Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-09-28
2001-01-16
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S287200, C435S287900, C435S288700, C356S301000
Reexamination Certificate
active
06174677
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to non-radioactive DNA gene probe systems, biosensors and methods for gene identification, particularly to non-radioactive gene probes, biosensors and methods for hybridization, detection and identification of hybridized oligonucleotides based on surface-enhanced Raman scattering (SERS) label detection, and more particularly to advanced SER gene probe systems, biosensors, detection systems and methods for detecting hybridized SER gene probes utilizing substrates and membranes suitable for hybridization or blotting materials.
BACKGROUND OF THE INVENTION
There is currently strong interest in the development of nonradioactive deoxyribonucleic acid (DNA) probes for use in a wide variety of applications, such as gene identification, gene mapping, DNA sequencing, medical diagnostics, and biotechnology. Among the various methods for gene identification, technologies using radioactive labels are currently the most widely used. Radioactive label techniques suffer from several disadvantages however. The principal isotope used, Phosphorus-32, has a limited shelflife because it has a 14-day half-life. Secondly, because there is one principal label for gene probes, DNA can only be probed for one sequence at a time. Due to material limitations, probing immobilized DNA with different
3
P-labeled sequences can only be performed a few (3-4) times. Therefore, the researcher must have idea about the sequence prior to probing. In addition to these inconveniences, the potential safety hazard associated with use of radioactive materials makes the technology undesirable. Shipping, handling and waste disposal of radioactive materials are strictly regulated by federal and state guidelines.
Recently, luminescence labels such as fluorescent or chemiluminescent labels have been developed for gene detection. Although sensitivities achieved by luminescence techniques are adequate, alternative techniques with improved spectral selectivities must be developed to overcome the need for radioactive labels and the poor spectral specificity of luminescent labels.
Spectroscopy is an analytical technique concerned with the measurement of the interaction of radiant energy with matter and with the interpretation of the interaction both at the fundamental level and for practical analysis. Interpretation of the spectra produced by various spectroscopic instrumentation has been used to provide fundamental information on atomic and molecular energy levels, the distribution of species within those levels, the nature of processes involving change from one level to another, molecular geometries, chemical bonding, and interaction of molecules in solution. Comparisons of spectra have provided a basis for the determination of qualitative chemical composition and chemical structure, and for quantitative chemical analysis.
Vibrational spectroscopy is a useful technique for characterizing molecules and for determining their chemical structure. The vibrational spectrum of a molecule, based on the molecular structure of that molecule, is a series of sharp lines which constitutes a unique fingerprint of that specific molecular structure.
One particular spectroscopic technique, known as Raman spectroscopy, utilizes the Raman effect, which is a phenomenon observed in the scattering of light as it passes through a material medium, whereby the light suffers a change in frequency and a random alteration in phase. When exciting optical energy of a single wavelength interacts with a molecule, the optical energy scattered by the molecule contains small amounts of optical energy having wavelengths different from that of the incident exciting optical energy. The wavelengths present in the scattered optical energy are characteristic of the structure of the molecule, and the intensity of this optical energy is dependent on the concentration of these molecules.
Raman spectroscopy is a spectrochemical technique that is complementary to infrared spectroscopy, and has been an important analytical tool due to its excellent specificity for chemical group identification. Raman spectroscopy provides a means for obtaining similar molecular vibrational spectra over optical fibers using visible or near infrared light that is transmitted by the optical fibers without significant absorption losses. In Raman spectroscopy, monochromatic light is directed to a sample and the spectrum of the light scattered from the sample is determined.
Raman spectroscopy is a useful tool for chemical analysis due to its excellent capability of chemical group identification. One limitation of conventional Raman spectroscopy is its low sensitivity, often requiring the use of powerful and costly laser sources for excitation. However, a renewed interest has recently developed among Raman spectroscopists as a result of observation that Raman scattering efficiency can be enhanced by factors of up to 108 or more when a compound is adsorbed on or near special metal surfaces. Spectacular enhancement factors due to the microstructured metal surface scattering process is responsible for increasing the intrinsically weak normal Raman scattering (NRS). The technique associated with this phenomenon is known as surface-enhanced Raman scattering (SERS) effect which can increase the Raman signal as well as the resonance Raman signal significantly. When the laser excitation wavelength occurs in the ultraviolet absorption band, the Raman signal of the analyte is enhanced and often called resonance Raman scattering (RRS) signal. The Raman enhancement process is believed to result from a combination of several electromagnetic and chemical effects between the molecule and the metal surface.
Deoxyribonucleic acid is the main carrier of genetic information in most living organisms. DNA is essentially a complex molecule built up of deoxyribonucleotide repeating units. Each unit comprises a sugar, phosphate, and a purine or pyrimidine base. The deoxyribonucleotide units are linked together by the phosphate groups, joining the 3′ position of one sugar to the 5′ position of the next. The alternate sugar and phosphate residues form the backbone of the molecule, and the purine and pyrimidine bases are attached to the backbone via the 1′ position of the deoxyribose. This sugar-phosphate backbone is the same in all DNA molecules. What gives each DNA its individuality is the sequence of the purine and pyrimidine bases. Peptide nucleic acid (PNA) is a DNA analog that combines sequence specific binding to genetic targets with biostability and synthetic versatility (M. Ericson,
Nucleosides and Nucleotides,
16 (1997), p. 617). Molecular probes (DNA, RNA and PNA) having a SERS label provide an excellent combination of detection sensitivity and spectral selectivity, important for many bioassays.
OBJECTS OF THE INVENTION
It is an object of the subject invention to provide a SER gene probe detection system for the detection and identification of biotargets such as Deoxyribonucleic acid (DNA), Ribonucleic acid (RNA) and Peptide nucleic acid (PNA) for the detection of bacteria and viruses and for medical diagnostics.
It is another object of the invention to provide methods for using a SER gene probe for hybridization, detection and identification of hybridized target oligonucleotides such as DNA, RNA and PNA related to bacteria, viruses and genetic material in a variety of samples such as environmental, biological samples or clinical samples.
It is still yet another object of the invention to provide methods for using a SER gene probe for hybridization, detection and identification of hybridized target oligonucleotides such as DNA, RNA and PNA related to bacteria, viruses and genetic material in a variety of samples using substrates suitable for hybridization, membranes and blotting materials.
It is a further object of the invention to provide methods for using a SER gene probe for use in diagnostics of genetic disease, DNA polymorphism, drug screening and drug discovery.
It is still a further object of the invention to provide methods for usin
Chakrabarti Arun
Fredman Jeffrey
Stafford Shelley L.
UT-Battelle LLC
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