Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-29
2003-07-22
Allen, Marianne P. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C702S020000, C435S091100, C435S091200, C435S069100
Reexamination Certificate
active
06596484
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention is directed to methods for simultaneous identification of differentially expressed mRNAs, as well as measurements of their relative concentrations.
An ultimate goal of biochemical research ought to be a complete characterization of the protein molecules that make up an organism. This would include their identification, sequence determination, demonstration of their anatomical sites of expression, elucidation of their biochemical activities, and understanding of how these activities determine organismic physiology. For medical applications, the description should also include information about how the concentration of each protein changes in response to pharmaceutical or toxic agents.
Let us consider the scope of the problem: How many genes are there? The issue of how many genes are expressed in a mammal is still unsettled after at least two decades of study. There are few direct studies that address patterns of gene expression in different tissues. Mutational load studies (J. O. Bishop, “The Gene Numbers Game,”
Cell
2:81-86 (1974); T. Ohta & M. Kimura, “Functional Organization of Genetic Material as a Product of Molecular Evolution,”
Nature
223:118-119 (1971)) have suggested that there are between 3×10
4
and 10
5
essential genes.
Before cDNA cloning techniques, information on gene expression came from RNA complexity studies: analog measurements (measurements in bulk) based on observations of mixed populations of RNA molecules with different specificities in abundances. To an unexpected extent, early analog complexity studies were distorted by hidden complications of the fact that the molecules in each tissue that make up most of its mRNA mass comprise only a small fraction of its total complexity. Later, cDNA cloning allowed digital measurements (i.e., sequence-specific measurements on individual species) to be made; hence, more recent concepts about mRNA expression are based upon actual observations of individual RNA species.
Brain, liver, and kidney are the mammalian tissues that have been most extensively studied by analog RNA complexity measurements. The lowest estimates of complexity are those of Hastie and Bishop (N. D. Hastie & J. B. Bishop, “The Expression of Three Abundance Classes of Messenger RNA in Mouse Tissues,”
Cell
9:761-774 (1976)), who suggested that 26×10
6
nucleotides of the 3×10
9
base pair rodent genome were expressed in brain, 23×10
6
in liver, and 22×10
6
in kidney, with nearly complete overlap in RNA sets. This indicates a very minimal number of tissue-specific mRNAs. However, experience has shown that these values must clearly be underestimates, because many mRNA molecules, which were probably of abundances below the detection limits of this early study, have been shown to be expressed in brain but detectable in neither liver nor kidney. Many other researchers (J. A. Bantle & W. E. Hahn, “Complexity and Characterization of Polyadenylated RNA in the Mouse Brain,”
Cell
8:139-150 (1976); D. M. Chikaraishi, “Complexity of Cytoplasmic Polyadenylated and Non-Adenylated Rat Brain Ribonucleic Acids,”
Biochemistry
18:3249-3256 (1979)) have measured analog complexities of between 100-200×10
6
nucleotides in brain, and 2-to-3-fold lower estimates in liver and kidney. Of the brain mRNAs, 50-65% are detected in neither liver nor kidney. These values have been supported by digital cloning studies (R. J. Milner & J. G. Sutcliffe, “Gene Expression in Rat Brain,”
Nucl. Acids Res.
11:5497-5520 (1983)).
Analog measurements on bulk mRNA suggested that the average mRNA length was between 1400-1900 nucleotides. In a systematic digital analysis of brain mRNA length using 200 randomly selected brain cDNAs to measure RNA size by northern blotting (Milner & Sutcliffe, supra), it was found that, when the mRNA size data were weighted for RNA prevalence, the average length was 1790 nucleotides, the same as that determined by analog measurements. However, the mRNAs that made up most of the brain mRNA complexity had an average length of 5000 nucleotides. Not only were the rarer brain RNAs longer, but they tended to be brain specific, while the more prevalent brain mRNAs were more ubiquitously expressed and were much shorter on average.
These concepts about mRNA lengths have been corroborated more recently from the length of brain mRNA whose sequences have been determined (J. G. Sutcliffe, “mRNA in the Mammalian Central Nervous System,”
Annu. Rev. Neurosci,
11:157-198 (1988)). Thus, the 1-2×10
8
nucleotide complexity and 5000-nucleotide average mRNA length calculates to an estimated 30,000 mRNAs expressed in the brain, of which about ⅔ are not detected in liver or kidney. Brain apparently accounts for a considerable portion of the tissue-specific genes of mammals. Most brain mRNAs are expressed at low concentration. There are no total-mammal mRNA complexity measurements, nor is it yet known whether 5000 nucleotides is a good mRNA-length estimate for non-neural tissues. A reasonable estimate of total gene number might be between 50,000 and 100,000.
What is most needed to advance by a chemical understanding of physiological function is a menu of protein sequences encoded by the genome plus the cell types in which each is expressed. At present, protein sequences can be reliably deduced only from cDNAs, not from genes, because of the presence of the intervening sequences (introns) in the genomic sequences. Even the complete nucleotide sequence of a mammalian genome will not substitute for characterization of its expressed sequences. Therefore, a systematic strategy for collecting transcribed sequences and demonstrating their sites of expression is needed. Such a strategy would be of particular use in determining sequences expressed differentially within the brain. It is necessarily an eventual goal of such a study to achieve closure; that is, to identify all mRNAs. Closure can be difficult to obtain due to the differing prevalence of various mRNAs and the large number of distinct mRNAs expressed by many distinct tissues. The effort to obtain it allows one to obtain a progressively more reliable description of the dimensions of gene space.
Studies carried out in the laboratory of Craig Venter (M. D. Adams et al., “Complementary DNA Sequencing: Expressed Sequence Tags and Human Genome Project,”
Science
252:1651-1656 (1991); M. D. Adams et al., “Sequence Identification of 2,375 Human Brain Genes,”
Nature
355:632-634 (1992)) have resulted in the isolation of randomly chosen cDNA clones of human brain mRNAs, the determination of short single-pass sequences of their 3′-ends, about 300 base pairs, and a compilation of some 2500 of these as a database of “expressed sequence tags.” This database, while useful, fails to provide any knowledge of differential expression. It is therefore important to be able to recognize genes based on their overall pattern of expression within regions of brain and other tissues and in response to various paradigms, such as various physiological or pathological states or the effects of drug treatment, rather than simply their expression in a single tissue.
Other work has focused on the use of the polymerase chain reaction (PCR) to establish a database. Williams et al. (J. G. K. Williams et al.; “DNA Polymorphisms Amplified by Arbitrary Primers Are Useful as Genetic Markers,”
Nucl. Acids Res.
18:6531-6535 (1990)) and Welsh & McClelland (J. Welsh & McClelland, “Genomic Fingerprinting Using Arbitrarily Primed PCR and a Matrix of Pairwise Combinations of Primers,”
Nucl. Acids Res.
18:7213-7218 (1990)) showed that single 10-mer primers of arbitrarily chosen sequences, i.e., any 10-mer primer off the shelf, when used for PCR with complex DNA templates such as human, plant, yeast, or bacterial genomic DNA, gave rise to an array of PCR products. The priming events were demonstrated to involve incomplete complementarity between the primer and the template DNA. Presumably, partially mismatched primer-binding sites are randomly distributed thro
Erlander Mark G.
Hasel Karl W.
Sutcliffe J. Gregor
Allen Marianne P.
Fitting Thomas
Mahatan C.
The Scripps Research Institute
LandOfFree
Method for simultaneous sequence-specific identification and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for simultaneous sequence-specific identification and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for simultaneous sequence-specific identification and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3095986