Fluorescent oligonucleotides and uses thereof

• US 6,838,244 B1
• Filed: 05/18/2001
• Issued: 01/04/2005
• Est. Priority Date: 05/19/2000
• Status: Active Grant

• Current Assignee: Monsanto Technology LLC (Bayer AG)
• Original Assignee: Monsanto Technology LLC (Bayer AG)
• Inventors: Zhou, Jian S., Li, Wan-Liang Robert
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/859,736
• Time in Patent Office: 1,327 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.51, 536/22.1, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32, 536/26.6, 422/50, 422/52, 422/56, 422/55, 422/57, 702/20
• US Class Current: 435/6.12
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide

Abstract

Oligonucleotide molecules labeled with a plurality of fluorophores of one or more types embedded in the backbone of said oligonucleotide, wherein at least one of said fluorophores is not located at either the 3′ or 5′ terminus of said oligonucleotide. The invention further provide methods for using the subject labeled oligonucleotides in detecting biological molecules, sequencing DNA molecules and particularly generating cDNA molecules for characterizing the differential expression of genes.

Claims

1. An oligonucleotide molecule labeled with a plurality of fluorophores of one or more types embedded in the backbone of said oligonucleotide, wherein at least one of said fluorophores is not located at either the 3′

or 5′

terminus of said oligonucleotide.

2. The labeled oligonucleotide of claim 1, wherein said oligonucleotide is a branched oligonucleotide.

3. The labeled oligonucleotide of claim 1, wherein said oligonucleotide is an unbranched oligonucleotide.

4. The labeled oligonucleotide of claim 1, having a structure selected from the group consisting of:

(a) F

N_iF

N_jF′

N_iF′

N_jF

_s

_r

wherein N is a nucleotide unit;

F and F′

are respectively a first and a second fluorophore which is attached to a sugar moiety of N through a sugar linkage such that the first and second fluorophores are constituents of a polymeric chain;

i and j are integers ranging from 2 to 7;

s is 1; and

r is an integer ranging from 1 to 5;

wherein N is a nucleotide unit;

F and F′

are respectively a first and a second fluorophore which is covalently attached to a sugar moiety of N through a sugar linkage such that Fs and F'"'"'s are constituents of a first and a second polymeric chain;

Y is the first polymeric chain having the structure FN_iF

N_jF′

N_iF′



N_jFN_iF

_p

_q;

N′

is a nucleotide having a modified heterocyclic nitrogenous base having a linking group thereon for covalently attaching the first polymeric chain Y to N′

;

i,j and k are integers ranging from 2 to 7;

p is 0 or 1; and

m are integers ranging from 1 to 3;

q is an integer ranging from 0 to 5; and

n is an integer ranging from 1 to 5; and

(c) F

N_iF

_r

wherein N is a nucleotide unit;

F is a fluorophore which is attached to a sugar moiety of N through a sugar linkage such that the fluorophore is a constituent of a polymeric chain;

i is an integer ranging from 2 to 7; and

r is an integer ranging from 2 to 5.

5. The labeled oligonucleotide of claim 1, wherein the oligonucleotide sequence comprises an oligo dT sequence.

6. The labeled oligonucleotide of claim 5, wherein the oligo-dT sequence comprises between about 10 and about 50 deoxythymidine bases.

7. The labeled oligonucleotide of claim 1, wherein all of said fluorophores have the same structure.

8. The labeled oligonucleotide of claim 1, wherein all of said fluorophores do not have the same structure.

9. The labeled oligonucleotide of claim 8, wherein said fluorophores are capable of generating two distinguishable fluorescent signals at two distinguished wavelengths.

10. A set of labeled oligonucleotides with a plurality of fluorophores of one or more types embedded in the backbone of said oligonucleotides, wherein at least one of said fluorophores is not located at either the 3′

or 5′

terminus of said oligonucleotides.

11. The set of labeled oligonucleotides of claim 10, wherein said set comprises four labeled oligonucleotides, wherein the oligonucleotides do not have the same structure.

12. The set of labeled oligonucleotides of claim 11, wherein said set of labeled oligonucleotides is capable of being used for sequencing nucleic acid molecules.

13. The set of labeled oligonucleotides of claim 11, further comprising (1) a first labeled oligonucleotide capable of generating a first pair of distinguishable fluorescent signals at a first pair of two distinguishable wavelengths;

(2) a second labeled oligonucleotide capable of generating a second pair of distinguishable fluorescent signals at a second pair of two distinguishable wavelengths;

(3) a third labeled oligonucleotide capable of generating a third pair of distinguishable fluorescent signals at a third pair of two distinguishable wavelengths;

(4) a fourth labeled oligonucleotide capable of generating a fourth pair of distinguishable fluorescent signals at a fourth pair of two distinguishable wavelengths;

wherein, said first, second, third and fourth pairs of distinguishable fluorescent signals are different from each other; and

said first, second, third and fourth pairs of distinguishable wavelengths are different from each other.

14. The set of labeled oligonucleotides of claim 10, wherein at least one of said labeled oligonucleotides have the structure selected from the group consisting of:

(a) F

N_iF

N_jF′

N_iF′

N_jF

_s

_r

wherein N is a nucleotide unit;

F and F′

are respectively a first and second fluorophore which is attached to the sugar moiety of N through a sugar linkage such that the first and second fluorophores are constituents of a polymeric chain;

i and j are integers ranging from 2 to 7;

s is 1; and

r is an integer ranging from 1 to 5;

wherein N is a nucleotide unit;

F and F′

are respectively a first and second fluorophore which is covalently attached to a sugar moiety of N through a sugar linkage such that F and F′

are constituents of a first and a second polymeric chain;

Y is the first polymeric chain having the structure FN_iF

N_jF′

N_iF′



N_jFN_iF

_p

_q;

N′

is a nucleotide having a modified heterocyclic nitrogenous base having a linking group thereon for covalently attaching the first polymeric chain Y to N′

;

i,j and k are integers ranging from 2 to 7;

p is 0 or 1; and

m are integers ranging from 1 to 3;

q is an integer ranging from 0 to 5; and

n is an integer ranging from 1 to 5; and

(c) F

N_iF

_r

wherein N is a nucleotide unit;

F is a fluorophore which is attached to a sugar moiety of N through a sugar linkage such that the fluorophore is a constituent of a polymeric chain;

i is an integer ranging from 2 to 7; and

r is an integer ranging from 2 to 5.

15. The set of claim 13, wherein each of the first through fourth pairs of distinguishable wavelengths comprises a combination of two distinguishable wavelengths of a group of 4 to 8 distinguishable wavelengths.

16. The set of claim 13, wherein each of the first through fourth pairs of distinguishable wavelengths comprises a combination of two distinguishable wavelengths of a group of 4 distinguishable wavelengths.

17. The set of labeled oligonucleotides of claim 10, wherein said set comprises 2 different labeled oligonucleotides.

18. The set of claim 17, wherein any or all of said labeled oligonucleotides have the structure selected from the group consisting of:

(a) F

N_iF

N_jF′

N_iF′

N_jF

_s

_r

wherein N is a nucleotide unit;

F and F′

are respectively a first and second fluorophore each of which is covalently attached to a sugar moiety of N through a sugar linkage such that the first and second fluorophores are constituents of a polymeric chain;

i and j are integers ranging from 2 to 7;

s is 1; and

r is an integer ranging from 1 to 5;

wherein N is a nucleotide unit;

F and F′

are respectively a first and a second fluorophores each of which is covalently attached to the sugar moiety of N through a sugar linkage such that Fs and F'"'"'s are constituents of a first and a second polymeric chains;

Y is the first polymeric chain having the structure FN_iF

N_jF′

N_iF′



N_jFN_iF

_p

_q;

N′

is a nucleotide having a modified heterocyclic nitrogenous base having a linking group thereon for covalently attaching the first polymeric chain Y to N′

;

i,j and k are integers ranging from 2 to 7;

p is 0 or 1; and

m are integers ranging from 1 to 3;

q is an integer ranging from 0 to 5; and

n is an integer ranging from 1 to 5; and

(c) F

N_iF

_r

wherein N is a nucleotide unit;

F is a fluorophore which is attached to a sugar moiety of N through a sugar linkage such that the fluorophore is a constituent of a polymeric chain;

i is an integer ranging from 2 to 7; and

r is an integer ranging from 2 to 5.

19. The set of labeled oligonucleotides of claim 17, wherein said wherein the oligonucleotide sequence comprises an oligo dT sequence.

20. The labeled oligonucleotide of claim 19, wherein the oligo-dT sequence comprises between about 10 and about 50 deoxythymidine bases.

21. The set of labeled oligonucleotides of claim 17, wherein said first fluorescent label is capable of generating a first and second fluorescent signal respectively at a first and a second distinguishable wavelength and said second fluorescent label is capable of generating a second and third fluorescent signal respectively at a second and a third distinguishable wavelength.

22. A kit for use in generating fluorescently labeled cDNA molecules from a RNA sample comprising an oligonucleotide molecule labeled with a plurality of fluorophores of one or more types embedded in the backbone of said oligonucleotide molecule, wherein at least one of said fluorophores is not located at either the 3′

or 5′

terminus of said oligonucleotide molecule.

23. The kit of claim 22, wherein said labeled oligonucleotide comprises a fluorescent label attached to the 5′

end of the oligonucleotide primer, the labeled oligonucleotide having a structure selected from the group consisting of;

(a) F

N_iF

N_jF′

N_iF′

N_jF

_s

_r

wherein N is a nucleotide unit;

F and F′

are respectively a first and a second fluorophore which is covalently attached to the sugar moiety of N through a sugar linkage such that the first and second fluorophores are constituents of a polymeric chain;

i and j are integers ranging from 2 to 7;

s is 1; and

r is an integer ranging from 1 to 5;

wherein N is a nucleotide unit;

F and F′

are respectively a first and a second fluorophore which is covalently attached to the sugar moiety of Ns through a sugar linkage such that F and F′

are constituents of a first and a second polymeric chains;

Y is the first polymeric chain having the structure FN_iF

N_jF′

N_iF′



N_jFN_iF

_p

_q;

N′

is a nucleotide having a modified heterocyclic nitrogenous base having a linking group thereon for covalently attaching the first polymeric chain Y to N′

;

i,j and k are integers ranging from 2 to 7;

p is 0 or 1; and

m are integers ranging from 1 to 3;

q is an integer ranging from 0 to 5; and

n is an integer ranging from 1 to 5; and

wherein N is a nucleotide unit;

F is a fluorophore which is attached to a sugar moiety of N through a sugar linkage such that the fluorophore is a constituent of a polymeric chain;

i is an integer ranging from 2 to 7; and

r is an integer ranging from 2 to 5.

24. The kit of claim 22, wherein said kit further comprises a reverse transcriptase.

25. The kit of claim 22, wherein said kit further comprises deoxynucleotides.

26. The kit of claim 22, further comprising one or more of reverse transcription buffer, reverse transcriptase, dNTPs, PCR buffer, control RNA, glycogen, water and loading dye.

27. A kit capable of use for DNA sequencing, said kit comprising a first, second, third and fourth oligonucleotide labeled with a plurality of fluorophores of one or more types embedded in the backbone of said oligonucleotide, wherein at least one of said fluorophores is not located at either the 3′

or 5′

terminus of said oligonucleotide.

28. The kit of claim 27, further comprising (1) a first labeled oligonucleotide capable of generating a first pair of distinguishable fluorescent signals at a first pair of two distinguishable wavelengths;

(2) a second labeled oligonucleotide capable of generating a second pair of distinguishable fluorescent signals at a second pair of two distinguishable wavelengths;

(3) a third labeled oligonucleotide capable of generating a third pair of distinguishable fluorescent signals at a third pair of two distinguishable wavelengths;

(4) a fourth labeled oligonucleotide capable of generating a fourth pair of distinguishable fluorescent signals at a fourth pair of two distinguishable wavelengths;

wherein, said first, second, third and fourth pairs of distinguishable fluorescent signals are different from each other; and

said first, second, third and fourth pairs of distinguishable wavelengths are different from each other.

29. The kit of claim 27, wherein said first, second, third and fourth labeled oligonucleotides have a structure selected from the group consisting of:

(a) F

N_iF

N_jF′

N_iF′

N_jF

_s

_r

wherein N is a nucleotide unit;

F and F′

are respectively a first and a second fluorophore which is covalently attached to the sugar moiety of N through a sugar linkage such that the first and second fluorophores are constituents of a polymeric chain;

i and j are integers ranging from 2 to 7;

s is 1; and

r is an integer ranging from 1 to 5;

wherein N is a nucleotide unit;

F and F′

are respectively a first and a second fluorophore which is covalently attached to the sugar moiety of Ns through a sugar linkage such that F and F′

are constituents of a first and a second polymeric chains;

Y is the first polymeric chain having the structure FN_iF

N_jF′

N_iF′



N_jFN_iF

_p

_q;

N′

is a nucleotide having a modified heterocyclic nitrogenous base having a linking group thereon for covalently attaching the first polymeric chain Y to N^′

;

i,j and k are integers ranging from 2 to 7;

p is 0 or 1; and

m are integers ranging from 1 to 3;

q is an integer ranging from 0 to 5; and

n is an integer ranging from 1 to 5; and

(c) F

N_jF

_i

wherein N is a nucleotide unit;

F is a fluorophore which is attached to a sugar moiety of N through a sugar linkage such that the fluorophore is a constituent of a polymeric chain;

i is an integer ranging from 2 to 7; and

r is an integer ranging from 2 to 5.

30. The kit of claim 28, wherein each of the first through fourth pairs of distinguishable wavelengths comprises a combination of two distinguishable wavelengths of a group of 4 to 8 distinguishable wavelengths.

31. The kit of claim 30, wherein each of the first through fourth pairs of distinguishable wavelengths comprises a combination of two distinguishable wavelengths of a group of 4 distinguishable wavelengths.

32. The kit of claim 28, wherein said kit further comprises polymerase.

33. The kit of claim 28, wherein said kit further comprises deoxynucleotides and dideoxynucleotides.