Probes labeled with energy transfer coupled dyes

• US 20030143594A1
• Filed: 11/04/2002
• Published: 07/31/2003
• Est. Priority Date: 02/01/1994
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Ju, Jingyue, Mathies, Richard, Glazer, Alexander
• Granted Patent: US 7,015,000 B2
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Fluorescent labels having at least one donor and at least one acceptor fluorophore bonded to a polymeric backbone in energy transfer relationship, as well as methods for their use, are provided. Of particular interest are the subject labels wherein the polymeric backbone is a nucleic acid and the donor fluorophore is bonded to the 5′ terminus of said nucleic acid. Such labels find use as primers in applications involving nucleic acid chain extension, such as sequencing, PCR and the like.

Claims

1. A fluorescent label comprising:

at least two fluorophores bonded to a polymeric backbone wherein at least one fluorophore is bonded to the terminal end base unit of said polymeric backbone and the other fluorophore(s) is bonded to a non-terminal monomeric unit of said polymeric backbone and said terminal bonded fluorophore is in energy transfer relationship with said other fluorophore(s).

2. The label according to claim 1, wherein said fluorophores comprise a donor and acceptor fluorophore and said donor absorbs light in the wavelength range of 350 to 800 nm and said acceptor emits light in the wavelength range of 450 to 1000 nm.

3. The label according to claim 2, wherein the difference between the emission maxima of said donor and acceptor fluorophores does not exceed 100 nm.

4. The label according to claim 1, wherein the distance in monomeric units of said polymeric backbone between said pair of fluorophores ranges from about 2 to 20.

5. The label according to claim 1, wherein the monomeric units of said polymeric backbone comprise a base selected from the group consisting of purines, pyrimidines and hybridizing analogues thereof.

6. The label according to claim 5, wherein said polymeric backbone is an oligonucleotide.

7. The label according to claim 5, wherein said polymeric backbone is a peptide nucleic acid.

8. The label according to claim 5, wherein said fluorophores comprises a donor and acceptor fluorophore and said donor fluorophore is bonded to the 5′

terminus of said oligonucleotide

9. The label according to claim 5, wherein said fluorophores comprise a donor and acceptor fluorophore and said acceptor fluorophore is bonded to the 5′

terminus of said oligonucleotide.

10. A fluorescent label comprising:

at least two fluorophores comprising a donor and acceptor fluorophore bonded to an oligonucleotide, wherein one of said fluorophores is bonded to the 5′

terminus of said oligonucleotide and the other of said fluorophores is bonded to a modified nucleotide from 2 to 20 nucleotides 3′

of said 5′

bonded fluorophore and said 5′

bonded fluorophore is in energy transfer relationship with said other fluorophore.

11. The label according to claim 10, wherein said donor absorbs light in the wavelength range of 350 to 800 nm and said acceptor emits light in the wavelength range of 450 to 1000 nm.

12. The label according to claim 10, wherein said oligonucleotide is from 3 to 30 nucleotides.

13. The label according to claim 10, wherein said donor and acceptor fluorophores are bonded to said oligonucleotide through a linker arm.

14. The label according to claim 10, wherein the difference between the emission maxima of said donor and acceptor fluorophores does not exceed 100 nm.

15. The label according to claim 10, wherein said 5′

bonded fluorophore is bonded to the phosphate group of the 5′

nucleotide.

16. The label according to claim 10, wherein said 5′

bonded fluorophore is bonded to the sugar group of the 5′

nucleotide.

17. The label according to claim 10, wherein said 5′

bonded fluorophore is bonded to a modified base of the 5′

nucleotide.

18. A fluorescent label comprising:

a donor and acceptor fluorophore having a difference in emission maxima that does not exceed 100 nm bonded to an oligonucleotide, wherein one of said donor and acceptor fluorophores is bonded to the 5′

terminus of said oligonucleotide and the other of said fluorophores is bonded to a modified nucleotide from 2 to 20 nucleotides 3′

of said 5′

bonded fluorophore.

19. The label according to claim 18, wherein at least one of said donor and acceptor fluorophores is a xanthene compound.

20. The label according to claim 18, wherein said xanthene compound is fluorescein or rhodamine.

21. The label according to claim 18, wherein said 5′

bonded fluorophore is bonded to the sugar group of the 5′

nucleotide of said oligonucleotide.

22. The label according to claim 18, wherein said 5′

bonded fluorophore is bonded to a modified base of the 5′

nucleotide.

23. The label according to claim 18, wherein said modified nucleotide is Amino Modifier C6 dT.

24. A fluorescent label comprising:

a donor and acceptor fluorophore having a difference in emission maxima that does not exceed 100 nm bonded to an oligonucleotide, wherein one of said donor and acceptor fluorophores is bonded to the sugar group of the 5′

nucleotide of said oligonucleotide and the other of said fluorophores is bonded to a modified nucleotide from 2 to 15 nucleotides 3′

of 5′

bonded fluorophore, wherein said modified nucleotide is Amino Modifier C6 dT.

25. The label according to claim 24, wherein said donor is FAM and said acceptor is selected from the group consisting of JOE, TAMRA, ROX, 6-carboxyrhodamine-6G and 5-carboxyrhodamine-6G.

26. A fluorescent label having a donor and acceptor fluorophore bonded to an oligonucleotide, wherein said donor is bonded to the sugar group of the 5′

nucleotide of said oligonucleotide and acceptor is bonded to a modified nucleotide 3′

of said 5′

bonded fluorophore, said label described by the formula;

D-N-A

wherein;

D is FAM;

N is the number of bases between said donor and acceptor fluorophore and ranges from 2 to 14; and

A is FAM.

27. A fluorescent label having a donor and acceptor fluorophore bonded to an oligonucleotide, wherein said donor is bonded to the sugar group of the 5′

nucleotide of said oligonucleotide and acceptor is bonded to a modified nucleotide 3′

of said 5′

bonded fluorophore, said label described by the formula;

D-N-A

wherein;

D is FAM;

N is the number of bases between said donor and acceptor fluorophore and ranges from 2 to 14; and

A is JOE.

28. A fluorescent label having a donor and acceptor fluorophore bonded to an oligonucleotide, wherein said donor is bonded to the sugar group of the 5′

nucleotide of said oligonucleotide and acceptor is bonded to a modified nucleotide 3′

of said 5′

bonded fluorophore, said label described by the formula;

D-N-A

wherein;

D is FAM;

N is the number of bases between said donor and acceptor fluorophore and ranges from 2 to 14; and

A is TAMRA.

29. A fluorescent label having a donor and acceptor fluorophore bonded to an oligonucleotide, wherein said donor is bonded to the sugar group of the 5′

nucleotide of said oligonucleotide and acceptor is bonded to a modified nucleotide 3′

of said 5′

bonded fluorophore, said label described by the formula;

D-N-A

wherein;

D is FAM;

N is the number of bases between said donor and acceptor fluorophore and ranges from 2 to 14; and

A is ROX.