DNA detection method using molecular beacon with the use of monomer emission/excimer emission switching of fluorescent molecule

• US 8,105,772 B2
• Filed: 08/18/2004
• Issued: 01/31/2012
• Est. Priority Date: 09/11/2003
• Status: Expired due to Fees

• Current Assignee: Japan Science & Technology Trading Co. Limited
• Original Assignee: Japan Science & Technology Trading Co. Limited
• Inventors: Inouye, Masahiko, Fujimoto, Kazuhisa, Shimizu, Hisao
• Primary Examiner(s): Whisenant, Ethan
• Application Number:
  US10/571,133
• Publication Number:
  US 20090018320A1
• Time in Patent Office: 2,722 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• 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 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

It is intended to provide a means and a method whereby a molecule for DNA detection at an elevated accuracy can be provided, different form the existing DNA detection system with the use of a molecular beacon. A molecular beacon wherein fluorescent organic groups capable of forming an excimer are bonded to the 3′ and 5′ ends of a single-stranded oligonucleotide to be hybridizable with a subject oligonucleotide and the switching of monomer emission/excimer emission is utilized; and a method of detecting SNP by using this molecular beacon.

Claims

1. A method of detecting a complementary strand DNA comprising:

determining whether fluorescent organic groups, which modify both ends of a first oligonucleotide, switch from excimer emission to monomer emission upon hybridization of the first oligonucleotide and a subject oligonucleotide,

wherein detection of the switch to monomer emission indicates that the subject oligonucleotide is complementary to the first oligonucleotide.

3. The method of detecting a complementary strand DNA of claim 1 wherein the subject oligonucleotide sequence consists of 15-22 bases.

4. The method of detecting a complementary strand DNA of claim 3 wherein the subject oligonucleotide sequence consists of 17-20 bases.

2. A method of detecting a complementary strand DNA comprising:

hybridizing a hair-pin loop region of a single stranded oligonucleotide of a molecular beacon and a subject oligonucleotide sequence, wherein both ends of the single stranded oligonucleotide are modified with the same kind of fluorescent organic groups, and

determining whether the fluorescent organic groups of the molecular beacon switch from excimer emission to monomer emission,

wherein detection of the switch to monomer emission indicates that the subject oligonucleotide is complementary to the hair-pin loop region of the single stranded oligonucleotide of the molecular beacon.

5. A method of detecting a SNP in a subject oligonucleotide comprising:

determining whether fluorescent groups, which modify both ends of a first oligonucleotide switch from excimer emission to monomer emission upon hybridization of the first oligonucleotide and a subject oligonucleotide,

wherein failure to detect the switch to monomer emission indicates that the subject oligonucleotide sequence comprises a SNP.

7. The method of detecting the SNP of claim 5 wherein the subject oligonucleotide sequence consists of 15-22 bases.

8. The method of detecting the SNP of claim 7 wherein the subject oligonucleotide sequence consists of 17-20 bases.

6. A method of detecting a SNP in a subject oligonucleotide sequence comprising:

hybridizing a hair-pin loop region of a single stranded oligonucleotide of a molecular beacon and a subject oligonucleotide sequence, wherein both ends of the single stranded oligonucleotide are modified with the same kind of fluorescent organic groups, and

determining whether the fluorescent organic groups of the molecular beacon switch from excimer emission to monomer emission,

wherein failure to detect the switch to monomer emission indicates that the subject oligonucleotide sequence comprises a SNP.

9. A method of detecting a complementary strand DNA or a subject oligonucleotide comprising a SNP

hybridizing a hair-pin loop region of a single stranded oligonucleotide of a molecular beacon and a subject oligonucleotide sequence, wherein said subject oligonucleotide optionally comprises a SNP, and wherein both ends of the single stranded oligonucleotide are modified with the same kind of fluorescent organic groups, and

determining whether the fluorescent organic groups of the molecular beacon switch from excimer emission to monomer emission,

wherein failure to detect the switch to monomer emission indicates that the subject oligonucleotide comprises a SNP and wherein detection of the switch to monomer emission indicates that the subject oligonucleotide is complementary to the oligonucleotide in the hair-pin loop region.

10. The method of claim 9 wherein the fluorescent organic group is a residue of pyrene or its derivative.

11. The method of claim 9 wherein the single-stranded oligonucleotide has 24-34 bases.

12. The method of claim 11 wherein the single-stranded oligonucleotide has 26-32 bases.

13. The method of claim 9 wherein the hair-pin loop region consists of 15-22 bases.

14. The method of claim 13 wherein the hair-pin loop region consists of 17-20 bases.

15. The method of claim 9 wherein the fluorescent organic group is bonded to both 5′

and 3′

ends of the single-stranded oligonucleotide via a spacer with an appropriate length.

16. The method of claim 15 wherein at least one of the spacers contains a (CH₂)_2-8 group.

17. The method of claim 9, wherein the subject oligonucleotide sequence consists of 15-22 bases.

18. The method of claim 17, wherein the subject oligonucleotide sequence consists of 17-20 bases.