Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-08-23
2003-12-02
Ketter, James (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500
Reexamination Certificate
active
06657053
ABSTRACT:
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
Changes in gene expression are important determinants of normal cellular physiology, including cell cycle regulation, differentiation and development, and they directly contribute to abnormal cellular physiology, including developmental anomalies, aberrant programs of differentiation and cancer (1-4). In these contexts, the identification, cloning and characterization of differentially expressed genes will provide relevant and important insights into the molecular determinants of processes such as growth, development, aging, differentiation and cancer. A number of procedures can be used to identify and clone differentially expressed genes. These include, subtractive hybridization (5-10), differential RNA display (DDRT-PCR) (3,4, 11,12), RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) (13,14), representational difference analysis (RDA) (15), serial analysis of gene expression (SAGE) (16,17), electronic subtraction (18,19) and combinatorial gene matrix analyses (20).
Since first introduced by Liang and Pardee (11), DDRT-PCR has gained wide popularity in analyzing and cloning differentially expressed genes. In DDRT-PCR, total RNAs or mRNAs from two or more cell types (or cells grown under different conditions, cells representing different stages of development, cells treated with agents modifying cellular physiology, etc.) are reverse-transcribed with two-base-pair anchored oligo dT primers, which divide mRNA populations into 12 cDNA subgroups. Then, each cDNA subgroup is amplified by PCR with one of 20 arbitrary 10-mer 5′ primers and a 3′ anchored primer and the PCR-amplified cDNA fragments are resolved in DNA sequencing gels. The combinations of primers are designed not only to yield a detectable size and number of bands, but also to display nearly the complete repertoire of mRNA species.
DDRT-PCR is a powerful methodology in which a vast number of mRNA species (>20,000, if no redundancy occurs) can be analyzed with only a small quantity of RNA (about 5 &mgr;g) (11). DDRT-PCR is often the method of choice when the RNA source is limiting, such as tissue biopsies. A direct advantage of DDRT-PCR is the ability to identify and isolate both up- and down-regulated differentially expressed genes in the same reaction. Furthermore, the DDRT-PCR technique permits the display of multiple samples in the same gel, which is useful in defining specific diagnostic alterations in RNA species and for temporally analyzing gene expression changes. However, the DDRT-PCR technique is not problem free. Difficulties encountered when using standard DDRT-PCR include, a high incidence of false positives and redundant gene identification, poor reproducibility, biased gene display and lack of functional information about the cloned cDNA. Furthermore, poor separation can mask differentially expressed genes of low abundance under the intense signals generated by highly expressed genes. The generation of false positives and redundancy can be highly problematic, resulting in an inordinate expenditure of resources to confirm appropriate differential expression and uniqueness of the isolated cDNAs. The cDNAs must be isolated from the gels in pure form (contamination of bands with multiple sequences complicates clone identification), reamplified, placed in an appropriate cloning vector, analyzed for authentic differential expression and finally sequenced. These limitations of the standard DDRT-PCR approaches emphasize the need for improvements in this procedure to more efficiently and selectively identify differentially expressed genes.
A number of modifications and improvements of the DDRT-PCR approach have been described (21-23). Single anchor or degenerate two-base anchor oligo dT primers can be used to streamline the massive numbers of reverse transcription and PCR reactions required for validation of cDNAs as well as to reduce false positives (24,25). Reproducibility can be improved by lengthening the arbitrary 5′ primers to accommodate a convenient restriction site followed by two cycles of PCR with successive low- and high-stringency annealing temperatures (25,26). DDRT-PCR with inosine-containing 5′ arbitrary primers can also increase reproducibility of this approach (27). However, since these modifications have only been analyzed using a subset of primers, further studies are necessary to validate these modifications of DDRT-PCR with additional primers and in several model systems.
In addition to genomic DNA contamination, mispriming, PCR artifacts, the high incidence of false positives and redundancy is also ascribed to poor separation between bands and the complexity of the templates amplified (28). Furthermore, poor separation can mask differentially expressed genes of low abundance under the intense signals generated by highly expressed genes. By enriching for unique cDNAs and removing common ones, it should in principle be possible to enrich for low abundant gene products and significantly decrease the complexity of amplified sequences. In addition, the sequence bias of DDRT-PCR should also be reduced by decreasing template complexity. These assumptions serve as the basis for the development of reciprocal subtraction differential RNA display (RSDD).
Subtractive hybridization, in which hybridization between tester and driver is followed by selective removal of common gene products, enriches for unique gene products in the tester cDNA population and reduces the abundance of common cDNAs (9). A subtracted cDNA library can be analyzed to identify and clone differentially expressed genes by randomly picking colonies or by differential screening (29-31). Although subtractive hybridization has been successfully used to clone a number of differentially expressed genes (5-7,10), this approach is both labor-intensive and does not result in isolation of the full spectrum of genes displaying altered expression (9,18).
In principle, DDRT-PCR performed with subtracted RNA or cDNA samples represents a powerful strategy to clone up and down-regulated gene products. This approach should result in the enrichment of unique sequences and a reduction or elimination of common sequences. This scheme should also result in a consistent reduction in band complexity on a display gel, thereby permitting a clearer separation of cDNAs resulting in fewer false positive reactions. Additionally, it should be possible to use fewer primer sets for reverse transcription and PCR reactions to analyze the complete spectrum of differentially expressed genes. Of particular importance for gene identification and isolation, rare gene products that are masked by strong common gene products should be displayed by using subtraction hybridization in combination with DDRT-PCR. In addition, the DDRT-PCR approach with subtractive libraries could also prove valuable for efficiently screening subtracted cDNA libraries for differentially expressed genes. However, even though subtraction hybridization plus DDRT-PCR appears attractive for the reasons indicated above, a previous attempt to use this approach has proven of only marginal success in consistently reducing the complexity of the signals generated, compared with the standard DDRT-PCR scheme (32).
We presently describe a reciprocal subtraction differential RNA display (RSDD) approach that efficiently and consistently reduces the complexity of DDRT-PCR and results in the identification and cloning of genes displaying anticipated differential expression.
SUMMARY OF THE INVENTION
This invention provides a method for identifying differentially expressed nucleic acids between two samples, comprising: (a) selecting a first and second nucleic acid sample, wherein the nucleic acid samples contain a repertoire of nucleic acids; (b) performing reciprocal subtraction between
Baker & Botts L.L.P.
Ketter James
The Trustees of Columbia University in the City of New York
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