Tripartite molecular beacons

• US 20040086879A1
• Filed: 12/19/2002
• Published: 05/06/2004
• Est. Priority Date: 12/20/2001
• Status: Abandoned Application

• Current Assignee: Mcmaster University
• Original Assignee: Mcmaster University
• Inventors: Li, Yingufu, Nutiu, Razvan
• Application Number:
  US10/322,605
• Publication Number:
  US 20040086879A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

Tripartite molecular beacons (TMBs), are disclosed that are readily adaptable to high throughput applications. Each tripartite molecular beacon comprises three oligonucleotide components. The first oligonucleotide forms a hairpin stem and loop structure and the second and third oligonucleotides each comprise a sequence complementary to opposite strands of the hairpin stem. The second oligonucleotide has a fluorophore attached thereto and the third oligonucleotide has a quencher attached thereto.

Claims

1. A tripartite probe comprising:

a)a first oligonucleotide having a first end segment, a second end segment and a probe segment intermediate said first and second end segments;

b)a second, fluorescent-labeled oligonucleotide (F-DNA) hybridized to said first end segment; and

c)a third, quencher-modified oligonucleotide (Q-DNA) hybridized to said second end segment,

wherein said first end segment and said second end segment have complementary regions capable of forming the first oligonucleotide into a stem-loop structure.

2. A probe according to claim 1, wherein the first end segment comprises a first oligonucleotide-binding segment and a first complementarity segment adjacent to said first oligonucleotide-binding segment, and the second end segment comprises a second complementarity segment complementary to said first complementarity segment and a second oligonucleotide-binding segment adjacent to said second complementarity segment and wherein said F-DNA hybridizes to said first oligonucleotide-binding segment and said Q-DNA hybridizes to said second oligonucleotide-binding segment.

3. A probe according to claim 1, wherein said probe segment comprises a sequence complementary to a target sequence.

4. The probe of claim 3, wherein, in the absence of a target sequence, said first complementarity segment and said second complementarity sequence hybridize to form a duplex, thereby bringing the F-DNA and the Q-DNA into proximity whereby fluorescence from the F-DNA is quenched by the Q-DNA.

5. The probe of claim 3, wherein, in the presence of a target sequence, said probe segment binds to said target sequence and forms a probe-target duplex, thereby spatially separating the F-DNA and the Q-DNA whereby fluorescence from the F-DNA can be detected.

6. The probe of claim 5, wherein the melting point of the probe-target duplex is higher than the melting point of the stem formed between the complementarity regions.

7. The probe of claim 1, wherein said fluorophore is covalently linked to one end of said second oligonucleotide.

8. The probe of claim 1, wherein said second oligonucleotide comprises at least one fluorescent nucleotide analog.

9. The probe of claim 1, wherein said third oligonucleotide has a quencher moiety attached at one end.

10. The probe of claim 1, wherein said third oligonucleotide incorporates quenching nucleotides.

11. A kit for the detection of a target sequence, said kit comprising:

i) a loop oligonucleotide (L-DNA) comprising a probe sequence and complementary sequences on each side of said probe sequence;

ii) a fluorescent labeled oligonucleotide capable of hybridizing to said loop oligonucleotide on one side of said probe sequence; and

iii) a quencher modified oligonucleotide capable of hybridizing to the loop oligonucleotide on the other side of the probe sequence.

12. A kit according to claim 11, wherein said probe sequence comprises a sequence complementary to a target sequence.

13. A kit according to claim 11, wherein said probe sequence comprises a restriction enzyme cloning site.

14. A method of preparing an array for detection of nucleic acid sequences comprising the steps of:

i) providing a loop oligonucleotide having a probe sequence and complementary end segments capable of forming a stem-loop structure;

ii) immobilizing said loop oligonucleotide on a surface;

iii) incubating said surface with a fluorophore labeled oligonucleotide complementary to a first region of said loop oligonucleotide and a quencher modified oligonucleotide complementary to a second region o f said loop oligonucleotide wherein said fluorophore labeled oligonucleotide and said quencher modified oligonucleotide hybridize to said loop oligonucleotide and fluorescence is detected when said probe sequence binds to a complementary target sequence.

15. A method according to claim 14, wherein said loop oligonucleotide is immobilized on the surface through free DNA ends.

16. A method according to claim 14, wherein said loop oligonucleotide, said fluorophore labeled oligonucleotide and said quencher modified oligonucleotide are combined prior to immobilization on the surface.

17. A method according to claim 14, wherein the fluorophore labeled oligonucleotide and the quencher-modified oligonucleotide are added after the loop oligonucleotide is immobilized.

18. A method according to claim 14 wherein said fluorophore labeled oligonucleotide and said quencher modified oligonucleotide are added sequentially.