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

• US 20090018320A1
• Filed: 08/18/2004
• Published: 01/15/2009
• Est. Priority Date: 09/11/2003
• Status: Active Grant

• Current Assignee: Japan Science & Technology Trading Co. Limited
• Original Assignee: Japan Science & Technology Trading Co. Limited
• Inventors: Fujimoto, Kazuhisa, Shimizu, Hisao, Inouye, Masahiko
• Granted Patent: US 8,105,772 B2
• Time in Patent Office: Days
• US Class Current: 536/24.300
• 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 molecular beacon wherein the both ends of a single-stranded oligonucleotide having a hair-pin loop structure are modified with the same kind of fluorescent organic groups.

2. A molecular beacon of claim 1 wherein the fluorescent organic group is that capable of forming an excimer.

3. A molecular beacon of claim 2 wherein the fluorescent organic group is a residue of pyrene or its derivative.

4. A molecular beacon of claim 1 wherein the single-stranded oligonucleotide has 24-34 bases.

5. A molecular beacon of claim 4 wherein the single-stranded oligonucleotide has 26-32 bases.

6. A molecular beacon of claim 1 wherein the hair-pin loop region consists of 15-22 bases.

7. A molecular beacon of claim 6 wherein the hair-pin loop region consists of 17-20 bases.

8. A molecular beacon of claim 1 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.

9. A molecular beacon of claim 1 wherein at least one of the spacer contains a methylene group having 2-8 carbon atoms.

10. A method for the synthesis of a molecular beacon of claim 1 wherein a phosphoroamidite derivative of the fluorescent organic group is used as a derivative for modification of the 5′

end, and a succinimidyl derivative of the fluorescent organic group is used as a derivative for modification of the 3 end.

11. A method of claim 10 wherein the phosphoroamidite derivative and succinimidyl derivative are synthesized from a methyl ester of the fluorescent organic group.

12. A method of claim 11 wherein the phosphoroamidite derivative and succinimidyl derivative are synthesized with the use of a methyl ester of 3-pyrenylpropionic acid.

13. A method of claim 1 wherein the fluorescent organic group is bonded to the 3′

end by reacting the succinimidyl derivative with an amino group at the 3′

end.

15. A method of detecting a complementary stand DNA wherein the switching from excimer emission to monomer emission of the fluorescent organic group in a molecular beacon is detected, which is derived from hybridization between an oligonucleotide sequence in a hair-pin region of the molecular beacon of claim 1 and the subject oligonucleotide sequence.

19. A method of detecting SNP wherein the switching from excimer emission to monomer emission of the fluorescent organic group in a molecular beacon is detected, which is derived from hybridization between an oligonucleotide sequence in a hair-pin region of the molecular beacon of claim 1 and the subject oligonucleotide sequence.

14. A method of detecting a complementary stand DNA wherein a subject oligonucleotide sequence is detected with the use of switching from excimer emission to monomer emission of a fluorescent organic group.

16. A method of detecting a complementary stand DNA of claim 14 wherein the subject oligonucleotide sequence consists of 15-22 bases.

17. A method of detecting a complementary stand DNA of claim 16 wherein the subject oligonucleotide sequence consists of 17-20 bases.

18. A method of detecting SNP wherein SNP comprised in a subject oligonucleotide sequence is detected with the use of switching from excimer emission to monomer emission of a fluorescent organic group.

20. A method of detecting SNP of claim 18 wherein the subject oligonucleotide sequence consists of 15-22 bases.

21. A method of detecting SNP of claim 20 wherein the subject oligonucleotide sequence consists of 17-20 bases.