Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-12
2003-04-08
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S091210, C435S091410, C435S091500, C435S091510
Reexamination Certificate
active
06544741
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATIONS
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STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
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INCORPORATION BY REFERENCE
Each of the applications and patents cited in this text, as well as each document or reference cited in each of these applications and patents (including during the prosecution of each issued patent; “application cited documents”), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference in their entirety. More generally, documents or references are cited in this text; and, each of these documents or references (“herein-cited documents or references”), as well as each document or reference cited in each of the herein-cited documents or references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference. Further, various references are cited by their WWW addresses and the content of these references are also expressly incorporated herein by reference.
FIELD OF THE INVENTION
The present invention discloses methods and materials that efficiently normalize cDNA libraries. The present invention also discloses methods and materials for aiding subtractive/differential hybridization and other normalization procedures. The methods and materials can be packaged in the form of a kit. The present invention supports a wide variety of genetic applications, including the isolation, identification and analysis of genes, the analysis and diagnosis of disease states, the study of cellular differentiation, and gene therapy.
BACKGROUND OF THE INVENTION
Approximately 20,000 genes are expressed in a typical mammalian tissue. However, not all genes are expressed in equal copy numbers. A wide range of gene expression patterns and/or levels are found among different cell types, or during stages of development.
Genes that are transcribed in many copies are categorized as “highly expressed genes” or “high copy number genes.” High copy number genes are often associated with maintenance of basic cellular functions, and are therefore known as “house-keeping” genes. Transcription of house-keeping genes is usually constitutive.
Genes that are transcribed in fewer copies are categorized as “moderate or rarely expressed genes” or “medium or low copy number genes.” Transcription of medium or low copy number genes is often subject to regulation, giving rise to differential patterns of expression. A regulated or restricted pattern of expression can be indicative of a unique gene function. For example, expression of VEGF, which plays a critical role in formation of the vascular endothelium, is restricted to the vascular endothelium. Mice that lack this gene have an embryonic lethal phenotype. De-regulation of differential gene expression is associated with many diseases, most notably, cancer.
Cloning of low copy number genes is difficult. Cloning involves screening of genetic libraries, such as cDNA or genomic libraries, using a polynucleotide complementary to the target gene. Prohibitive levels of background in many cDNA libraries lead to repetitive screening and/or sequencing of large numbers of clones. When cDNA libraries are used, hybridization and related screening procedures would be optimized by reducing the amount of high copy number genes or “background” in the library. Presently, there are significant problems with the techniques available for improving the efficiency of cDNA library screening.
For example, a rat liver cDNA library is dominated by fewer than fifty highly/moderately expressed genes, which in turn constitute nearly 50% of the cloned genes (http://www.ncbi.nlm.nih.gov/UniGene/lib.cgi?ORG=Rn&LID=31) and create a large “background” against which low copy number genes must be selected. Consequently, rarely expressed genes, which are often the focus of research efforts to isolate disease genes, are “buried” among the background.
“Normalization” procedures reduce the redundancy of highly expressed genes, or background, in cDNA libraries, thereby increasing the relative amount of transcripts represented by rarely expressed genes. Previous normalization procedures concern annealing opposite strands of nucleic acids. That is, the higher the concentration of a nucleic acid fragment, the higher the probability that it will anneal to its complementary fragment. Thus, annealing occurs more rapidly to a high copy number transcript than a low copy number transcript.
Soares et al. (1994) Proc. Natl. Acad. Sci. USA 91:9228-32 concerns such a procedure, where the unannealed, more rarely expressed single-stranded nucleic acid population is separated from the more highly expressed double-stranded population. The separation method involves hydroxyapatite column chromatography, wherein the double-stranded DNA selectively binds to hydroxyapatite. The single-stranded DNA is recovered from the flow-through fraction, processed and cloned in bacteria. Despite claims of high “normalization efficiency”, this method is cumbersome to use, requires high amounts of input DNA, involves several reaction steps, and results in a loss of material from failure to fully elute single-stranded DNA from the column. The end result is an incomplete reduction of genetic redundancy (or poor normalization). http://www.ncbi.nlm.nih.gov/dbEST/index.html provides public cDNA libraries.
Another normalization method concerns digestion by restriction endonucleases, wherein the preferential target is double-stranded DNA. This method is disclosed in “Normalizing cDNA libraries using the Eppendorf Thermomixer” by Scheinert & Schalk; Bernhard-Nocht-Inst. of Tropical Medicine, Virology Dept., Hamburg, Del.; Eppendorf products, application catalogue and http://www.eppendorf.com/prepa/page8.html.
Another method is enzymatic degrading subtraction (EDS) for construction of subtractive libraries from PCR amplified cDNA. Zeng et al. (1994) Nucl. Acids Res. 22:4381-4385. The tester DNA is blocked by thionucleotide incorporation, the rate of hybridization is accelerated by phenol-emulsion reassociation, and the driver cDNA and double-stranded hybrid molecules are enzymatically removed by digestion with exonucleases III and VII rather than by physical partitioning. Here, double-stranded DNA represents the more highly expressed genes, having a higher probability for annealing to its complementary fragment. EDS has been used to construct a substance library enriched for cDNAs expressed in adult but not embryonic rat brains.
Yet another normalization method involves hybridization to genomic DNA coated onto beads. In genomic DNA, all genes are essentially present in the same copy number, and thus highly expressed genes will hybridize to genomic DNA in the same copy number as rarely expressed genes. Coche (1997) Met. Mol. Biol. 67:359-369. However, this method suffers from many of the same shortcomings as hydroxyapatite-column-chromatography separation methods discussed above. Consequently, this method is not widely used for normalization, but for selecting cDNA encoded by a chromosome or genomic DNA fragment of interest.
Suppression Subtractive Hybridization (SSH) and other subtraction methods preserve the copy number difference in the subtracted population leading to redundancy. A modified SSH method was developed by Diachenko et al. (1999) Met. Enzymol. 303:349-380. The modified SSH attempts normalization and subtraction in one reaction.
In the end, hybridization-based methods that depend on the efficiency of hybridization and the sensitivity of the separation/selection techniques, are affected by or influenced by variability in sources of RNA, and thus lack reproducibility in practice. Moreover, current normalization methods are not recommended for full-length library preparation because they either require high temperature treatment to denature and renature the DNAs, which break longer
Browdy and Neimark PLLC
Myers Carla J.
Quark Biotech, Inc.
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