Multiplex probes

• US 10,370,707 B2
• Filed: 10/09/2014
• Issued: 08/06/2019
• Est. Priority Date: 10/09/2013
• Status: Active Grant

• Current Assignee: Fluoresentric, Inc.
• Original Assignee: Fluoresentric, Inc.
• Inventors: Caplin, Brian
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/510,939
• Publication Number:
  US 20150099659A1
• Time in Patent Office: 1,762 Days
• 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 method of detecting a target nucleic acid sequence, comprising:

a) providing

i) a downstream oligonucleotide probe, comprising at least 10 base pairs, having a sequence that is complementary to a first sequence of a target oligonucleotide and that has a cleavable sequence, and

ii) an upstream oligonucleotide primer, comprising at least 10 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, and

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

end, wherein the Tm of the downstream oligonucleotide probe, the Tm of the downstream oligonucleotide primer, and the Tm of the upstream oligonucleotide primer are each within about 15°

C. of the target nucleic acid Tm, and wherein the downstream oligonucleotide primer and the upstream oligonucleotide primer are designed to allow thermal cycling to be conducted within a 15°

C. temperature range;

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, wherein thermal cycling is conducted at least in part within said 15°

C. temperature range.

2. The method 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 method of claim 1, wherein the downstream oligonucleotide probe includes at least one label that is cleaved by nuclease activity.

4. The method 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 method of claim 1, wherein the upstream oligonucleotide primer and the downstream oligonucleotide probe are hybridized to the target oligonucleotide.

6. The method 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 method 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 method 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 method 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 method of claim 1, wherein the downstream oligonucleotide probe includes an upstream label and a downstream label.

11. The method 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 method 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 method 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 method of claim 1, wherein the downstream oligonucleotide probe is a hybrid hairpin/cleaved probe.

15. The method of claim 1, wherein the downstream oligonucleotide probe comprises 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

wherein the upstream oligonucleotide primer comprises 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.