Probe-antiprobe compositions and methods for DNA or RNA detection

• US 8,076,067 B2
• Filed: 08/15/2007
• Issued: 12/13/2011
• Est. Priority Date: 08/15/2006
• Status: Active Grant

• Current Assignee: Genetag Technology, Inc.
• Original Assignee: Genetag Technology, Inc.
• Inventors: Shafer, David A.
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US11/893,323
• Publication Number:
  US 20090209434A1
• Time in Patent Office: 1,581 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/23.1, 536/24.3
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2537/162 Helper probe
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded

Abstract

The invention provides novel compositions and methods for detecting unlabeled nucleic acid targets using labeled polynucleotide probes and partially complementary antiprobes. The interaction of probes, antiprobes and targets result in signaling changes that indicate target frequency. This novel detection mechanism is called a DNA detection switch, and it enable end-point detection, microarray detection and real-time PCR detection of a variety of nucleic acid targets including microbial species and subspecies, drug resistant mutants, and pathogenic strains.

Claims

1. A universal probe-antiprobe composition to detect a target nucleotide sequence in a sample, the composition comprising:

(a) a universal linker/primer comprising

(i) a 5′

universal linker sequence, wherein the 5′

universal linker sequence is not complementary to any sequence in the sample; and

(ii) a 3′

primer sequence complementary to the target nucleotide sequence;

(b) a universal probe comprising

(i) a universal primer sequence that substantially corresponds to the universal linker sequence of the universal linker/primer; and

(ii) a first labeling component; and

(c) a universal antiprobe comprising

(i) a sequence complementary to a partial sequence of the universal probe; and

(ii) a second labeling component;

the composition suitable for real-time amplification and detection of a specific target sequence.

2. The universal probe-antiprobe composition of claim 1, comprising a universal primer sequence that is not complementary to any sequence in a natural organism.

3. The universal probe-antiprobe composition of claim 1, wherein the universal probe is a self-quenching universal probe comprising sequentially:

(i) a 5′

fluorescent labeling compound;

(ii) a cytidine-rich sequence comprising about 2 to about 8 cytidines; and

(iii) a 3′

universal primer sequence; and

wherein the composition does not comprise a universal antiprobe.

4. The universal probe-antiprobe composition of claim 1, wherein the composition comprises a common universal probe and antiprobe and two or more universal linker/primers;

wherein the two or more universal linker/primers comprise different primer sequences specific to different target sites and a common universal linker sequence;

wherein the common universal probe produces a combined detection signal to avoid false a negative in a multiplex amplification of different targets.

5. The universal probe-antiprobe composition of claim 1, wherein the composition comprises two or more different universal probes and anti probes and two or more universal linker/primers;

wherein the two or more universal linker/primers comprise different primer sequences specific to different target sites and different universal linkers specific to the different universal probes and antiprobes;

comprising different labeling components effective to produce a pattern of two or more signals that confirm detection to avoid a false positive in a multiplex amplification of different targets.

6. The universal probe-antiprobe composition of claim 1, wherein false negative or false positive tests are avoided by (i) employing a common self-quenching universal probe with two or more universal linker/primers that prime, amplify and label different targets with the same signal;

or by (ii) employing two or more self-quenching universal probes and universal linker/primers that prime, amplify and label different targets with different signals.

7. The universal probe-antiprobe composition of claim 1, wherein the universal primer/probe comprises a sequence comprising one of SEQ ID NOS:

1 to 16.

8. The composition of claim 1, wherein the composition exhibits a first signaling state when the first and second labeling components are in proximity due to binding of the universal probe and the universal anti probe, and a second signaling state when the first and second labeling components are dissociated due to binding of the universal probe to the target nucleotide sequence, and wherein the first signaling state switches to the second signaling state relative to the presence and frequency of the target nucleotide sequence in the sample.

9. The composition of claim 8, wherein the first and second labeling components comprise a FRET donor-acceptor pair that is a fluorescent donor compound paired with a fluorescent acceptor moiety, wherein the fluorescent acceptor moiety comprises either a longer wavelength fluorescent compound, a quencher compound, or a guanine-rich sequence segment having about 2 to about 8 guanines;

wherein the signaling state of the composition is modulated by the proximity of the donor and acceptor labeling components.

10. The composition of claim 8, wherein the first and second labeling components are selected from the group consisting of a FRET donor-acceptor pair, FAM, TET, HEX, JOE, VIC, ROX, NED, Texas Red, Yakima Yellow, BHQI, BHQ2, BHQ3, Iowa Black FQ, Iowa Black RQ, TAMRA, DABCYL, ElleQuencher, Eclipse Dark Quencher, Methyl Red, DisperseBlue3, Bodipy 493/503, the Cy dyes Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, the AlexaFluor dyes 488, 532, 546, 555, 568, 594, 610, 647, and 680, the PromoFluor dyes 488, 555, 590, 633, 647, and 680, a fluorescent compound, a quencher compound, a color dye compound, a quantum dot, a silver or nanogold compound, and a guanine-rich sequence segment.

11. The composition of claim 10, wherein the first and second labeling components are a FRET donor-acceptor pair.

12. The composition of claim 11, wherein the labeling configuration of the FRET donor acceptor pair is selected from the group consisting of a 5′

donor-labeled probe and a 3′

acceptor-labeled antiprobe;

a 3′

donor-labeled probe and a 5′

acceptor-labeled antiprobe;

a 3′

acceptor-labeled probe and a 5′

donor-labeled antiprobe;

a 5′

acceptor-labeled probe and a 3′

donor-labeled antiprobe;

a 5′ and

3′

donor-labeled probe and a 5′ and

3′

acceptor-labeled antiprobe;

a 5′ and

3′

acceptor-labeled probe and a 5′ and

3′

donor-labeled antiprobe;

a probe with 5′

donor-label and 3′

acceptor-label and an antiprobe with 3′

acceptor-label and 5′

donor-label;

a probe with 5′

acceptor-label and 3′

donor-label and an antiprobe with 3′

donor-label and 5′

acceptor-label;

a probe with a central donor-label and an antiprobe with a central acceptor-label; and

a probe with a central acceptor-label and an antiprobe with central donor-label.