Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-11
2002-07-02
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S025400, C536S023100
Reexamination Certificate
active
06413720
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to the study of gene expression. The invention is more particularly related to methods for isolating newly synthesized RNA, and for using such RNA to evaluate the effect of various stimuli on gene expression.
BACKGROUND OF THE INVENTION
To understand a biological process at the molecular level, it is important to be able to identify genes that are differentially expressed in response to a stimulus. Some differentially expressed genes have been identified by isolating total mRNA before and after exposure to a stimulus, and identifying particular mRNAs that are present at different levels in the two mRNA pools. However, many such genes remain undetected because these pools of total mRNA contain mostly uninformative transcripts, due to the relatively long half-lives of most mRNAs (on the order of hours) within terminally differentiated eukaryotic cells.
Various differential and subtractive methods have been employed to facilitate the discovery of new differentially expressed genes. However, these techniques usually require considerable effort. In an attempt to facilitate the isolation of newly synthesized RNAs, phosphothioate analogues of nucleoside triphosphates have been employed (see, e.g., Melvin et al.,
Eur. J. Biochem.
92:373-379, 1978; Woodford et al.,
Anal. Biochem.
171:166-172, 1988; and Ono and Kawakami,
J. Biochem.
81:1247-1252, 1977). Following incubation with such analogues, newly synthesized mRNAs can be isolated by organomercury affinity chromatography. Such techniques can permit the isolation of newly synthesized RNA, but the analogues can be toxic and can reduce the efficiency of transcription.
Accordingly, there is a need in the art for improved methods for preferentially isolating newly synthesized mRNAs, and for evaluating changes in gene expression as cells or tissues respond to stimuli. The present invention fulfills these needs and further provides other related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides methods for isolating populations of mRNAs that are enriched for newly synthesized transcripts, and for evaluating the effect of stimuli on cellular gene expression. Within one aspect, the present invention provides methods for isolating a population of mRNAs that is enriched for newly synthesized mRNA, comprising the steps of: (a) contacting a cell with a biotinylated rNTP analogue, such that the biotinylated rNTP analogue is incorporated into mRNA transcribed by the cell; (b) terminating transcription within the cell; and (c) separating biotinylated RNA from the cell, and therefrom isolating a population of mRNAs that is enriched for newly synthesized mRNA. Within certain embodiments, the biotinylated RNA may be separated from the cell by: (i) isolating RNA from the cell; (ii) contacting the RNA with streptavidin linked to a support material, such that biotinylated RNA binds to the streptavidin; and (iii) removing unbound RNA from the bound biotinylated RNA.
Within further aspects, the present invention provides methods for determining an effect of a stimulus on RNA transcription, comprising the steps of: (a) simultaneously or in either order, (i) contacting a cell with a biotinylated rNTP analogue, such that the biotinylated rNTP analogue is incorporated into mRNA transcribed by the cell; and (ii) exposing the cell to a stimulus; (b) terminating transcription within the cell; (c) determining a level of a biotinylated RNA in the cell; and (d) comparing the level in step (c) with a predetermined level for a similarly treated cell that is not exposed to the stimulus, and therefrom determining the effect of the stimulus on transcription of the RNA.
The present invention further provides, within other aspects, methods for identifying a gene that is differentially transcribed in cells exposed to a stimulus, comprising the steps of: (a) simultaneously or in either order, (i) contacting a cell with a biotinylated rNTP analogue, such that the biotinylated rNTP analogue is incorporated into mRNA transcribed by the cell; and (ii) exposing the cell to a stimulus; (b) terminating transcription within the cell; and (c) identifying a biotinylated RNA molecule that is present at a level that is altered relative to the level observed in similarly treated cells that are not exposed to the stimulus, and therefrom identifying a gene that is differentially transcribed in cells exposed to a stimulus.
Within other aspects, the present invention provides cDNA libraries prepared by a method comprising the step of reverse transcription of biotinylated mRNA prepared as described above; as well as isolated polypeptides produced by in vitro translation of a biotinylated mRNA molecule prepared as described above.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.
REFERENCES:
patent: 5888727 (1999-03-01), Lund et al.
patent: 0 480 408 (1992-04-01), None
patent: WO 97/07244 (1997-02-01), None
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Hiriyana et al. “Electron microscopic visualization of sites of nascent DNA synthesis by Streptavidin-gold binding to biotinylated nucleotides incorporated in vivo”. J. Cell. Biol. Vol. 107, p. 33-44, 1988.*
Fenn and Herman, “Direct Quantitation of Biotin-Labeled Nucleotide Analogs in RNA Transcripts,”Analytical Biochemistry190:78-83, 1990.
Melvin et al., “Incorporation of 6-Thioguanosine and 4-Thiouridine into RNA,”Eur. J. Biochem.92:373-379, 1978.
Ono and Kawakami, “Separation of Newly-Synthesized RNA by Organomercurial Agarose Affinity Chromatography,”J. Biochem.81:1247-1252, 1977.
Roy et al., “Biotinylated RNA probes of the detection of potato spindle tuber viroid (PSTV) in plants,”Journal of Virological Methods23:149-155, 1989.
Woodford et al. “Selective Isolation of Newly Synthesized Mammalian mRNA after in Vivo labeling . . . ” Analytical Biochemistry, 171, 1988, p. 166-172.*
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McInnes et al. “Non-Racioactive Photobiotin-loabeled probes detect single copy genes and low abundance mRNA” Bio/Technology, vol. 5, 1987, p. 269-272.*
Brogi et al. “Indirect Angiogenic Cytokines Upregulate VEGF and bFGF Gene expression . . . ” Circulation, vol. 90, No. 2, 1994, p. 649-652.*
Srivastava et al. “Purifying Nascent mRNA from Nuclear Run-On Assays . . . ” BioFeedback, vol. 15, No. 2, 1993, p. 226-227.*
Dynal, Biomagnetic Techniques in Molecular Biology, 3rd Edition, 1998.*
Miller et al., “5'Mercuri UTP As A Substrate For Transcription,”Analytical Biochemistry123:94-100, 1982, Abstract Only.
Van De Walle et al., “Species of RNA Synthesized During Early Germination In The Radicle Of The Lentil Vicia-Lens Embryo,”Physiologia Plantarum57:181-188, 1983, Abstract Only.
Chan Kyle W. H.
Pardinas Jose R.
Fredman Jeffrey
Goldberg Jeanine
Pennie & Edmonds LLP
Signal Pharmaceuticals Inc.
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