Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-11-02
2003-11-18
Sisson, B. L. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C435S287200, C422S068100, C422S069000, C422S070000, C536S023100, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06649378
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the general fields of molecular biology and medical science, and specifically to a method of extracting nucleic acid, amplifying specific target sequences, and detecting amplified nucleic acid sequences in a self-contained device. This application thus describes a self-contained device capable of rapid and accurate detection of target nucleic acid sequences.
2. Description of the State of the Art
The use of nucleic acid probe tests based on hybridization in routine clinical laboratory procedures is hindered by lack of sensitivity. The ability to amplify nucleic acids from clinical samples has greatly advanced nucleic acid probe technology, providing the sensitivity lacking in earlier versions of non-isotopic assays. Sensitivity afforded by oligonucleotide probe tests utilizing nucleic acid amplification now exceeds that of any other method. Nucleic acid amplification procedures can detect a single copy of a specific nucleic acid sequence. Routine detection and identification of specific gene sequences has extremely broad applications in a number of settings and industries.
The major barrier for the transfer of technology to routine field testing is the absence of an economical and easy-to-use system or apparatus. In order to compete in today's cost conscious environment, genetic based testing must provide for high throughput while incorporating adequate controls and safeguards to prevent false positive results due to sample cross-contamination.
Current technology involves several steps, although recent developments are directed toward automating systems for detection of the amplified target sequence. The first step, extraction of nucleic acids, is accomplished in a variety of ways, for example, phenol extraction, chaotropic reagent extraction, chromatographic purification such as purification on silica membranes (WO 95/01359, specifically incorporated herein) and ultracentrifugation (Maniatis, et al.,
Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982) specifically incorporated herein by reference). Phenol is a well-established health hazard and requires special handling for waste removal. The extraction method is also tedious and labor intensive. Ultracentrifugation often requires the use of expensive and hazardous chemicals as well as the use of sophisticated and costly equipment. The process often requires long run times, sometimes involving one or more days of centrifugation. The easiest and fastest method is separation using chromatography purification.
The second step, the amplification of the target nucleic acid, employs a variety of enzymes known as polymerases and ligases. Polymerase chain reaction (PCR) is the most commonly used amplification technique. The general principles and conditions for amplification of nucleic acids using PCR are quite well known in the art, the details of which are provided in numerous references including U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,965,188, all to Mullis, et al., and all of which are specifically incorporated herein by reference. Thus, the details of PCR technology are not included herein. Other approaches include ligase chain reaction, Q&bgr; replicase, strand displacement assay (SDA), transcription mediated iso CR cycling probe technology, nucleic acid sequence-based amplification (NASBA) and cascade rolling circle amplification (CRCA).
A current protein detection technology for antigen-antibody assays involves the use of microparticles. Furthermore, a variety of microparticle strategies for dip-stick detection in antigen-antibody assays are currently available, for example, a currently marketed at-home pregnancy test (U.S. Pat. No. 5,141,850 to Cole et al., specifically incorporated herein by reference). Such tests use dyed particles that form a visible line following a specific antigen-antibody reaction.
The third and final step, detection of amplified nucleic acid for clinical use relies largely on hybridization of the amplified product and detection with a probe labeled with a variety of enzymes and luminescent reagents. U.S. Pat. No. 5,374,524 to Miller, which is specifically incorporated herein by reference, describes a nucleic acid probe assay that combines nucleic acid amplification and solution hybridization using capture and reporter probes. These techniques require multiple reagents, several washing steps, and specialized equipment for detection of the target nucleic acid. Moreover, these techniques are labor intensive and require technicians with expertise in molecular biology.
The use of probes comprised of oligonucleotide sequences bound to microparticles is well known and illustrated in the prior art. The mechanism for attachment of oligonucleotides to microparticles in hybridization assays and for the purification of nucleic acids is also well known. European Patent No. 200133, which is specifically incorporated herein, describes the attachment of oligonucleotides to water-insoluble particles less than 50 micrometers in diameter used in hybridization assays for the capture of target nucleotides. U.S. Pat. No. 5,387,512 to Wu, which is specifically incorporated herein by reference, describes the use of oligonucleotide sequences covalently bound to microparticles as probes for capturing PCR amplified nucleic acids. U.S. Pat. No. 5,328,825 to Findlay, which is specifically incorporated herein by reference, also describes an oligonucleotide linked by way of a protein or carbohydrate to a water-insoluble particle. The oligonucleotide probe is covalently coupled to the microparticle or other solid support. The sensitivity and specificity of all of the above-reference patents is based on hybridization of the oligonucleotide probe to the target nucleic acid.
The use of incorporated non-radioactive labels into amplification reactions for the detection of nucleic acids is also well known in the art. Nucleic acids modified with biotin (U.S. Pat. No. 4,687,732 to Ward et al.; European Patent No. 063879; both of which are specifically incorporated herein by reference), digoxigenin (European Patent No. 173251, which is specifically incorporated herein) and other haptens have also been used. For example, U.S. Pat. No. 5,344,757 to Graf, which is specifically incorporated herein by reference, uses a nucleic acid probe containing at least one hapten as a label for hybridization with a complementary target nucleic acid bound to a solid membrane. The sensitivity and specificity of these assays is based on the incorporation of a single label in the amplification reaction which can be detected using an antibody specific to the label. The usual case involves an antibody conjugated to an enzyme. Furthermore, the addition of substrate generates a colorimetric or fluorescent change which can be detected with an instrument.
Still, the above-described approaches are labor intensive with many steps and washes; require special and costly equipment for the detection of the target nucleic acid; require trained staff; and take several hours to complete. Several patents have issued which deal with automation of the processes of amplification and subsequent detection of the amplicon. These patents use specialized equipment and are still based on the principle of hybridization and immunoassay technology. For example, European Patent No. 320308, which is specifically incorporated herein by reference, describes a system detecting target nucleic acids amplified by the ligase chain reaction.
Nucleic acid probe technology has developed rapidly in recent years as the scientific community has discovered its value for detection of various diseases, organisms or genetic abnormalities. Amplification techniques have provided the sensitivity to qualitatively determine the presence of even minute quantities of nucleic acid. The drawback to wide spread use of this technology is the possibility of cross contamination of samples since the test is so sensitive. The cost of nucleic acid based tes
Gerdes John C.
Kozwich Diane L.
Hogan & Hartson LLP
O'Rourke Sarah S.
Petersen Steven C.
Sisson B. L.
Xtrana, Inc.
LandOfFree
Self-contained device integrating nucleic acid extraction,... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Self-contained device integrating nucleic acid extraction,..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Self-contained device integrating nucleic acid extraction,... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3174313