Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-05-22
2001-06-26
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100
Reexamination Certificate
active
06251591
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
The invention relates to a method for detecting the concentration of a nucleobase-containing sequence in a sample. In particular, the invention relates to a method for detecting such a concentration using laser-induced fluorescent spectroscopy.
2. Description of Related Art
A popular method for detecting the concentration of nucleic acids in a sample employs ultraviolet spectroscopy to monitor the absorption of the sample at 260 nm. The bases of nucleic acids are chromophores having an ultraviolet radiation absorption maximum at about 260 nm. Thus, the amount of ultraviolet radiation absorbed at 260 nm is an indication of the concentration of nucleic acids in a sample.
However, this method for detecting nucleic acid concentration does not discriminate between different nucleic acid segments in the sample. The method merely provides information regarding the total amount of all nucleic acids in the sample. This can be particularly undesirable when the method is being used to determine the amount of a specific nucleic acid sequence in a sample that inadvertently contains unknown quantities of contaminating nucleic acid sequences.
Antisense probes have been used to obtain qualitative information regarding target nucleobase-containing sequences. See, e.g., U.S. Pat. No. 5,166,330 to Engels etal., U.S. Pat. No. 4,469,863 to Ts'o et al., U.S. Pat. No. 5,539,082 to Nielsen et al. and U.S. Pat. No. 5,503,980 to Cantor, Perry-O'Keefe et al., “Peptide Nucleic Acid Pre-Gel Hybridization: An Alternative to Southern Hybridization,” 93 Proc. Natl. Acad. Sci. USA 14670 (December 1996), and Smulevitch et al., “Enhancement of Strand Invasion by Oligonucleotides Through Manipulation of Backbone Charge, ” 14 Nature Biotechnology 1700 (December 1996) (disclosed in Landsdorp, “Close Encounters of the PNA Kind,” 14 Nature Biotechnology 1653 (December 1996)).
Many types of sample analyses, including certain of the aforementioned antisense probe type assays, rely upon the fluorescent properties of a marker. See, e.g., U.S. Pat. No. 5,594,138 to Dykstra et al., U.S. Pat. No. 4,963,477 to Tchen, U.S. Pat. No. 5,538,848 to Livak et al., U.S. Pat. No. 4,220,450 to Maggio, U.S. Pat. No. 5,332,659 to Kidwell, and U.S. Pat. No. 5,674,698 to Zarling et al., and K. H. Andy Choo, Ed., “In Situ Hybridization Protocols,” Chapters 2 and 4 (Humana Press, Totowa, N.J., 1994).
Fluorescence occurs when a molecule excited by light of one wavelength returns to the ground state by emitting light of a longer wavelength. The exciting and emitted light, being of different wavelengths, can be separated from one another using optical filters, a camera or a CCD. Fluorescence has been used to visualize certain molecules (and hence structures) by light microscopy for many years, and is also used in other analytical techniques, such as flow cytometry. Further, the emission of fluorescence showing different colors can be detected by a human eye, a camera, a charge coupled device (CCD) or a photomultiplier.
Until the present invention, however, it has not been possible to rapidly and specifically test for the concentration of a particular nucleotide sequence in solution using a method which does not destroy the sample, is less hazardous to laboratory personnel than radiation based assays, does not require the cost and delay of separating unhybridized probes from hybridization complexes, does not require the provision of quenching agents, does not require the provision of enzymes, does not require the provision of multiple interactive reporting moieties on, or in the vicinity of, each probe, does not require the provision of up-converting labels, and is readily automated.
All references and prior patent applications cited herein are incorporated herein by reference in their entireties.
SUMMARY OF THE INVENTION
The invention addresses at least the foregoing deficiencies in the art in providing a method for determining a concentration of at least one single stranded or double stranded nucleobase-containing target sequence in a fluid medium, said method comprising:
adding to said fluid medium antisense probes capable of forming hybridization complexes with said at least one nucleobase-containing target sequence;
separating unhybridized antisense probes from said hybridization complexes to form a test medium;
irradiating said test medium with a laser beam having a wavelength which excites fluorescent markers in said hybridization complexes and causes said fluorescent markers to emit fluorescent light;
measuring an intensity of said emitted fluorescent light; and
comparing said measured intensity with a reference intensity to determine a concentration of said at least one target sequence in said fluid medium,
wherein said measured intensity is proportional to said concentration, and wherein said method other than said separating step is entirely conducted without binding said antisense probes, said at least one nucleobase-containing target sequence or said hybridization complexes to a solid support or gel.
The invention also provides a method for determining a concentration of at least one single stranded or double stranded nucleobase-containing target sequence in a fluid medium, said method comprising:
adding to said fluid medium antisense probes capable of forming a hybridization complex with said at least one nucleobase-containing target sequence;
irradiating said fluid medium with a laser beam having a wavelength which excites fluorescent markers in said hybridization complex and causes said fluorescent markers to emit fluorescent light;
measuring an intensity of said emitted fluorescent light; and
comparing said measured intensity with a reference intensity to detect whether said fluid medium contains said at least one target sequence,
wherein said measured intensity is inversely proportional to said concentration, and said method is conducted without separating unhybridized probes from hybridization complexes prior to said signal detecting, and without providing a signal quenching agent on said antisense probes or on said at least one nucleobase-containing target sequence.
The invention thus provides methods for rapidly, economically and efficiently determining the concentration of a target nucleobase-containing in a sample using laser induced fluorescence of antisense probes. The methods can be used, e.g., to determine the concentration of a contaminant in a sample as a part of a system of quality control.
The methods can be conducted without separating unhybridized probes from the hybridization complexes prior to signal detecting, without providing a signal quenching agent on, or in the vicinity of, the probe or the nucleobase-containing sequence, without the use of enzymes, and without the use of up-converting labels.
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patent
Nie Eileen Xiao-Feng
Wu Yuan Min
Caesar Rivise Bernstein Cohen & Pokotilow Ltd.
Jones W. Gary
Lorne Park Research, Inc.
Souaya Jehanne
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