# US 10,370,707 B2 · Multiplex probes

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- Number: US 10,370,707 B2
- Document ID: US10370707B2
- Status: Granted
- Kind: B2
- Application number: 14/510,939
- Filed: 2014-10-09
- Published: 2015-04-09
- Granted: 2019-08-06
- Earliest claimed: 2013-10-09
- Assignees: Fluoresentric, Inc.
- Applicants: Fluoresentric, Inc
- Inventors: Brian Caplin
- Web page: https://patentgrep.com/patent/US10370707B2

## CPC

- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

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.

### Claim 13 (depends on 1)

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.

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 1)

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.