Chemistry: molecular biology and microbiology – Apparatus – Including measuring or testing
Reexamination Certificate
2000-06-02
2002-05-21
Whisenant, Ethan C. (Department: 1655)
Chemistry: molecular biology and microbiology
Apparatus
Including measuring or testing
C435S285200, C435S287100, C435S006120, C435S091100, C422S068100
Reexamination Certificate
active
06391624
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to an improved probe which detects the presence of biological material and, more particularly, a probe designed to detect the presence of preselected DNA or RNA segments which are characteristic of a particular biological agent at very low levels.
BACKGROUND OF THE INVENTION
The ability to remotely and automatically detect the presence of specific molecular structures, and particularly the presence of a biologically active structures, is desirable in connection with a wide variety of applications. In environmental and battlefield applications, it is desirable to provide probes which can rapidly detect the presence of highly virulent bacteria, viruses or other organisms which pose a threat to human health. Remote detection of the presence of characteristic deoxyribonucleic acid (DNA) of an organism or virus would enable a highly accurate identification of a biological agent without entering the immediate environment harboring the substance. In view of the potential for the agent to be widely dispersed in air or water, it is also desirable to be able to detect the presence of potential pathogens at very low levels. The detection of pathogens at very low levels can provide an early warning so that responsive protective measures may be quickly implemented. For example, the remote detection of an environmentally released cloud of a biological agent is of vital importance to the military.
A further application where the rapid and accurate detection of low levels of DNA is desirable is in connection with the diagnosis of disease. Biologically active agents, including bacteria and viruses which may cause disease, can be identified by their unique DNA sequences. The rapid and accurate identification of a pathological biological agent from a culture, biopsy or blood sample can assist in the diagnosis of disease and accordingly facilitate an appropriate treatment of a patient by health care professionals at the earliest opportunity. The rapid and accurate detection of the presence of particular segments of DNA or RNA (ribonucleic acid) segments also has applications in connection with genetic screening and in connection with new drug development. For example, probes may be employed for a rapid determination of viral titer in samples after being treated with antiviral or anti-bacterial drugs under investigation to assess their efficacy.
Conventional techniques for identification of unknown biological agents which are frequently invoked include assays which employ antigen-antibody reactions. Although the antibody-antigen assays provide a rapid response, they are not particularly accurate, because often antibodies are not highly specific will bind with more than one antigen. Furthermore, all such assays are not particularly sensitive when only very low levels of the antigen are present. Sensitivity is an assay's ability to make a detection at very low levels of sample material. In some circumstances it is feasible to culture samples containing unknown organisms to increase the volume available for subsequent inspection and testing by trained laboratory technicians, however these procedures are time consuming, expensive and often not practical. Other traditional assay techniques involve the basic physical measurements of the molecular structures of an unknown sample by optical techniques, mass spectroscopy or nuclear magnetic resonance. These physics-based detection techniques attempt to identify the unknown material by pattern matching against the large databases of known material parameters that have been accumulated over the years. Although these techniques can provide a rapid response, these chemical and biological sensors are often large (e.g., table-top to room-sized), are not highly sensitive or accurate, and require expensive laboratory equipment.
One problem with employing these conventional detection technologies is that they require relatively large volumes of the unknown agent, and due to collection techniques, or because the concentration in the sample of a target organism or virus is very low, the identification methods are not sensitive enough to provide for an accurate positive identification. For example, the concentration of the target biological agent in a sample may be very small in view of a wide dispersal and consequent dilution of the agent in air or water. A further problem and concern with conventional detection technologies is the time necessary to make a positive and accurate identification. The ability to make a real time identification of highly virulent agents would enable the implementation of an immediate and appropriate response.
The advent of polymerase chain reaction or PCR technology has significantly advanced many of the problems associated with the problems with very small sample sizes. The PCR process provides a relative rapid and accurate manner to amplify any DNA within a sample and the process has been incorporated into automated systems for the amplification and detection of nucleic acid sequences for infectious agents. Development efforts are presently underway which combine the PCR process and miniaturization of mechanical components such as putting an electrophoretic, optical or mass spectrometer detector-on-a-chip along with fabricating the adjunct microfluidic components, such as tiny valves and pumps, which are necessary to configured an integrated DNA amplifier and detector. Other related amplification techniques include ligase chain reaction, strand displacement amplification, transcription-associated amplification and nucleic sequence-based amplifications.
Although PCR technology addresses some of the problems with insufficient or inadequate sample size and thereby can improve sensitivity, the integration of these process typically increases the time necessary to complete the identification process. Use of the PCR process essentially forecloses the ability to make detections in real time because it requires successive amplification steps. Further, although PCR provides a solution for problems associated with low sample volumes, the technology does not directly address the identification or detection step. Utilization of PCR technology can reduce the time and increase the accuracy compared with conventional assay techniques however the protocols remain complex, there are contamination problems and the process is time consuming and costly.
A number of gene detection techniques involve the hybridization of target DNA with a complementary nucleic acids sequence on a probe. In such ligase-assisted gene detection reactions, specific DNA or RNA sequences are investigated by using them as guides for the covalent joining of pairs of complementary probe molecules. Identification techniques which employ such hybridization steps generally have a high degree of accuracy and can provide a rapid identification of the biological agent. Although these hybridization techniques are highly specific, the low sensitivities of the probes remain a problem associated with these methods. For example, one approach using hybridization of complimentary nucleic acid sequences deals with the sensitivity problem employs PCR-based methodology to amplify the target nucleic acid sequence. The PCR process is followed by a biotinylation step. The amplified products are then captured on a substrate consisting of oligonucleotide-coated paramagnetic microparticles. The detection step employs an avidin-horseradish peroxidase conjugate. Hybridized molecules can be detected by binding to an avidin enzyme complex resulting in a colored product which indicates the presence and/or position of the biotinylated probe.
A further detection technique which employs the hybridization of nucleic acids involves the attachment of fluorescent or radioactive markers on DNA molecules. Hybridization proceeds with oligonucleotides attached to a chip where the locations of particular oligonucleotides have been previously identified. Any DNA in die hybridization mixture flows over the probe and forms a bond to any sites that mimic the opposi
Aitken Andrew C.
Lockheed Martin Corporation
Lu Frank
Venable
Whisenant Ethan C.
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