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

• US 20030175728A1
• Filed: 06/06/2002
• Published: 09/18/2003
• Est. Priority Date: 12/08/1999
• Status: Active Grant

• Current Assignee: ELITechGroup Inc. (ELITech Group SAS)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Afonina, Irina A., Belousov, Yevgeniy S.
• Granted Patent: US 7,205,105 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 continuous monitoring of polynucleotide amplification, comprising:

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

 

wherein MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore, and said ODN portion has a sequence complementary to a portion of said target sequence being amplified, to provide a mixture;

(b) incubating said mixture under conditions favorable for amplification of said polynucleotide; and

(c) continuously monitoring said amplification by monitoring the fluorescence produced upon conjugate hybridization to amplified target.

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[2,1-c][1,4]benzodiazepines.

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 nucleotides, said K is a polyalkylene glycol linker 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 linker having from 1 to 20 main chain atoms, with the proviso that said linker is sufficient to provide at least a 20% fluorescence enhancement when compared to the same conjugates in which K is a bond.

9. A method in accordance with claim 8, wherein said linker is sufficient to provide at least a 50% fluorescence enhancement when compared to the same conjugates in which K is a bond.

10. A method in accordance with claim 8, wherein said linker is sufficient to provide at least a 100% fluorescence enhancement when compared to the same conjugates in which K is a bond.

39. A method in accordance with claim 1, wherein two different oligonucleotide conjugates are combined with said sample, each of said conjugates having a different fluorophore.

11. A method for monitoring gene expression comprising:

(a) providing an array of oligonucleotide probes of different sequences,

(b) incubating a population of polynucleotides with the array under hybridization conditions, and

(c) determining to which of the oligonucleotide probes in the array the population hybridizes;

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

 

wherein MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore.

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[2,1-c][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, resorufms, 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 nucleotides, said K is a polyalkylene glycol linker 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 linker having from 1 to 20 main chain atoms, with the proviso that said linker is sufficient to provide at least a 20% fluorescence enhancement when compared to the same conjugates in which K is a bond.

14. A method in accordance with claim 13, wherein said linker is sufficient to provide at least a 50% fluorescence enhancement when compared to the same conjugates in which K is a bond.

15. A method in accordance with claim 14, wherein said linker is sufficient to provide at least a 100% fluorescence enhancement when compared to the same conjugates in which 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;

 

wherein MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore, said conjugate having a defined sequence under hybridization conditions, wherein one of the polynucleotides has a target sequence that is perfectly complementary to said oligonucleotide conjugate and at least one other of the polynucleotides has a target sequence having a single-nucleotide mismatch with the oligonucleotide conjugate; and

(b) determining the hybridization strength between each of the polynucleotides and the oligonucleotide conjugate.

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[2,1-c][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, resorufms, 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 nucleotides, said K is a polyalkylene glycol linker 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 linker having from 1 to 20 main chain atoms, with the proviso that said linker is sufficient to provide at least a 20% fluorescence enhancement when compared to the same conjugates in which K is a bond.

19. A method in accordance with claim 18, wherein said linker is sufficient to provide at least a 50% fluorescence enhancement when compared to the same conjugates in which K is a bond.

20. A method in accordance with claim 19, wherein said linker is sufficient to provide at least a 100% fluorescence enhancement when compared to the same conjugates in which K is a bond.

21. A method for detecting a target sequence in a polynucleotide, wherein the polynucleotide is present in a mixture of other polynucleotides, and wherein one or more of the other polynucleotides in the mixture comprise sequences that are related but not identical to the target sequence, the method comprising:

(a) contacting the mixture of polynucleotides with an oligonucleotide conjugate having the formula;

 

wherein MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore; and

wherein the conjugate forms a stable hybrid only with said target sequence that is perfectly complementary to the ODN portion of said conjugate, and the conjugate does not form a stable hybrid with any of the other polynucleotides; and

(b) measuring the fluorescence produced on hybrid formation, whereby hybrid formation indicates the presence of said target sequence.

22. A method in accordance with claim 21, 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[2,1-c][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.

23. A method in accordance with claim 22, wherein when said ODN portion has 12 nucleotides, said K is a polyalkylene glycol linker 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 linker having from 1 to 20 main chain atoms, with the proviso that said linker is sufficient to provide at least a 20% fluorescence enhancement when compared to the same conjugates in which K is a bond.

24. A method in accordance with claim 23, wherein said linker is sufficient to provide at least a 50% fluorescence enhancement when compared to the same conjugates in which K is a bond.

25. A method in accordance with claim 24, wherein said linker is sufficient to provide at least a 100% fluorescence enhancement when compared to the same conjugates in which K is a bond.

26. A method for distinguishing between wild-type, mutant and heterozygous target polynucleotides, said method comprising:

(a) contacting a sample containing a target polynucleotide with two probes wherein a first probe is specific for said wild-type target polynucleotide and a second probe is specific for said mutant target polynucleotide, each of said probes having a formula;

 

wherein MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore;

wherein said first and second probes have different fluorophores and each of said probes forms a stable hybrid only with the target sequence that is perfectly complementary to the ODN portion of said probes; and

(b) measuring the fluorescence produced on hybrid formation, whereby hybrid formation indicates the presence or absence of each of said wild-type, mutant and heterozygous target polynucleotides.

27. A method in accordance with claim 26, wherein the melting temperatures (T_m) for each hybrid produced between said first and second probes and their respective targets are within about 5°

C. of each other.

28. A method in accordance with claim 26, wherein the ODN portion of each of said probes is an oligonucleotide or modified oligonucleotide having from 8 to 18 bases or modified bases.

29. A method in accordance with claim 26, wherein the ODN portion of each of said probes is an oligonucleotide or modified oligonucleotide having from 10 to 15 bases or modified bases.

30. A method in accordance with claim 26, wherein the fluorophore portions of each of said probes are selected from the group consisting of 5-FAM™

, 6-FAM™

, TET™

, JOE™

, HEX™

, VIC™

, NED™

, TAMRA™

, ROX™ and

YY™

.

31. A method in accordance with claim 26, wherein the ODN portion of each of said probes contains at least one modified base.

32. A method in accordance with claim 31, wherein each modified base is independently selected from the group consisting of

6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, 4-amino-1H-pyrazolo[3,4-d]pyrimidine, 1H-pyrazolo[5,4-d]pyrimidin-4(5H)-6(7H)-dione, 6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-hydroxyprop-1-yny)1-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-aminoprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-hydroxyprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-aminoprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 3-prop-1-ynyl-4,6-diaminopyrazolo[3,4-d]pyrimidine, 2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol, 3-(2-aminoethynyl)pyrazolo[3,4-d]pyrimidine-4,6-diamine, 5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione, 5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione, 6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one, 6-amino-5-(3-hydroxyprop-1-yny)-1,3-dihydropyrimidine-2-one, 6-amino-5-(3-aminoprop-1-yny)-1,3-dihydropyrimidine-2-one, 5-[4-amino-3-(3-methoxyprop-1-ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxymethyl)oxolan-3-ol, 6-amino-1-[4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-3-(3-methoxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, 4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, 6-Amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one, 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione, 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine, 3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine and 3-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.

33. A method in accordance with claim 26, wherein said sample is further contacted with a set of primers under amplification conditions and each of said primers contains from one to ten modified bases selected from the group consisting of 6-amino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one, 4-amino-1H-pyrazolo[3,4-d]pyrimidine, 1H-pyrazolo[5,4-d]pyrimidin-4(5H)-6(7H)-dione, 6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-hydroxyprop-1-yny)1-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 6-amino-3-(3-aminoprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidine-4-one, 4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-hydroxyprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 4-amino-3-(3-aminoprop-1-ynyl)pyrazolo[3,4-d]pyrimidine, 3-prop-1-ynyl-4,6-diaminopyrazolo[3,4-d]pyrimidine, 2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol, 3-(2-aminoethynyl)pyrazolo[3,4-d]pyrimidine-4,6-diamine, 5-prop-1-ynyl-1,3-dihydropyrimidine-2,4-dione, 5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidine-2,4-dione, 6-amino-5-prop-1-ynyl-3-dihydropyrimidine-2-one, 6-amino-5-(3-hydroxyprop-1-yny)-1,3-dihydropyrimidine-2-one, 6-amino-5-(3-aminoprop-1-yny)-1,3-dihydropyrimidine-2-one, 5-[4-amino-3-(3-methoxypro-1-ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxymethyl)oxolan-3-ol, 6-amino-1-[4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-3-(3-methoxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, 4-(4,6-Diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, 6-Amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one, 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidine-2,4-dione, 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine, 3Iodo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine, 3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine and 3-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.

34. An oligonucleotide conjugate having the formula:

35. An oligonucleotide conjugate of claim 34, wherein the ODN portion is a RNA, chimera, a PNA or a locked nucleic acid.

36. An oligonucleotide conjugate having the formula:

37. An oligonucleotide conjugate of claim 36, wherein the ODN portion is a RNA, chimera, a PNA or a locked nucleic acid.

38. An oligonucleotide conjugate of claim 36, wherein the target sequence has at least 50% adenine and thymine bases and said conjugate has sufficient modified base substitutions to provide an increase of stability of duplex formation of at least 5°

C., relative to a conjugate without said at least one modified base.