Real-time linear detection probes: sensitive 5'-minor groove binder-containing probes for PCR analysis

• US 7,485,442 B2
• Filed: 06/19/2006
• Issued: 02/03/2009
• Est. Priority Date: 12/08/1999
• Status: Expired due to Fees

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Belousov, Yevgeniy S., Dempcy, Robert O., Lokhov, Sergey G., Lukhtanov, Eugeny A., Kutyavin, Igor V., Afonina, Irina A.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/471,292
• Publication Number:
  US 20070003957A1
• Time in Patent Office: 960 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.3, 536/26.6
• US Class Current: 435/91.1
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07C 245/08 with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
• CPC Class Codes: C07D 519/00 Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/572 Five-membered rings
• CPC Class Codes: C07F 9/65522 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 29/0813 substituted by OH, O-C(=X)-R, O-C(=X)-X-R, O-R (X being O,S,NR; R being hydrocarbonyl)
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/1025 : labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 : Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures

Abstract

Oligonucleotide probes/conjugates are provided along with method for their use in assays to monitor amplification wherein the signal produced does not rely on 5′ nuclease digestion.

Claims

1. A method for monitoring the amplification of a target nucleic acid sequence, comprising:

(a) providing a mixture comprising a sample containing the target sequence, one or more oligonucleotide primers complementary to regions of the target sequence, a polymerizing enzyme, nucleotide substrates, and an oligonucleotide conjugate having a formula;

2. A method in accordance with claim 1, wherein said oligonucleotide conjugate is nuclease resistant and exhibits fluorescence in solution and prior to hybridization which is 50% or less of the fluorescence exhibited after hybridization to the target sequence.

3. A method in accordance with claim 2, wherein said MGB portion is selected from the group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1c][1,4]benzodiazepines analogs.

4. A method in accordance with claim 2, wherein said Fl portion is a fluorophore having an emission wavelengths of from about 400 nm to about 800 nm, said fluorophore being selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs.

5. A method in accordance with claim 2, wherein said Q portion is a member selected from the group consisting of mono azo and bis azo dyes.

6. A method in accordance with claim 2, wherein said ODN portion of said conjugate is from 8-25 nucleotides in length.

7. A method in accordance with claim 2, wherein said ODN portion of said conjugate is from 8-15 nucleotides in length and K is a linker having a length of from 10-50 main chain atoms selected from the group consisting of C, O, N, S, P and Si.

8. A method in accordance with claim 7, wherein when said ODN portion has 12 or fewer nucleotides, said K is a polyalkylene glycol linking group having a length of from 8 to 25 main chain atoms;

when said ODN portion has from 13 to 15 nucleotides, said K is a polyalkylene glycol, ribose or deoxy ribose linking group having a length of from 8 to 25 main chain atoms; and

when said ODN portion has from 16-20 nucleotides, said K is a linking group having from 1 to 20 main chain atoms.

9. A method in accordance with claim 8, wherein fluorescence is 50% greater than when K is a bond.

10. A method in accordance with claim 8, wherein fluorescence is 100% greater than K is a bond.

21. A method in accordance with claim 8, wherein fluorescence is 20% greater than when K is a bond.

11. A method for monitoring gene expression comprising:

(a) providing an array of sites comprising oligonucleotide probes, wherein each site of the array comprises oligonucleotide probes having sequences different from the oligonucleotide probes on the other sites

(b) incubating a population of polynucleotides with the array under hybridization conditions sufficient for annealing complementary nucleic acid base pairs to one another; and

(c) determining site contains the probes to which the polynucleotides hybridized;

wherein one or more of the oligonucleotide probes is an oligonucleotide conjugate having the formula;

12. A method in accordance with claim 11, wherein said MGB portion is selected from the group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1c][1,4]benzodiazepines, said Fl portion is a fluorophore having an emission wavelengths of from about 400 nm to about 800 nm, said fluorophore being selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs, and said Q portion is a member selected from the group consisting of mono azo and bis azo dyes.

13. A method in accordance with claim 12, wherein when said ODN portion has 12 or fewer nucleotides, said K is a polyalkylene glycol linking group having a length of from 8 to 25 main chain atoms;

when said ODN portion has from 13 to 15 nucleotides, said K is a polyalkylene glycol, ribose or deoxy ribose linker having a length of from 8 to 25 main chain atoms; and

when said ODN portion has from 16-20 nucleotides, said K is a linking group having from 1 to 20 main chain atoms.

14. A method in accordance with claim 13, wherein fluorescence is 50% greater than when K is a bond.

15. A method in accordance with claim 13, wherein fluorescence is 100% greater than when K is a bond.

22. A method in accordance with claim 13, wherein fluorescence is 20% greater than when K is a bond.

16. A method for discriminating between polynucleotides which differ by a single nucleotide, the method comprising:

(a) separately incubating each of at least two polynucleotides with an oligonucleotide conjugate having the formula;

17. A method in accordance with claim 16, wherein said MGB portion is selected from the-group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1c][1,4]benzodiazepines, said Fl portion is a fluorophore having an emission wavelengths of from about 400 nm to about 800 nm, said fluorophore being selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and BODIPY analogs, and said Q portion is a member selected from the group consisting of mono azo- and bis azo-dyes.

18. A method in accordance with claim 17, wherein when said ODN portion has 12 or fewer nucleotides, said K is a polyalkylene glycol linking group having a length of from 8 to 25 main chain atoms;

when said ODN portion has from 13 to 15 nucleotides, said K is a polyalkylene glycol, ribose or deoxy ribose linking group having a length of from 8 to 25 main chain atoms; and

when said ODN portion has from 16-20 nucleotides, said K is a linking group having from 1 to 20 main chain atoms.

19. A method in accordance with claim 18, wherein fluorescence is 50% greater than when K is a bond.

20. A method in accordance with claim 18, wherein fluorescence is 100% greater than K is a bond.

23. A method in accordance with claim 18, wherein fluorescence is 20% greater than when K is a bond.