hybrid is a DNA;

DNA hybrid.

9. The method of claim 1, wherein the second probe has a detectable label.

10. The method of claim 9, wherein the label is selected from the group consisting of a chemical, an enzymatic, a radioactive, a fluorescent, a luminescent and a FRET label.

11. The method of claim 1, further comprising a step of crosslinking the stern region of the probe that has not been hybridized wit the target after the formation of the intermolecular hybrid but before the addition of the detectably labeled secondary probe.

12. The method of claim 11, wherein the crosslinking step is effected via addition of a crosslinking agent subsequent to hybridization of the probe with the target nucleotide sequence.

13. The method of claim 1, wherein the hairpin probe further comprises an element or a modification that facilitates intramolecular crosslinking of the probe upon a suitable treatment.

14. The method of claim 13, wherein the element is a crosslinking agent that is activated by chemical or photoactive treatment.

15. The method of claim 1, wherein the secondary probe specifically hybridizes with a portion of the target nucleotide sequence that is not involved in the hybridization of the target nucleotide sequence and the probe.

16. The method of claim 1, wherein the secondary probe specifically hybridizes with a portion of the target nucleotide sequence that is involved in the hybridization of the target nucleotide sequence and the probe and the nucleotide sequence in the probe that is complementary to said portion of the target nucleotide sequence is removed concurrently with the addition of the secondary probe.

17. The method of claim 16,wherein the hairpin structure in the probe is formed between a DNA strand that contains methylphosphonates and a RNA strand that is complementary to the target nucleotide sequence and wherein the RNA strand, after forming a hybrid with the target nucleotide sequence but before the addition of the secondary probe, is removed by a RNase H treatment.

18. The method of claim 1, wherein the secondary probe specifically hybridizes with a portion of the probe that is involved in the formation of the intramolecular hybrid but is not involved in the formation of the first intermolecular hybrid with the target nucleotide sequence and wherein said portion of the probe dissociates as a single strand after the formation of the first intermolecular hybrid.

19. The method of claim 1, wherein the secondary probe is a specific probe.

20. (Amended) The method of claim 1, wherein the probe is a degenerate probe.

21. The method of claim 1, wherein the probe is immobilized on a solid support.

22. The method of claim 1, wherein a plurality of the probes immobilized on a solid support is used.

23. The method of claim 1, wherein the sample is a biosample.

24. The method of claim 1, wherein a plurality of samples is assayed simultaneously.

25. The method of claim 1, wherein the oligonucleotide probe and the target nucleotide sequence is contacted at a temperature from about 4°

C. to about 90°

C.

26. The method of claim 1, wherein the oligonucleotide probe and the target nucleotide sequence is contacted for a time from about 1 minute to about 60 minutes.

27. The method of claim 1, wherein the probe further comprises an element that is sensitive to nuclease digestion or a modification that is resistant to nuclease digestion.

28. The method of claim 27, wherein the nuclease digestion is effected via a restriction enzyme.