Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-10
2004-05-25
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100
Reexamination Certificate
active
06740492
ABSTRACT:
FIELD OF THE INVENTION
The invention described herein generally relates to the field of biomolecule detection. More specifically, the invention concerns novel compositions and methods for the detection of a biomolecule using cell or viral propagation as an indicator of the presence and location of the biomolecule.
BACKGROUND OF THE INVENTION
In order to understand the complexity of many biological systems, it necessary to use molecular detection techniques that enable a high degree of sensitivity and specificity. A number of organic stains have been adapted for the detection of electrophoretically separated proteins, for example, including Bromphenol Blue, Fast Green (Food Green 3) and Amido Black (Acid Black 1). (Durrem,
J. Am. Chem. Soc.
72:2943 (1950) and Grassman and Hannig,
Z. Physiol. Chem.
290:1 (1952)). Of the organic stains, Coomassie Blue has proved to be one of the most sensitive. (Fazekas De St. Groth et al.,
Biochim. Biophys. Acta
71:377 (1963) and Meyer and Lamberts,
Biochim. Biophys. Acta
107:144 (1965)).
Fluorescent stains, such as fluorescamine are also used to detect proteins and have been shown to detect as little as 6 nanograms of myoglobin. (Ragland et al.,
Anal. Biochem.
59:24 (1974) and Pace et al.,
Biochem. Biophys. Res. Commun.
57:482 (1974)). A related compound, 2-methoxy-2,4-diphenyl-3(2H)-Furanone (MDPH), has the same speed and simplicity of reaction as fluorescamine and can detect as little as one nanogram of protein. Currently, the most sensitive technique for staining proteins is silver staining. In ideal conditions silver staining can detect as little as 0.01 nanogram of protein. (Merril et al.,
Proc. Natl. Acad. Sci. USA
76:4335 (1979) and Switzer et al.,
Anal Biochem.
98:231 (1979)).
Although organic stains, fluorescent stains, and silver staining are quite suitable for many molecular biological applications, the detection of a protein with a high degree of sensitivity is difficult. For example, 0.01 nanogram of a protein of molecular weight 30,000 Da represents 200,000,000 molecules of protein. Since the average number of molecules of a specific protein per cell is 5,000, the limit of current methods of protein detection is on the order of several thousand cells.
Similarly, the detection of nucleic acid sequences with a high degree of sensitivity is difficult. To perform in situ hybridization and Southern and Northern hybridization techniques, for example, several nanograms of target nucleic acid are needed and detection can require several weeks of exposure to autoradiography film. The capacity to amplify specific segments of nucleic acid, made possible by the Polymerase Chain Reaction (PCR), at least in theory, allows an investigator to detect a single molecule of nucleic acid. In practice, however, PCR with minute quantities of template is extremely difficult due to the proclivity of contamination of the sample reaction. (Erlich, ed.,
PCR Technology, Principles and Applications for DNA Amplification
, Freeman & Co., publishers, New York, page 4 (1992)). In view of the foregoing, and notwithstanding the various efforts exemplified in the prior art, there remains a need for compositions and methods for highly sensitive biomolecule detection.
BRIEF SUMMARY OF THE INVENTION
Compositions and methods based on the discovery of a highly sensitive biomolecule detection system are disclosed. Generally, the biomolecule detection system described herein uses cell growth and/or viral propagation as a marker for the presence and location of a target biomolecule. That is, a cell or viral particle that displays a molecule that binds to the target biomolecule can be used to identify the presence and location of very small amounts of a target molecule disposed on a support by monitoring cell or viral propagation. Accordingly, many embodiments are practiced by providing a target biomolecule disposed on a support, contacting said target biomolecule with a cell or virus that displays a molecule that binds to said target biomolecule, removing any non-bound or non-specifically bound cells or virus, culturing the cells bound to the target biomolecule or infecting host cells with the virus-bound target, and determining the presence or location of the target biomolecule by detecting the presence of cell growth or plaques.
In one embodiment, for example, a method of detecting the presence of a biomolecule on a support is accomplished by providing a support having disposed thereon a biomolecule and contacting the biomolecule with a collection of phage, wherein individual phage in the collection have a phage-expressed binding protein so that the collection of phage in aggregate comprises a collection of phage-expressed binding proteins and wherein contact of the biomolecule and the collection of phage results in a non-bound population of phage and a bound population of phage. Subsequently, the non-bound population of phage is removed in a manner that retains the bound population of phage and the bound population of phage is placed together with a host for the phage under conditions that permit the bound phage to infect the host so as to produce a replicated population of phage. The presence of the biomolecule is then determined by detecting the replicated population of phage.
In several aspects of this embodiment, the biomolecule is selected from the group consisting of a lipid, a carbohydrate, a protein, and a nucleic acid and the biomolecule is separated by a one-dimensional or a two-dimensional procedure. Additionally, the support can be selected from the group consisting of a gel, a membrane, a filter, a paper, a chromatography matrix, and a chromatography resin and the gel can have a plastic backing. Preferably, a suspension of phage is contacted with the biomolecule and the unbound population of phage are removed by washing with a buffer. The phage suspension and washing buffer can contain blocking agents including but not limited to, casein, nonfat milk, bovine serum albumin, gelatin, tRNA, and a non-ionic detergent (e.g. Tween or NP-40) to reduce non-specific binding and facilitate removal of non-bound phage. Once the non-bound and non-specifically bound phage are removed, the bound population of phage is replicated on a lawn of host bacteria. The presence of the target protein is then detected by observing bacterial cell lysis. This aspect can also include a step whereby at least one phage from the replicated population of phage is isolated and the phage is incorporated into a pharmaceutical product, a biotechnological tool, or a diagnostic kit.
Another method of detecting the presence of a biomolecule on a support involves providing a support having a biomolecule disposed thereon and contacting the biomolecule with a collection of phage, wherein individual phage in the collection are joined to a protein that can bind to the biomolecule so that the collection of phage in aggregate comprises a collection of proteins that can bind to the biomolecule and, wherein contact of the biomolecule and the collection of phage results in a non-bound population of phage and a bound population of phage. After binding, as above, the non-bound population of phage is removed in a manner that retains the bound population of phage and the bound population of phage is placed together with a host for the phage under conditions that permit the bound phage to infect the host so as to produce a replicated population of phage. The replicated population of phage is then detected and, from this detection step, the presence of the biomolecule is determined.
In some aspects of this embodiment, the protein is joined to the phage by a linker including, but not limited to, avidin or strepavidin or a derivative thereof. Further, the biomolecule can be selected from the group consisting of a lipid, a crabohydrate, a protein, and a nucleic acid and the biomolecule can be separated by a one-dimensional or a two-dimensional procedure. The support can be selected from the group consisting of a gel, a membrane, a filter, a paper, a chromatography matrix, and a chromatography resin
Knobbe Martens Olson & Bear LLP
The United States of America as represented by the Department of
Whisenant Ethan
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