Method of identifying gene transcription patterns

Details Method of identifying gene transcription patterns Method of identifying gene transcription patterns

1999-03-05

2002-10-08

Sisson, Bradley L. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S183000, C435S320100, C536S023500

Reexamination Certificate

active

06461814

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method of identifying gene transcription patterns in a cell or tissue.
BACKGROUND OF THE INVENTION
Expressed Sequence Tag (EST) programs have provided DNA sequence information for a substantial proportion of expressed human genes (Fields, C. et al.,
Nature Genetics
7: 345-346 (1994)) in the human genome. However, DNA sequence information alone is insufficient for a complete understanding of gene function and regulation.
Because only a fraction of the full genetic repertoire is expressed in a cell at any given time, and because gene expression effects cell phenotype, tools to qualitatively and quantitatively monitor gene transcription are needed.
Classical qualitative and quantitative techniques such as northern blotting and nuclease protection assays are accurate and quantitative, but cannot provide information quickly enough to generate global gene expression profiles.
More recent approaches include sequence analysis of random isolates from cDNA libraries, Polymerase Chain Reaction (PCR) and hybridization-array-based methodologies, but each of these methods has limitations.
High-density microarray hybridization of RNA or cDNA corresponding to known genes (Ramsay, G.
Nature Biotechnology
16: 40-44 (1998)) is a fast method for parallel analysis of global gene expression. This method, however, is limited to known genes and the number of genes in a single microarray is limited as well.
Sequencing of random isolates from cDNA libraries to generate ESTs provides quantitative results, but is a daunting task. (Adams, M. D., et al.,
Science
252: 1651-1656 (1991); Adams, M. D. et al.,
Nature
355: 632-634 (1992)). Within cDNA libraries, the frequency of a cDNA clone should be proportional to the steady-state amount of that transcript in the RNA population of the cell or tissue from which the RNA was derived. (Okubo K. et al.,
Nature Genetics
2: 173-179 (1992); Lee, N. H. et al.,
Proc Natl Acad Sci USA
92: 8303-8307 (1995)). This approach, however, requires DNA sequencing efforts beyond the capacity of most laboratories.
PCR-based methods can generate DNA fragments from mRNA pools which differ in size and sequence enabling their separation and identification to form an expression profile. Profiles from different cell or tissue populations to detect differentially expressed genes. This method has been used to establish databases of mRNA fragments. (Williams, J. G. K.,
Nucl. Acids Res.
18:6531 (1990); Welsh, J., et al.
Nucl. Acids Res.,
18:7213 (1990); Woodward, S. R., Mamm. Genome, 3:73 (1992); Nadeau, J. H.,
Mamm. Genome
3:55 (1992)). Some have sought to adapt these methods to compare mRNA populations between two or more samples (Liang, P. et al.
Science
257:967 (1992); See also Welsh, J. et al.,
Nucl. Acid Res.
20:4965 (1992); Liang, P., et al.,
Nucl. Acids Res.,
3269 (1993), and WO 95/13369, Published May 18, 1995. Differential Display and Amplified Fragment Length Polymorphism (AFLP) (Liang P. and Pardee, A. B.
Science
257: 967-971 (1992)), (Vos, P. et al.,
Nucleic Acids Res.
23: 4407-4414. (1995)), for example, can provide gene expression information at the appropriate speed and scale, but these methods can suffer from a lack of precision and reproducibility due to their susceptibility to quantitative PCR artifacts.
Recently, a variation of PCR for a random cDNA sequencing approach was described by Velculescu et al. (Velculescu, V. E. et al.,
Science
270:484 (1995)). This technique, called Serial Analysis of Gene Expression (SAGE), generates short, defined sequences from cDNAs which are randomly ligated in a tail-to-tail fashion and amplified by PCR to form “di-tags”. These di-tags are then concatenated into arrays which are cloned and analyzed by DNA sequencing. Because each sequencing template contains identifiable tags corresponding to many genes, the potential throughput of SAGE exceeds traditional cDNA sequencing, allowing gene transcription profiling in many laboratories.
However, the results for SAGE, like any other PCR process is influenced by factors other than starting template abundance. Sequence-specific differences in “amplification efficiency” are known to give rise to artifactual differences in product yield. That is, the quantity of PCR product may differ in the absence of real differences in starting template. For example, amplification of the same template preparation produces product yields that can vary by as much as 6-fold (Gilliand et al. PCR Protocols. Academic Press, pp 60-69 (1990)). Hence, any PCR-based method that attempts to infer starting template abundance from the quantity of product generated by amplification requires stringent co-amplification controls.
Thus, there is a need for a simple and reproducible method for detecting and quantifying gene transcription, identifying genes, and gene transcription patterns and frequency in individual cells or tissues, which is free from PCR and other artifacts, provides for unknown genes, and yet is fast enough to allow speedy detection and comparison between samples.
In order to circumvent the problems found in the art, we have developed a cDNA tag-based technique called TALEST (Tandem Arrayed Ligation of Expressed Sequence Tags) that avoids PCR amplification artifacts. The technique provides a ~25-fold increase in throughput relative to random cDNA sequencing approaches to gene expression profiling.
SUMMARY OF THE INVENTION
This invention provides an improved method of obtaining short DNA “tag” sequences which allows for determination of the relative abundance of a gene transcript within a given mRNA population.
This invention provides a method of obtaining an array of tags.
This invention provides a method of identifying patterns of gene transcription.
This invention provides a method of detecting differences in gene transcription between two or more mRNA populations.
This invention provides a method of determining the frequency of individual gene transcription in an mRNA population.
This invention provides a method of screening for the effects of a drug on a cell or tissue.
This invention provides a method of detecting the presence of a stress, whether disorder, disease, the onset or proceeding of development or differentiation, exogenous substance (chemical, cofactor, biomolecule or drug), condition (including environmental conditions, such as heat, osmotic pressure, or the like), receptor activity (whether due to a ligand in a receptor or otherwise), abberant cellular condition (including mutation, unusual copy number or the like) in a target organism.
This invention provides a method of isolating a gene.
This invention provides a kit for obtaining a tag or an array of tags.
DETAILED DESCRIPTION OF THE INVENTION
To aid the skilled artisan in understanding this invention the following definitions are provided, where they deviate from the terms commonly used in the art.
“A pattern of gene transcription” as used herein, means the set of genes within a specific tissue or cell type that are transcribed or expressed to form RNA molecules. Which genes are expressed in a specific cell line or tissue and at what level the genes are expressed will depend on factors such as tissue or cell type, stage of development of the cell, tissue, or target organism and whether the cells are normal or transformed cells, such as cancerous cells. For example, a gene is expressed at the embryonic or fetal stage in the development of a specific target organism and then becomes non-expressed as the target organism matures. Or, as another example, a gene is expressed in liver tissue but not in brain tissue of an adult human. In another example, a gene is expressed at low levels in normal lung tissue but is expressed at higher levels in diseased lung tissue. “A punctuating restriction endonuclease” as used herein, means a restriction endonuclease having a probability of recognizing a sequence within each copy of cDNA. Preferably, the punctuating endonuclease recognizes a sequence consisting of less than six bases. More preferably the punctuating endonucleas

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