Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-27
2001-04-17
Whisenant, Ethan (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06218114
ABSTRACT:
BACKGROUND OF THE INVENTION
Exploring regulated gene expression in complex biological systems often requires the ability to monitor expression of a large number of expressed genes in a simple, inexpensive assay. Nucleic acid hybridization on filter membranes (e.g., nitrocellulose or nylon) are familiar to most researchers practicing molecular biology, as is autoradiography, which has been the standard detection method for measuring gene expression via hybridization. True-color signals generated by chromogen-converting enzymes yield more information than just intensity, the focus of many known detection methods. Based on how the human eye works, true color can be separated into three components: hue, saturation, and brightness. Alternatively, true color can be separated into the subtractive primary colors cyan, magenta, and yellow. A slight change in any of the three components results in a difference often observable by the human eye.
SUMMARY OF THE INVENTION
In general, the invention relates to a method of detecting a differentially expressed gene by providing a first sample of nucleic acids representing a first population of RNA transcripts and a second sample of nucleic acids representing a second population of RNA transcripts. The nucleic acids in the first sample is labeled (e.g., end-labeled or internally labeled) with a first member of a first specific binding pair, and the nucleic acids in the second sample is labeled (e.g., end-labeled or internally labeled) with a first member of a second specific binding pair. The labeled nucleic acids in each sample are then hybridized to an excess of copies of a gene-specific sequence from a DNA library. The hybridized nucleic acids in each sample are further labeled by binding a second member of the first specific binding pair to the first member of the first specific binding pair and binding a second member of the second specific binding pair to the first member of the second specific binding pair, in which the second member of the first specific binding pair has an activity to convert a first chromogenic substrate (e.g., X-gal) into a first chromogen and the second member of the second specific binding pair has an activity to convert a second chromogenic substrate (e.g, Fast Red/Naphthol AS-MX) into a second chromogen. The first chromogenic substrate and the second chromogenic substrate are contacted with the second member of the first specific binding pair and the second member of the second specific binding pair, respectively. As a result, the first chromogenic substrate and the second chromogenic substrate are converted into the first chromogen and the second chromogen, respectively, and the amounts of the first chromogen and the second chromogen relative to each other are determined. A difference in the amounts of the first chromogen and the second chromogen indicates that the gene-specific sequence is differentially expressed in the first population of RNA transcripts and the second population of RNA transcripts.
In one variation of the assay, the nucleic acids in the first sample and the nucleic acids in the second sample are mixed together after the nucleic acids in the first sample are labeled with the first member of the first specific binding pair and the nucleic acids in the second sample are labeled with the first member of the second specific binding pair. In this case, the first members of the first and second specific binding pairs are different. For example, the first member of the first specific binding pair includes biotin and the first member of the second specific binding pair includes digoxigenin. In addition, the color of the first chromogen is different from the color of the second chromogen.
Alternatively, the nucleic acids in the first sample and the nucleic acids in the second sample are not mixed together and the color of the first chromogen can be the same as the color of the second chromogen.
The invention also relates to a method of determining a copy number of a gene transcript by providing a sample nucleic acid representing the gene transcript and adding a predetermined amount of a known nucleic acid to the sample nucleic acid. The nucleic acids in the sample are stoichiometrically labeled with a first member (e.g., biotin or digoxigenin) of a specific binding pair and separately hybridized to (1) an excess of copies of a sequence specific to the gene transcript and (2) an excess of copies of a sequence specific to the known nucleic acid. The hybridized sample nucleic acid and known nucleic acid are labeled by binding a second member of the specific binding pair to the first member of the specific binding pair, the second member of the specific binding pair having an activity to convert a chromogenic substrate (e.g., X-gal or Fast Red TR/Naphthol AS-MX) into a chromogen. The chromogenic substrate is then contacted with the second member of the specific binding pair, thereby converting the chromogenic substrate into the chromogen. The copy number of the gene transcript in the sample is determined by comparing (1) the amount of chromogen produced from hybridizing the labeled sample nucleic acid to an excess of copies of a sequence specific to the gene transcript to (2) the amount of chromogen produced from hybridizing the labeled known nucleic acid to an excess of copies of a sequence specific to the known nucleic acid.
The specific binding pairs described can be any combination of at least two molecules which bind to each other with high specificity and, preferably, with high affinity and avidity. Such pairs are well known in the art, including biotin/streptavidin.
The methods of the invention require that a second member of each specific binding pair contain an activity that can convert a chromogenic substrate into a chromogen. For example, if streptavidin is the second member of a specific binding pair, streptavidin can further contain a &bgr;-galactosidase enzyme fused to the streptavidin moiety. The &bgr;-galactosidase activity would then convert the relatively colorless chromogenic substrate X-gal into a bluish chromogen. Other suitable activities are well known in the art, including alkaline phosphatase and horseradish peroxidase.
Other features or advantages of the present invention will be apparent from the following drawings, detailed description, and claims. All references cited herein are incorporated by reference.
REFERENCES:
patent: 4825388 (1989-04-01), Dailey et al.
patent: 4996142 (1991-02-01), Al-Hakim et al.
patent: 5316906 (1994-05-01), Haugland et al.
patent: 5338843 (1994-08-01), Quante et al.
patent: 5445934 (1995-08-01), Fodor et al.
patent: 5480791 (1996-01-01), Fujita et al.
patent: 5595726 (1997-01-01), Magda et al.
patent: 5690894 (1997-11-01), Pinkel et al.
patent: 5700637 (1997-12-01), Southern
patent: 5721102 (1998-02-01), Vo-Dinh
patent: 5723320 (1998-03-01), Dehlinger
patent: 5800992 (1998-09-01), Fodor et al.
patent: 5804382 (1998-09-01), Sytkowski et al.
patent: 6040138 (2000-03-01), Lockhart et al.
The Sigma Catalog, p. 82 (1993 Edition).*
Bers et al., “Protein and Nucleic Acid Blotting and Immunobiochemical Detection”, Bio Techniques 3:276-288, 1985.
Lee et al., “A Simplified High Speed Multicolor Immunoblotting Method”, Analytical Biochemistry 175:30-35, 1988.
Liang et al., “Differential Display of Eukaryotic Messenger RNA by Means of the Polymerase Chain Reaction”, Science 257:967-971, 1992.
Schena et al., “Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray”, Science 270:467-470, 1995.
Velculescu et al., “Serial Analysis of Gene Expression”, Science 270:484-487, 1995.
Chang Fu
Chen Jeremy J. W.
Chu Yi-Wen
Peck Konan
Wu Cheng-Wen
Academia Sinica
Fish & Richardson P.C.
Whisenant Ethan
LandOfFree
Methods for detecting differentially expressed genes does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for detecting differentially expressed genes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for detecting differentially expressed genes will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2476456