Nucleic acid hybridization assay utilizing tricyclic target...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C536S023100, C536S024300

Reexamination Certificate

active

06492120

ABSTRACT:

FIELD OF THE INVENTION
This invention relates generally to nucleic acid-based diagnostic assays for detecting, identifying and/or quantitating target sequences in a sample. More specifically, the invention utilizes sequential cleavage of nucleic acids to achieve target and signal amplification through cycling reactions.
BACKGROUND OF THE INVENTION
Nucleic acid hybridization assays are useful in the detection of particular nucleic acid sequences of interest, also referred to as “target sequences”. These target sequences may be characteristic of a particular disease-associated gene, or they may be specific for various organisms or cell types. Accordingly, detection and identification of a particular target sequence can provide diagnostically significant information.
The ability to detect target nucleic acid sequences in a sample by hybridization between a target nucleic acid and a complementary “probe” nucleic acid is the cornerstone of nucleic acid-based diagnostic technologies. These assays can generally be characterized as either “heterogeneous” or “homogeneous.” Heterogeneous assays depend on the ability to separate hybridized from non-hybridized nucleic acids. One such assay involves immobilization of either the target or probe nucleic acid on a solid support so that non-hybridized nucleic acids which remain in the liquid phase can be easily separated after completion of the hybridization reaction (Southern,
J. Mol. Biol
., 98: 503-517 (1975)).
In comparison, homogeneous assays depend on means other than physical separation for distinguishing between hybridized and non-hybridized nucleic acids. The avoidance of a separation step renders homogeneous assays easier to use and, therefore, more desirable. One such homogeneous assay relies on the use of a label attached to a probe nucleic acid that is only capable of generating signal when the target is hybridized to the probe (Nelson, et al.,
Nonisotopic DNA Probe Techniques
, Academic Press, New York, N.Y., pages 274-310 (1992)).
One of the most significant obstacles to the development of nucleic acid-based diagnostic assays has historically been a lack of sensitivity. In particular, when the number of copies of the target nucleic acid in a sample are limited, sensitivity becomes very important. Many strategies have been designed to overcome this obstacle, with variable success. Among the most successful strategies are those that involve either target amplification or signal amplification. Target amplification involves increasing sensitivity by exponentially multiplying the number of copies of target sequences in a sample. Examples of target amplification techniques include the polymerase chain reaction, or “PCR” (Saiki, et al.,
Science
239: 487-491 (1988), and ligase chain reaction, or “LCR” (Wu, et al.,
Genomics
4: 560-569 (1990)).
Another method for increasing sensitivity is to amplify the signal generated by a single target/probe hybridization event. This can be accomplished by using branched probes, each capable of generating multiple detectable signals (Urdea, et al.,
Clin. Chem
. 39: 725-726 (1993)), or by utilizing cycling probe technology or “CPT,” which relies on the ability to generate multiple detectable probes from a single target sequence (Bekkaoui, et al.,
BioTechniques
, 20: 240-248 (1996)).
Still another method for increasing the sensitivity of nucleic acid-based diagnostics employs a cascade reaction to amplify signal. U.S. Pat. No. 4,699,876 discloses a heterogeneous assay utilizing a probe with an enzyme activator attached thereto. Once the probe binds to the target and unhybridized probe is removed, the enzyme activator initiates a signal generation cascade that produces amplified signal levels.
Although a variety of assays exist for detecting nucleic acid, the expanding applications of such assays provides a continuous need for further assay innovation to achieve improved sensitivity and ease of use.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a highly sensitive tricyclic assay system for measuring a target nucleic acid from a sample added to an assay medium, comprising:
a) a non-diffusible first probe-reporter complex, wherein the first probe comprises a nucleotide sequence that is complementary to the target nucleic acid, and wherein the fist probe and the target nucleic acid have sufficient complementarity to hybridize to form a first cleavage site;
b) a first cleavage reagent that recognizes and cleaves the first cleavage site thus forming a diffusable probe fragment-reporter complex and releasing the target nucleic acid into the assay medium;
c) a non-diffusible first target mimic that is functionally separated from the probe reporter complex, wherein the first target mimic comprises a nucleotide sequence that is complementary to the probe, and wherein the first target mimic and probe fragment have sufficient complementarity to hybridize to form a second cleavage site; and
d) a second cleavage reagent, which may be the same or different from the first cleavage reagent, that recognizes and cleaves the second cleavage site thus forming a diffusible target mimic fragment and releasing the probe fragment reporter complex into the assay medium;
wherein the target mimic fragment has sufficient complementarity to hybridize to a second probe;
wherein the target nucleic acid after release into the assay medium following cleavage of the first probe remains capable of binding to a second probe; and
wherein the probe fragment has sufficient complementarity to hybridize to a second target mimic.
As used herein, the term “complementary” is not intended to be limited to cases in which two nucleic acids that are hybridized are perfectly complementary. Accordingly, when one nucleic acid is referred to as being “complementary” to the other nucleic acid, this term is inclusive of pairs of nucleic acids that, when hybridized together, have one (or more) mismatch(es), so long as they have sufficient complementarity to form stable duplexes.
Other aspects of the invention are described throughout the specification and claims.


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