Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-26
2003-03-04
Whisenant, Ethan C. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06528256
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is in the field of molecular and cellular biology. In general, the invention is related to a method for the identification and isolation of specific genetic sequences or genetic markers from the genomic DNA or cDNA of an organism. In particular, the invention is related to a method whereby a DNA fragment from a first sample of genomic DNA or cDNA, not found in a second sample of genomic DNA or cDNA, may be identified and isolated via a series of digestion, amplification, purification and sequencing steps. This invention has utility in the identification and isolation of genomic DNA or cDNA sequences that may serve as genetic markers for use in a variety of medical, forensic, industrial and plant breeding procedures.
2. Related Art
Genomic DNA
In examining the structure and physiology of an organism, tissue or cell, it is often desirable to determine its genetic content. The genetic framework (i.e., the genome) of an organism is encoded in the double-stranded sequence of nucleotide bases in the deoxyribonucleic acid (DNA) which is contained in the somatic and germ cells of the organism. The genetic content of a particular segment of DNA, or gene, is only manifested upon production of the protein which the gene ultimately encodes. In order to produce a protein, a complementary copy of one strand of the DNA double helix (the “sense” strand) is produced by polymerase enzymes, resulting in a specific sequence of messenger ribonucleic acid (mRNA). This mRNA is then translated by the protein synthesis machinery of the cell, resulting in the production of the particular protein encoded by the gene. There are additional sequences in the genome that do not encode a protein (i.e., “noncoding” regions) which may serve a structural, regulatory, or unknown function. Thus, the genome of an organism or cell is the complete collection of protein-encoding genes together with intervening noncoding DNA sequences. Importantly, each somatic cell of a multicellular organism contains the full complement of genomic DNA of the organism, except in cases of focal infections or cancers, where one or more xenogeneic DNA sequences may be inserted into the genomic DNA of specific cells and not into other, non-infected, cells in the organism. As noted below, however, the expression of the genes making up the genomic DNA may vary between individual cells.
cDNA and cDNA Libraries
Within a given cell tissue or organism, there exist myriad mRNA species, each encoding a separate and specific protein. This fact provides a powerful tool to investigators interested in studying genetic expression in a tissue or cell—mRNA molecules may be isolated and further manipulated by various molecular biological techniques, thereby allowing the elucidation of the full functional genetic content of a cell, tissue or organism.
One common approach to the study of gene expression is the production of complementary DNA (cDNA) clones. In this technique, the mRNA molecules from an organism are isolated from an extract of the cells or tissues of the organism. This isolation often employs solid chromatography matrices, such as cellulose or hydroxyapatite, to which oligomers of deoxythymidine (dT) have been complexed. Since the 3′ termini on all eukaryotic mRNA molecules contain a string of deoxyadenosine (dA) bases, and since dA binds to dT, the mRNA molecules can be rapidly purified from other molecules and substances in the tissue or cell extract. From these purified mRNA molecules, cDNA copies may be made using the enzyme reverse transcriptase, which results in the production of single-stranded cDNA molecules. The single-stranded cDNAs may then be converted into a complete double-stranded DNA copy of the original mRNA (and thus of the original double-stranded DNA sequence, encoding this mRNA, contained in the genome of the organism) by the action of a DNA polyinerase. The protein-specific double-stranded cDNAs can then be inserted into a plasmid, which is then introduced into a host bacterial cell. The bacterial cells are then grown in culture media, resulting in a population of bacterial cells containing (or in many cases, expressing) the gene of interest.
This entire process, from isolation of mRNA to insertion of the cDNA into a plasmid to growth of bacterial populations containing the isolated gene, is termed “cDNA cloning.” If cDNAs are prepared from a number of different mRNAs, the resulting set of cDNAs is called a “cDNA library,” representing the different functional (i.e., expressed) genes present in the source cell, tissue or organism. Genotypic analysis of these cDNA libraries can yield much information on the structure and function of the organisms from which they were derived.
DNA Fingerprinting
To determine the genotype of an organism, tissue or cell, a variety of molecular biological techniques are employed. These techniques allow researchers, clinicians, forensic scientists and others to probe for the presence of specific genes in the samples which are being studied. The results of such analyses may be useful to researchers in examining the phylogenetic relationship between two organisms, to clinicians in determining whether an individual is infected with a particular disease or is a carrier of a disease-related gene, and to forensic scientists in analyzing crime scene evidence such as blood or other tissues.
A technique often used in such genotypic analysis is known as DNA fingerprinting. This technique relies on the digestion of the DNA of an organism, tissue or cell with a restriction endonuclease enzyme which cleaves the DNA sample into fragments of discrete length. Due to the specificity with which different restriction endonucleases cleave their DNA substrates, a given set of enzymes will always produce the same results, in terms of fragment number and size (the term “size” as used herein is defined as the length and/or molecular weight of a given restriction fragment), from a given DNA sample. The restriction fragments may then be resolved by a variety of techniques such as size exclusion chromatography, gel electrophoresis, or attachment to a variety of solid matrices. Most commonly, gel electrophoresis is performed, and the restriction fragments are resolved into a series of bands on the gel via their differential mobilities within the gel (which is inversely related to fragment size). The pattern of these bands within the gel is specific for a given DNA sample, and is often referred to as the “fingerprint” of that sample.
When the DNA fingerprints of closely related organisms, tissues or even cells are compared, these fingerprints are often quite similar. However, subtle differences between the fingerprints may be observed. These differences, termed “DNA polymorphisms,” tend to increase in number (i.e., the fingerprints become more dissimilar) as DNA samples from more distantly related or unrelated organisms are compared. This technique of examining such Restriction Fragment Length Polymorphisms, or “RFLPs,” has been used for a number of years in genotypic analysis of eukaryotes such as plants (Tanksley, S. D. et al.,
Bio/Technology
7:257-264 (1989)) and animals, including humans (Botstein, D. et al.,
Am. J. Hum. Genet
. 32:314-331 (1980)). In fact, RFLP analysis is being used in combination with other techniques in molecular biology to determine the complete structure (i.e., the “map”) of the human genome (See, e.g., Donis-Keller, H. et al.,
Cell
51:319-337 (1987)). In this way, RFLP analysis can be used to determine the relationship, or lack thereof, between specific organisms, tissues or cells by a simple comparison of differences in their DNA fingerprints.
DNA Amplification
One early drawback to the use of RFLP analysis, however, was its requirement for larger amounts of DNA than are typically available in the samples to be analyzed. In addition, complex genomic samples are often difficult to analyze by RFLP, as a multitude of different DNA molecules are simultaneously fragmented and resolved. As a means of overcoming these d
Kuo Jonathan
Li Wu-Bo
Lin Jhy-Jhu
Invitrogen Corporation
Sterne Kessler Goldstein & Fox P.L.L.C.
Whisenant Ethan C.
LandOfFree
Methods for identification and isolation of specific... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods for identification and isolation of specific..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for identification and isolation of specific... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3014460