Multiplex VGID

Details Multiplex VGID Multiplex VGID

1999-01-15

2002-07-16

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S097000, C536S024100, C536S025400, C436S518000, C436S501000, C436S528000

Reexamination Certificate

active

06420111

ABSTRACT:

1. FIELD OF THE INVENTION
2. BACKGROUND OF THE INVENTION
2.1. CHARACTERISTICS OF DISEASE AND OTHER PHENOTYPES
2.2. GENE IDENTIFICATION BY POSITIONAL CLONING
2.2.1. LINKAGE MAPPING
2.2.2. CHROMOSOMAL LOCALIZATION
2.2.3. FURTHER REFINEMENT
2.2.4. FROM LOCUS TO GENE
2.3. MISMATCH REPAIR
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE DRAWINGS
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. GENETIC HETEROGENEITY
5.1.1. GENETIC HETEROGENEITY IN CELL LINES
5.1.2. GENETIC HETEROGENEITY IN TISSUES
5.2. TWO APPROACHES FOR THE VGID
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METHOD
5.2.1. FIRST APPROACH: CELL LINES OR SOLE TISSUE SAMPLE
5.2.2. SECOND APPROACH: SAMPLES FROM ORGANISMS HAVING CONSANGUINITY
5.3. MISCELLANEOUS METHODS USED IN CONJUNCTION WITH THE VGID
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METHOD
5.3.1. DNA AMPLIFICATION
5.3.2. ADJUSTING STRINGENCY
5.4. PHENOTYPE SELECTION TO OPTIMIZE THE VGID
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METHOD
5.4.1. TISSUE SAMPLE COLLECTION
5.4.2. CELL CULTURE
5.5. TROUBLESHOOTING THE VGID
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METHOD
5.6. ASSAYS FOR PHENOTYPE SELECTION
5.7. DISEASES, DISORDERS, AND OTHER PHENOTYPES
5.8. LINKING OLIGONUCLEOTIDES TO SPECIFIC BINDING LIGANDS
5.9. ANTIBODIES AND DERIVATIVES THEREOF
5.10. ANTIBODY COLUMNS FOR SORTING NUCLEIC ACIDS
5.11. DETECTION OF ANTIBODIES AGAINST PEPTIDE-LABELED OLIGONUCLEOTIDES
6. EXAMPLE: USE OF THE VGID
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METHOD TO IDENTIFY hDinP GENES
6.1. INTRODUCTION
6.2. MATERIALS AND METHODS
6.3. RESULTS
6.4. DISCUSSION
6.5. APPLICATION OF THE VGID
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METHOD TO A COMPLEX, MULTISTAGE SYSTEM
6.5.1. EXAMPLE OF A COMPLEX SYSTEM-BREAST CANCER
6.5.2. ANALYSIS OF A COMPLEX SYSTEM USING MULTIPLEX VGID
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1. FIELD OF THE INVENTION
The present invention relates generally to the field of genomics. More particularly, the present invention relates to a method for gene identification beginning with user-selected input phenotypes. The method is referred to generally as the ValiGene
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Gene Identification method, or the VGID
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method. The method employs nucleic acid mismatch binding protein chromatography to effect a molecular comparison of one phenotype with others. Genes are identified as having a specified function, or as causing or contributing to the cause or pathogenesis of a specified disease, or as associated with a specific phenotype, by virtue of their selection by the method. Identified genes may be used in development of reagents, drugs and/or combinations thereof useful in clinical or other settings for prognosis, diagnosis and/or treatment of diseases, disorders and/or conditions. The method is equally suited for gene identification for agricultural, bio-engineering, medical, veterinary, and many other applications. When more than two source populations of nucleic acids are simultaneously compared, the method may be referred to as multiplex VGID
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.
2. BACKGROUND OF THE INVENTION
Identification of a particular genotype responsible for a given phenotype is an essential goal underlying gene-based medicine because it affords a rational departure point for the development of successful strategies for disease management, therapy and even cure. While, by one recent estimate, only two percent (2%) of the human genome has yet been sequenced, perhaps more than 50% of expressed human genes are at least partially represented in existing databases (Duboule, D., Oct. 24, 1997, Editorial: The Evolution Of Genomics,
Science
278, 555). It is therefore quite clear that understanding functional interactions among the products of expressed genes represents the next great challenge in medicine and biology. This pursuit has been referred to as “functional genomics,” although this term is perhaps too broad to have a clear meaning (Heiter, P. and Boguski, M., Oct. 24, 1997, Functional Genomics: It's All How You Read It,
Science
278, 601-602). Nevertheless, it is the prevailing view that functional genomics generally describes “ . . . a transition or expansion from the mapping and sequencing of genomes . . . to an emphasis on genome function.” (Id.). Further, this new emphasis will require “ . . . creative thinking in developing innovative technologies that make use of the vast resource of structural genomics information.” Perhaps the best definition of functional genomics is “ . . . the development and application of global (genome-wide or system-wide) experimental approaches to assess gene function by making use of the information provided by structural genomics.” (Id., emphasis added).
One of the major advantages of the present invention is the circumvention of large-scale sequencing in determining functional relationships among genes. The VGID
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method of the present invention is a straightforward yet very powerful genetic comparison or subtraction technique. Functional information is obtained from global (i.e. genome-wide) expressed gene comparison of two or more user-defined phenotypes using mismatch binding protein chromatography. With the VGID
SM
method, disease genes may be identified over a time period of weeks, unlike the years required to succeed using positional cloning.
2.1. Characteristics of Disease and Other Phenotypes
Genetic diseases and other genetically-determined phenotypes, irrespective of mode of inheritance, can be due to single or multiple lesions (i.e. mutations) affecting one gene or more than one gene simultaneously. Genetic heterogeneity (i.e. a difference in DNA sequence), by definition, characterizes all diseases which have a genetic component. Genetic diseases can be further categorized among four broad genotypic groups, as described below.
A mono-allelic disease is characterized as having a mutation in a single allele of a single gene. This disease group is the simplest in terms of genetic analysis since mono-allelic diseases arise, by definition, from a unique lesion affecting a single gene. Mono-allelic diseases have also been described as displaying “molecular monomorphism,” which is another way of saying that a single molecular defect in a single gene accounts for the disease phenotype. Since such genetic lesions are unique, they are invariably “causative” of the disease in question. For a mono-allelic disease, only a few affected individuals need to undergo genetic analysis to attribute a given mutation to a disease phenotype. That is, large familial studies are not required to identify the disease-causing gene. Only a few examples of such diseases are known. One example is sickle cell anemia, which is due to a single base substitution (i.e. A→T) in the gene encoding hemoglobin. This base substitution changes the respective codon from GAG to GTG, ultimately resulting in a glutamate-to-valine amino acid substitution at position six of the hemoglobin &bgr; chain molecule and the characteristic, devastating sickle-shaped erythrocyte.
A polyallelic disease is characterized as having several different mutations arising independently in a single gene. Here, each independent mutation event gives rise to a different disease allele. A significant proportion of all genetic disease is thought to result in this way. Because such de novo mutations are so frequent, polyallelism is a very common characteristic of genetic disease. Duchenne's muscular dystrophy (DMD), Becker's myopathy, and cystic fibrosis (CF) are well-known examples of polyallelic diseases (see e.g. McKusick, Mendelian Inheritance in Man, Catalog of Autosomal Dominant, Autosomal Recessive, and X-Linked Phenotypes, 10th Edition, 1992, The Johns Hopkins University Press, Baltimore, Md.). Polyallelism may arise in at least two ways. First, each new case of a disease may arise from an independent mutation event in the target gene. For example, in DMD, at least 30% of cases present novel mutations in the dystrophin gene which differ from all previously-characterized mutations. Second, selective fixation of different founder-effect mutations contributes to the occurrence of polyallelism. One example of this is the &bgr;-thallasemias in which the world population of affected individuals presents remarkably high polyallelism, but local populations are characterized by limited allelic heterogeneity.
Non-allelic genetic disease is characterized as ha

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