MULTIPLEX PROBES

• US 20150099659A1
• Filed: 10/09/2014
• Published: 04/09/2015
• Est. Priority Date: 10/09/2013
• Status: Active Grant

• Current Assignee: Fluoresentric, Inc.
• Original Assignee: Fluoresentric, Inc.
• Inventors: Caplin, Brian
• Granted Patent: US 10,370,707 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2537/119 Triple helix formation
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

Methods and reagents suitable for conducing polymerase chain reaction are described. In particular, the disclosure provides probes and primers that are suitable in dynamic flux amplification procedures. In aspects, the disclosure provides long oligonucleotide probes and primers, as well as triplex forming probes and primers, which function within the narrow Tm ranges used with dynamic flux amplification. However, embodiments are also provided wherein the probes and primers taught herein can be utilized in standard PCR.

Claims

1. A nucleic acid system, comprising:

a) a downstream oligonucleotide probe, comprising at least 50 base pairs, having a sequence that is complementary to a first sequence of a target oligonucleotide and that has a cleavable sequence; and

b) an upstream oligonucleotide primer, comprising at least 50 base pairs, having a sequence that is complementary to a second sequence of the target oligonucleotide and that has an initial nucleic acid polymerase binding site,

wherein the downstream oligonucleotide probe is configured for a polymerase to cleave mononucleotides or small oligonucleotides at its 5′

end, and

wherein the Tm of the downstream oligonucleotide probe and the upstream oligonucleotide primer is within about 15°

C. of the target nucleic acid Tm.

2. The nucleic acid system of claim 1, wherein the downstream oligonucleotide probe includes a cleavable sequence that is cleaved by polymerization-independent cleavage by a polymerase that binds with the initial nucleic acid polymerase binding site.

3. The nucleic acid system of claim 1, wherein the downstream oligonucleotide probe includes at least one label that is cleaved by nuclease activity.

4. The nucleic acid system of claim 1, wherein the downstream oligonucleotide probe includes a 5′

end having a reporter and having a quencher downstream of the reporter.

5. The nucleic acid system of claim 1, wherein the upstream oligonucleotide primer and the downstream oligonucleotide probe are hybridized to the target oligonucleotide.

6. The nucleic acid system of claim 1, wherein the downstream oligonucleotide probe and the upstream oligonucleotide primer anneal to the target oligonucleotide at a sufficiently close nucleotide distance for a nucleic acid polymerase to contact the 5′

end of the downstream oligonucleotide probe when binding to the initial nucleic acid polymerase binding site of the upstream oligonucleotide primer.

7. The nucleic acid system of claim 1, wherein the upstream oligonucleotide primer and the downstream oligonucleotide probe are cooperatively configured for the downstream oligonucleotide probe to be cleaved by polymerization-independent cleavage by a polymerase that binds with the initial nucleic acid polymerase binding site of the upstream oligonucleotide primer.

8. The nucleic acid system of claim 1, wherein the downstream oligonucleotide probe and the upstream oligonucleotide primer anneal to the target oligonucleotide at a sufficiently far nucleotide distance for a nucleic acid polymerase to not contact a 5′

end of the downstream oligonucleotide probe when the nucleic acid polymerase binds to the initial nucleic acid polymerase binding site of the upstream oligonucleotide primer.

9. The nucleic acid system of claim 1, wherein the upstream oligonucleotide primer and the downstream oligonucleotide probe are cooperatively configured for the downstream oligonucleotide probe to be cleaved by polymerization-dependent cleavage by a polymerase after polymerization.

10. The nucleic acid system of claim 1, wherein the downstream oligonucleotide includes an upstream label and a downstream label.

11. The nucleic acid system of claim 1, wherein the downstream oligonucleotide probe comprises a fluorescent dye and quencher located interchangeably on the 5′

or 3′

end of said probe, such that when the probe is in solution the signal from the fluorescent dye is suppressed by the quencher.

12. The nucleic acid system of claim 1, wherein the downstream oligonucleotide probe comprises a fluorescent dye and quencher located interchangeably on the 5′

or 3′

end of said probe, such that when binding of the upstream oligonucleotide primer and downstream oligonucleotide probe to the target oligonucleotide occurs, along with the binding of a polymerase to the initial nucleic acid polymerase binding site, the polymerase will cleave either the fluorescent label or the quencher of the downstream oligonucleotide probe.

13. The nucleic acid system of claim 1, wherein one of the upstream oligonucleotide primer or downstream oligonucleotide probe include a bathophenanthroline-RU II complex as a label, and the other of the upstream oligonucleotide primer or downstream oligonucleotide probe includes an energy donor molecule.

14. The nucleic acid system of claim 1, wherein the downstream oligonucleotide probe is a hybrid hairpin/cleaved probe.

15. A method of detecting a target nucleic acid sequence, comprising:

a) providing the nucleic acid system according to claim 1;

b) denaturing DNA having the target sequence;

c) hybridizing the upstream oligonucleotide primer and downstream oligonucleotide probe to the target oligonucleotide;

d) binding a polymerase to the initial nucleic acid polymerase binding site;

e) extending the upstream oligonucleotide primer via polymerization with the polymerase toward the downstream oligonucleotide probe;

f) cleaving nucleotides or small oligonucleotides of the downstream oligonucleotide probe; and

g) detecting a signal resulting from said cleaving.

44. A kit having two or more oligonucleotides of claims 1 and 16.

45. The kit of claim 44, wherein at least one of the oligonucleotides is a triplex forming oligonucleotide.

46. The kit of claim 44 including a TFR primer.

47. The kit of claim 44 including a TFO probe.

48. The kit of claim 44 including a TFR primer and a TFO probe.

49. The kit of claim 44 having polymerization reagents.

16. A multiplexing nucleic acid system, comprising:

a) a triplex forming oligonucleotide (TFO) primer having;

i. a hybridizing polynucleotide sequence that is designed to hybridize to a target sequence of a target oligonucleotide; and

ii. a triplex forming region (TFR) associated with the hybridizing polynucleotide sequence; and

b) a target nucleotide.

17. The multiplexing nucleic acid system of claim 16, further comprising a TFO probe that hybridizes to the TFR, thus forming a triplex, in a double stranded DNA sequence that was created during an amplification process of the target nucleotide sequence by the TFR primer.

18. The multiplexing nucleic acid system of claim 17, wherein the TFR probe includes a label moiety selected from the group consisting of:

a fluorescent moiety, radioactive moiety, color moiety, fluorescent reporter moiety, fluorescent quenching moiety, one of a pair of fluorescent resonance energy transfer moieties, and combinations thereof.

19. The multiplexing nucleic acid system of claim 16, wherein the TFR is at the 5′

end of the hybridizing polynucleotide sequence or wherein the TFR is proximal of the 5′

end of the hybridizing polynucleotide sequence.

20. The multiplexing nucleic acid system of claim 16, wherein the TFR is at the 3′

end of the hybridizing polynucleotide sequence or wherein the TFR is proximal of the 3′

end of the hybridizing polynucleotide sequence.

21. The multiplexing nucleic acid system of claim 16, wherein the TFR is at a location between the 5′

end and 3′

end of the hybridizing polynucleotide sequence.

22. The multiplexing nucleic acid system of claim 16, wherein the hybridizing polynucleotide sequence is about 50 bases or greater in length.

23. The multiplexing nucleic acid system of claim 16, wherein the hybridizing polynucleotide sequence ranges from about 20 to about 100 bases in length.

24. The multiplexing nucleic acid system of claim 16, further comprising a label linked to the hybridizing polynucleotide sequence.

25. The multiplexing nucleic acid system of claim 17, further comprising a cap at the 3′

end of the TFO probe that inhibits extension from the 3′

end.

26. The multiplexing nucleic acid system of claim 17, wherein the TFO probe is a triplex forming fluorescent probe (TFFP).

27. The multiplexing nucleic acid system of claim 17, wherein the TFO probe is a triplex forming fluorescent probe (TFFP) and wherein the TFFP anneals to amplified DNA only when the temperature of reaction is at or below annealing temperature of primers, and thereby the TFFP does not participate in polymerization reaction.

28. The multiplexing nucleic acid system of claim 17, wherein the TFO probe is a triplex forming fluorescent probe (TFFP) and the double stranded DNA has a receptor dye.

29. The multiplexing nucleic acid system of claim 28, when the TFFP anneals to amplified double stranded DNA, and a light is shone thereon, the donor dye on the TFFP resonates, as the donor dye resonates, it transfers energy to the acceptor dye located on the double stranded DNA, causing the acceptor dye to fluoresce at a particular wavelength, emitting light of a color that corresponds to that wavelength so as to indicates that the target sequence was present and has been amplified.

30. The multiplexing nucleic acid system of claim 17, wherein the double stranded DNA has a first label and wherein the TFO probe has a second label, wherein the first label and second label provide a detectable emission upon close association.

31. The multiplexing nucleic acid system of claim 16, further comprising a plurality of TFR primers with different target sequences, wherein the TFR primers have different labels.

32. The multiplexing nucleic acid system of claim 17, further comprising a plurality of TFR primers with different target sequences, wherein the TFR primers have different first labels, and the TFO probe includes a corresponding second label that causes fluorescence when associated with the different first labels.

33. The multiplexing nucleic acid system of claim 17, wherein the TFO probe is designed to anneal at approximately the same, or lower, temperature than a Tm of the TFR primers.

34. The multiplexing nucleic acid system of claim 16, wherein the TFR comprises a naturally occurring TFR sequence.

35. The multiplexing nucleic acid system of claim 17, further comprising one or more non-specific DNA binding dyes that bind with hybridized triplex DNA.

36. The multiplexing nucleic acid system of claim 17, further comprising one or more quadruplex binding dyes.

37. The multiplexing nucleic acid system of claim 17, wherein the TFO probe includes a fluorescent dye and quencher.

38. The multiplexing nucleic acid system of claim 17, wherein the TFO probe includes a fluorescent dye and quencher in a hairpin configuration.

39. The multiplexing nucleic acid system of claim 16, wherein the TFR primer creates strands of triplex forming DNA when target DNA includes a sequence having complementarity with the sequence of the TFR primer.

40. The multiplexing nucleic acid system of claim 16, wherein the target DNA includes a natural triplex forming region.

41. A method of amplifying a target nucleic acid sequence, comprising:

a) providing the multiplexing nucleic acid system of claim 16;

b) hybridizing the TFR primer to the target nucleotide, wherein the TFR primer includes a tag having one or more TFRs incorporated therein;

c) extending the TFR primer by polymerization with a polymerase;

d) de-hybridizing the extended TFR primer from the target oligonucleotide;

e) hybridizing a primer to the extended TFR primer;

f) extending the primer so as to form a complement of the extended TFR primer.

42. The method of claim 41, further comprising:

g) providing a TFO probe; and

h) detecting whether the TFO probe binds with a TFR region in the amplified target nucleotide.

43. The method of claim 42, wherein the TFO probe includes a label to facilitate detection of amplified target nucleotide.