Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-08-21
2004-01-27
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C424S488000, C424S486000, C436S501000, C536S023100
Reexamination Certificate
active
06682893
ABSTRACT:
BACKGROUND OF THE INVENTION
Rapid advances in the ability to accurately determine polynucleotide sequences, such as DNAs and RNAs from the genomes of organisms, has made possible the sequencing of huge quantities of polynucleotides. In recent years, the entire genomes of microorganisms, such as
Helicobacter pylori
, have been sequenced.
Traditional sequencing methods have relied on automated sequencing equipment which processes a polynucleotide strand one base at a time. A more recent approach, sequencing by hybridization (SBH), which could potentially increase sequencing throughput, relies on fragmenting a target polynucleotide into short segments; these short segments can be captured, for example on an ordered microarray of immobilized complementary single-stranded DNA probes, and the sequences of the target polynucleotide determined by analyzing the overlap of the sequences of the DNA probes bound to fragments of the target polynucleotide. See, for example, U.S. Pat. No. 5,525,464 to Drmanac et al. Microarrays of DNA attached to a solid support have been prepared, see, for example, U.S. Pat. No. 5,445,934 to Fodor et al.
Often, however, DNA microarrays are limited to analyzing nucleic acids in a single, fluid environment. An alternative to conventional DNA microarrays on a solid support is a microarray comprising biological molecules, such as DNA, attached to a matrix of crosslinked polymers known as gel pads. See, e.g., U.S. Pat. No. 5,552,270 to Khrapko et al. Gel pads provide the ability to customize the micro-environment surrounding the DNA in each individual gel pad, which makes possible more sophisticated experiments in micro-array format.
However, although gel pads have certain advantages over convention micro-arrays, new types of arrays, and methods for making them, are needed.
SUMMARY OF THE INVENTION
This invention features gel pad arrays, e.g., arrays on a support, and methods for making and using them. The arrays can be used for sequencing by hybridization (e.g., where the pads include nucleic acid strands immobilized within the gel matrix), for cell based assays (e.g., where the pads include, or are adjacent to and contacting, living cells), and for other uses which will be apparent to one of ordinary skill in the art.
In general, the invention features, a method of providing a gel having a substance disposed within the gel. The method includes:
(1) providing a substrate on which is disposed a gel, e.g., a gel pad or an array of gel pads, and wherein said gel is an intelligent gel, capable of existing in an expanded and a contracted state;
(2) contacting the intelligent gel, while in the expanded state, with the substance, e.g., a solute in a solution, and allowing the substance to enter the gel;
(3) causing the expanded intelligent gel to contract, wherein upon contraction molecules of the substance remain in the gel, thereby forming a gel having a substance disposed, e.g., concentrated or captured, within the gel.
In a preferred embodiment the substance can be: a nucleic acid, e.g., DNA, RNA, or a probe; a protein, e.g., an enzyme which modifies DNA, e.g., DNA polymerase; a particle; a cell; or a reactant.
Substances which can be disposed within a gel can include the following:
A molecule that is important for cell function, for example: a molecule that mediates the expression of specific genes, e.g., hormones, e.g., glucocorticoids; DNA subunits, e.g., nucleotides, e.g., dideoxy nucleotides; a molecule that donates a phosphate group, e.g., ATP; a carbohydrate; a protein; a nucleic acid; a lipid, e.g., a structure based in whole or in part on lipids, e.g., bilayer membrane;
A protein that is generated by a living cell, for example: a protein that interacts with the promoter of a gene, e.g., a transcription factor; a protein that interacts with the origin of replication, e.g., single-strand DNA binding protein; a protein associated with the cytoskeleton of a cell e.g., a matrix attachment protein; a protein associated with the membrane of a cell, e.g., a cell surface receptor; a protein associated with signal transduction pathways within a cell, e.g., the RAS family of proteins; a protein associated with RNA, e.g., heteronuclear RNA binding protein (hnRNP); a protein associated with an immune response, e.g., an antibody; a protein associated with the contraction of muscle, e.g., actin or myosin; a protein that is associated with the chromatin of a cell, e.g., a histone; a protein that mediates protein folding, e.g., a chaperone; a protein associated with cell cycle regulation, e.g., cyclin A;
Enzymes that are generated by living cells, for example: an enzyme that links two nucleic acid molecules together, e.g., a DNA or RNA ligase; an enzyme that cuts nucleic acids, e.g., a restriction enzyme that cuts DNA at the binding site (e.g. EcoR1), a type IIS restriction enzyme that cuts DNA 5′ or 3′ to the binding site (e.g.); an enzyme that modifies the linking number of a closed circular dsDNA molecule, e.g., a topoisomerase; an enzyme that modifies the ends of a chromosome, e.g., a telomerase; an enzyme comprised in whole or in part of RNA, e.g., a ribozyme; an enzyme that generates proteins from amino acid sububits, e.g., a ribosome; an enzyme that transfers phosphate groups onto a protein, e.g., a kinase; an enzyme that removes a phosphate from a protein, e.g., a dephosphorylase; an enzyme that generates a strand of RNA from a template nucleic acid, e.g., an RNA polymerase; an enzyme that generates a strand of DNA from a nucleic acid template, e.g., a DNA polymerase or a reverse transcriptase; an enzyme that functions as part of the DNA repair process, e.g., an enzyme that modifies mismatched base pairs in double-stranded DNA, e.g., an endonuclease or an exonuclease;
Cells, e.g., cells that can be cultured in vitro, for example, living cells, e.g., bacterial cells, e.g., bacterial cells that cause disease in humans (e.g.
Stapholococcus Aureus, E. coli
); living cells, e.g., eukaryotic cells, e.g., fungal cells, e.g., yeast; living cells, e.g., eukaryotic cells, e.g., mammalian cells, e.g., human cells, e.g., colon cancer cell, e.g., human cell lines derived from colon cancer cells, e.g., colo320 cells; cells useful in this assay include cells from nematodes, e.g.,
C. elegans;
flies, e.g.,
D. melanogaster;
mouse, e.g., laboratory strains of mouse; rat, e.g., laboratory strains of rat; chicken; cow; bovine; fish, e.g., zebra fish; feline, e.g., house cat; canine; rabbit, e.g., laboratory strains of rabbit; frogs, e.g.,
Xenopus laevis;
primates, e.g., humans or monkeys;
In preferred embodiments these cells will be modified with a foreign piece of DNA, e.g., a foreign DNA that incorporates itself into genomic DNA through the process of cloning. In other embodiments, foreign DNA enters the cell but is not incorporated into genomic DNA, e.g., the foreign DNA independently replicates in the cytosol, e.g., a plasmid. For example, the cells are modified with a foreign DNA that codes for a selective factor, e.g., a protein that enables the cell to resist a toxic chemical, e.g., an antibiotic, e.g., beta lactamase. Alternatively, the foreign DNA codes for a recombinant molecule, e.g., a recombinant protein, e.g., a fusion protein, e.g., an expressed fusion protein that contains a tag at one end of the molecule, e.g., a FLAG tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) or a repeat of the amino acid histidine (i.e. HIS tag) for capture of the recombinant protein. Alternatively, the foreign DNA codes for a molecule that imparts a property to the cell e.g., a nutritive property, e.g., the ability of the cell to grow in the absence of a molecule, such as an amino acid, in the growth media, e.g., a gene coding for a protein critical to the metabolic pathway leading to a specific amino acid;
Viruses, for example, viruses that invade human cells, e.g., human immunodeficiency virus (HIV) or herpes simplex; viruses that invade bacteria, e.g., bacteriophage; and
Proteinaceous agents that cause disease, for example, proteins that are associated with disease in human cells
Croker Kevin
Taylor Seth
Weber Shane
Fish & Richardson P.C.
Horlick Kenneth R.
Wilder Cynthia
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