Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-06-26
2003-05-13
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100
Reexamination Certificate
active
06562575
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
The present invention relates to assays for sensitive and specific detection of analytes in biological, environmental, pharmaceutical, or industrial samples. Such assays have broad applicability for detection of infectious agents, including bacteria, viruses, fungi, parasites, and other organisms, and for analyzing normal or aberrant genes or gene expression. These assays are useful in fields including human and veterinary medicine, water and environmental quality, food safety, identification of the source of nucleic acids found in forensic samples, as well as paternity testing, and for improvement of plant and animal agricultural products.
Throughout this application, various patents, published patent applications and other publications are referenced and citations provided for them. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
A variety of methods are used in the art to detect and analyze analytes in biological samples. These methods include, among others, methods to identify and distinguish polynucleotide sequences, such as nucleic acid hybridization, methods to increase the quantity of polynucleotides, such as polymerase chain reaction or PCR (U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188), nucleic acid sequence based amplification or NASBA (U.S. Pat. Nos. 5,409,818; 5,130,238; and 5,554,517), transcription-mediated amplification or TMA (U.S. Pat. No. 5,437,990), self-sustained sequence replication or 3SR (Fahy, et al., PCR Methods & Appl. 1: 25-33, 1991), ligation chain reaction or LCR (e.g., U.S. Pat. Nos. 5,494,810 and 5,830,711), continuous amplification reaction or CAR (U.S. Pat. No. 6,027,897), linked linear amplification of nucleic acids or LLA (U.S. Pat. No. 6,027,923) and strand displacement amplification or SDA (U.S. Pat. Nos. 5,455,166; 5,712,124; 5,648,211; 5,631,147), and methods to increase a signal produced in the presence of a polynucleotide, such as rolling circle amplification or RCA (U.S. Pat. No. 5, 854,033), cycling probe reaction or CPR (e.g., U.S. Pat. Nos. 4,876,187 and 5,011,769 and 5,660,988), branched chain amplification (e.g., U.S. Pat. Nos. 4,775,619 and 5,118,605 and 5,380,833 and 5,629,153). Many of these methods are discussed by Brow, et al. in U.S. Pat. No. 6,001,567, which is incorporated herein by reference.
Much work has also been done on using Q-beta (Q-beta) replicase, an RNA-dependent RNA polymerase for increasing the quantity of a polynucleotide or amplifying a signal produced in its presence (e.g., see U.S. Pat. Nos. 4,786,600; 4,957,858; 5,112,734; 5,118,801; 5,312,728; 5,356,774; 5,364,760; 5,472,840; 5,503,979; 5,556,751; 5,556,769; 5,602,001; 5,616,459; 5,620,851; 5,620,870; 5,629,156; 5,631,129; 5,652,107; 5,686,243; 5,750,338; 5,759,773; 5,763,171; 5,763,186; 5,780,273; 5,800,994; 5,807,674; 5,837,466; 5,871,976; 5,959,095; 6,001,570; European Patent Nos. 0266399; 0346594; 0386228; 0436644; 0473693). Some researchers believe it is the most sensitive system known.
Methods using Q-beta replicase have been proposed for detecting of a wide variety of analytes, including nucleic acids (DNAs and RNAs) and segments of nucleic acids; proteins, including glycoproteins and lipoproteins, enzymes, hormones, receptors, antigens, and antibodies; and polysaccharides. For example, Chu, et al. (U.S. Pat. Nos. 4,957,858 and 5,364,760) disclosed methods in which a substrate for Q-beta replicase was attached by various methods to an affinity molecule for an analyte. Following binding of the affinity molecule to the analyte and washing to remove unbound affinity molecules, the substrate was released from the affinity molecule by various method and was replicated by Q-beta replicase. Thus, replication of the substrate served as signaling system for the presence of the analyte. Most of the other work with Q-beta replicase has been limited to detecting nucleic acid analytes.
Q-beta replicase is remarkable because, from a small number of template strands, it can initiate in vitro synthesis of a large number of product strands (Haruna, I., and Spiegelman, S., Proc. Nat. Acad. Sci. USA, 54: 579-587, 1965; Science, 150: 884-886, 1965). A 100,000-fold increase in RNA can occur during a ten-minute reaction (Kramer, F. R., et al., J. Mol. Biol., 89: 719-736, 1974). This striking amplification is the consequence of an autocatalytic reaction mechanism. Single-stranded RNAs serve as templates for the synthesis of complementary single-stranded products. Both the product strand and the template strand are released from the replication complex and are free to serve as templates in subsequent rounds of synthesis. Consequently, the number of RNA strands increases exponentially as the reaction proceeds. The autocatalytic reaction proceeds at an exponential rate until the number of autocatalytically replicatable RNA molecules exceeds the number of active enzyme molecules in the reactions. After that point, the amount of autocatalytically replicatable RNA increases linearly with time. As a consequence, in reactions given a sufficient period of time to reach this linear phase (for example 15 minutes for 100 molecules), the amount of amplified product RNA will be directly related to the logarithm of the number of autocatalytically replicatable RNAs initially added (Lizardi, et al., Nature Biotechnology, 6: 1197-1202, 1988). Since the initial number of autocatalytically replicatable RNA probes is proportional to the amount of target, the amount of target present in the sample being examined may be quantified over a very wide range.
In vitro, Q-beta replicase can utilize a number of other RNA molecules besides the Q-beta genome as templates. One such template, termed midivariant RNA (MDV), discovered as a naturally occurring product in Q-beta replicase reactions, has been used for making amplifiable reporter probes for nucleic acid hybridization assays (e.g., U.S. Pat. No. 4,786,600) . These reporter probes were made by inserting a target-specific probe sequence into an MDV molecule in a site such that it: 1) permits the MDV probe to specifically hybridize to its intended target nucleic acid, and 2) remains replicatable by Q-beta replicase in spite of the additional probe sequence. The MDV serves as an amplifiable detection ligand. One billion or more progeny molecules can be produced from a single starting template recombinant MDV molecule in approximately 30 minutes. Thus, a very large number of detection ligands (MDV RNA molecules) can be produced from very few hybridized reporter probes.
Theoretically, this permits the development of extremely sensitive nucleic acid hybridization assays; that is, assays which are capable of detecting the presence of very few target molecules (or organisms) in a test sample. However, assay sensitivity is a function not only of the amount of signal that can be generated for a given amount of target nucleic acid, but also of the amount of “background” signal that is generated even in the absence of target nucleic acid. The presence of background limits the sensitivity of assays at low target concentrations. Target induced signal must be significantly greater than background in order for assays to be considered reliable. Background has been a serious problem for assays using Q-beta replicase, in part because even a single replicatable RNA molecule will be replicated by the enzyme at an exponential rate.
For example, although Lizardi et al. (Nature Biotechnology, 6: 1197-1202, 1988) showed that MDV-1 RNA with a sequence for a protozoan parasite embedded within it was capable of exponential amplification by Q-beta replicase, they concluded that “practical assays employing recombinant RNAs did not exist yet” using this method because nonspecifically bound probes served as templates for amplification. According to the methods and format used, the recombinant RNA probes bound
Epicentre Technologies Corporation
Horlick Kenneth R.
Quarles & Brady LLP
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