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Polynucleotide detection method employing self-reporting dual inversion probes

a detection method and probe technology, applied in the field of nucleic acid detection, can solve the problem that the design of the molecular beacon probe is naturally rendered more complicated than the one befor

Inactive Publication Date: 2006-11-02
GEN PROBE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Molecular beacon probe design is naturally rendered somewhat more complicated than the process of designing linear probes due to the added presence of the stem structure.

Method used

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  • Polynucleotide detection method employing self-reporting dual inversion probes
  • Polynucleotide detection method employing self-reporting dual inversion probes
  • Polynucleotide detection method employing self-reporting dual inversion probes

Examples

Experimental program
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Effect test

example 1

Quantifying Interactions Between Polynucleotide Targets and Probes

[0098] The 1034, 1094 and 1093 probes were independently synthesized by solid-phase phosphite triester chemistry using DABCYL-linked controlled pore glass and 5′ fluorescein-labeled phosphoramidite on a Perkin-Elmer (Foster City, Calif.) EXPEDITE model 8909 automated synthesizer. The inversion linkages in the 1034 and 1094 probes, like all of the inversion linkages described herein, were created using a combination of 5′-β-cyanoethyl and 3′-β-cyanoethyl phosphoramidites that were purchased from Glen Research Corporation (Sterling, Va.), Proligo (Boulder, Colo.) or Pierce Biotechnology (Rockford, Ill.). All of these probes were constructed using 2′-OMe nucleotide analogs in their target-complementary loop regions, and standard deoxyribonucleotides in their stem regions. Following synthesis, the probes were deprotected and cleaved from the solid support matrix and then purified using polyacrylamide gel electrophoresis ...

example 2

Target Polynucleotide Binding Triggers Signaling by Parallel-Stem Hybridization Probes

[0107] Individual samples containing the 1093 molecular beacon or the 1094 parallel-stem hybridization probe and either of the two polynucleotide targets described in Example 1 were hybridized and monitored for fluorescence emission. In these procedures the concentrations of the probes were held constant at 0.3 μM, while the concentration of the target varied from 0-3 μM. Parallel procedures were carried out using the 1059 and 1061 targets. In all instances the mixtures were heated to 60° C. for 30 minutes, cooled to 42° C. for 30 minutes, and finally cooled to room temperature (about 23° C.) for 30 minutes before reading fluorescence at room temperature. These thermal step procedures promoted interaction between the probe and the target polynucleotide in the samples that included a target polynucleotide. Fluorescence measurements were carried out using a FLUOROSKAN ASCENT fluorometer (Labsystems,...

example 3

Parallel-Stem Hybridization Probes and Molecular Beacons Exhibit Different Functional Characteristics in Hybridization Assays

[0113] Samples containing the 1093 molecular beacon, the 1094 parallel-stem hybridization probe, or a combination of these two probes were hybridized at one of several probe concentrations with 0-0.3 μM of the 1059 target polynucleotide essentially as described above. Samples containing the 1093 molecular beacon had probe concentrations of either 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM or 0.3 μM. Samples containing the 1094 parallel-stem hybridization probe had probe concentrations of 0.05 μM, 0.1 μM, 0.15 μM or 0.2 μM. Samples containing both the molecular beacon and the parallel-stem hybridization probe all had total probe concentrations of 0.3 μM, with different proportions of this total being due to each of the two probes. In all cases background fluorescence measurements from buffer controls were subtracted from the fluorescence signals measured for each sample...

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Abstract

Method of detecting a target polynucleotide based on the use of dual inversion hybridization probes having stem-and-loop structures, wherein the stem portion of the structure comprises a pair of interactive arms that are substantially prevented from interacting with target polynucleotides. The arms of the dual inversion hybridization probes interact in a conventional antiparallel fashion, but have backbone polarities opposite that of the target-complementary loop portion of the probe. Arm portions of the dual inversion probes do not substantially contribute to sequence-dependent stabilization of probe:target hybrids. Incorporating inversion linkages into the structures of these probes dramatically simplifies the process of designing stem-and-loop hybridization probes.

Description

RELATED APPLICATIONS [0001] This is a continuation of allowed U.S. patent application Ser. No. 10 / 388,918, filed Mar. 14, 2003, issued as U.S. Pat. No. 7,070,933, which is a continuation-in-part of U.S. patent application Ser. No. 10 / 259,272, filed Sep. 27, 2002, which claims the benefit of U.S. Provisional Application No. 60 / 325,600, filed Sep. 28, 2001. The entire disclosures of these related applications are hereby incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates to the field of nucleic acid detection. More specifically, the invention relates to labeled, unitary hybridization probes having stem-and-loop structures, wherein the stem comprises arm structures that cannot substantially interact with target sequences. BACKGROUND OF THE INVENTION [0003] Hybridization probes used for nucleic acid detection generally are single-stranded molecules complementary to a nucleic acid sequence sought to be detected (“target sequence”). Background description...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04
CPCC07H21/04C12Q1/6816C12Q1/6837C12Q1/6876C12Q2565/518C12Q2525/301
Inventor BROWNE, KENNETH
Owner GEN PROBE INC
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