Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-11
2004-02-03
Berman, Susan W. (Department: 1711)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S181000, C436S532000, C436S531000, C522S114000, C522S116000, C522S117000, C522S120000, C522S121000, C522S152000, C522S153000, C522S148000, C527S200000, C527S201000
Reexamination Certificate
active
06686161
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention provides solid supports (e.g., glass) and hydrogels (particularly hydrogel arrays present on a solid support) comprising one or more reactive sites for the attachment of biomolecules, as well as biomolecules comprising one or more reactive sites for attachment to solid supports and hydrogels. Desirably the polyacrylamide hydrogels are made from prepolymer (including especially polyacrylamide reactive polymers). The invention thus desirably provides novel compositions and methods for the preparation of biomolecules, solid supports, and hydrogels comprising reactive sites. In particular, the invention provides for preparation of solid supports, hydrogels, and hydrogel arrays wherein one or more biomolecules is attached by means of the reactive site present in the biomolecule(s) and the reactive site present on the solid support or polymer hydrogel in a photocycloaddition reaction.
BACKGROUND OF THE INVENTION
Acrylamide (CH
2
═CHCONH
2
; C.A.S. 79-06-1; also known as acrylamide monomer, acrylic amide, propenamide, and 2-propenamide) is an odorless, free-flowing white crystalline substance that is used as a chemical intermediate in the production and synthesis of polyacrylamides. These high molecular weight polymers have a variety of uses and further can be modified to optimize nonionic, anionic, or cationic properties for specified uses.
Polyacrylamide hydrogels are especially employed as molecular sieves for the separation of nucleic acids, proteins, and other moieties, and as binding layers to adhere to surfaces biological molecules including, but not limited to, proteins, peptides, oligonucleotides, polynucleotides, and larger nucleic acid fragments. The gels currently are produced as thin sheets or slabs, typically by depositing a solution of acrylamide monomer, a crosslinker such methylene bisacrylamide, and an initiator such as N,N,N′,N′-tetramethylethylendiamine (TEMED) in between two glass surfaces (e.g., glass plates or microscope slides) using a spacer to obtain the desired thickness of polyacrylamide. Generally, the acrylamide polymerization solution is a 4-5% solution (acrylamide/bisacrylamide 19/1) in water/glycerol, with a nominal amount of initiator added. The solution is polymerized and crosslinked either by ultraviolet (UV) radiation (e.g., 254 nm for at least about 15 minutes, or other appropriate UV conditions, collectively termed “photopolymerization”), or by thermal initiation at elevated temperature (e.g., typically at about 40° C.). Following polymerization and crosslinking, the top glass slide is removed from the surface to uncover the gel. The pore size (or “sieving properties”) of the gel is controlled by changing the amount of crosslinker and the % solids in the monomer solution. The pore size also can be controlled by changing the polymerization temperature.
In the fabrication of polyacrylamide hydrogel arrays (i.e., patterned gels) used as binding layers for biological molecules, the acrylamide solution typically is imaged through a mask during the UV polymerization/crosslinking step. The top glass slide is removed after polymerization, and the unpolymerized monomer is washed away (developed) with water leaving a fine feature pattern of polyacrylamide hydrogel, the crosslinked polyacrylamide hydrogel pads. Further, in an application of lithographic techniques known in the semiconductor industry, light can be applied to discrete locations on the surface of a polyacrylamide hydrogel to activate these specified regions for the attachment of an anti-ligand, such as an antibody or antigen, hormone or hormone receptor, oligonucleotide, or polysaccharide on the surface (e.g., a polyacrylamide hydrogel surface) of a solid support (PCT International Application WO 91/07087, incorporated by reference). Following fabrication of the hydrogel array, the polyacrylamide subsequently is modified to include functional groups for the attachment of moieties, and the moieties (e.g., DNA) later are attached.
Immobilization of biomolecules (e.g., DNA, RNA, peptides, and proteins, to name but a few) through chemical attachment on a solid support or within a matrix material (e.g., hydrogel, e.g., present on a solid support) has become a very important aspect of molecular biology research (e.g., including, but not limited to, DNA synthesis, DNA sequencing by hybridization, analysis of gene expression, and drug discovery) especially in the manufacturing and application of microarray or chip-based technologies. Typical procedures for attaching a biomolecule to a surface involve multiple reaction steps, often requiring chemical modification of the solid support itself, or the hydrogel present on a solid support, in order to provide a proper chemical functionality capable forming a covalent bond with the biomolecule. The efficiency of the attachment chemistry and strength of the chemical bonds formed are critical to the fabrication and ultimate performance of the microarray.
For polyacrylamide, or other hydrogel-based microarrays, the necessary functionality for attachment of biomolecules (e.g., such as a DNA oligonucleotide probe) presently is incorporated by chemical modification of the hydrogel through the formation of amide, ester, or disulfide bonds after polymerization and crosslinking of the hydrogel. An unresolved problem with this approach is the less than optimal stability of the attachment chemistry over time, especially during subsequent manufacturing steps, and under use conditions where the microarray is exposed to high temperatures, ionic solutions, and multiple wash steps. This may promote continued depletion in the amount of probe molecules present in the array through washing away of these molecules, and thus reduce the performance and limit the useful life of the microarray. A further problem is the low efficiency of the method.
Another approach that has been employed is the polymerization of a suitable “attachment co-monomer” into the polyacrylamide matrix that is capable of reacting with the DNA oligonucleotide probe. However, this also has limitations in that the incorporation of the attachment co-monomer as a third component of the matrix (i.e., along with acrylamide monomer and crosslinker) can give rise to problems during acrylamide polymerization, including interference in the matrix formation, and degradation of matrix properties (e.g., resulting in no polymerization, loss of mechanical integrity, and/or adhesion of the matrix to the solid support).
A more recent method has employed direct co-polymerization of an acrylamide-derivatized oligonucleotide has been described. For instance, Acrydite (Mosaic Technologies, Boston, Mass.) is an acrylamide phosphoramidite that contains an ethylene group capable of free radical polymerization with acrylamide. Acrydite-modified oligonucleotides are mixed with acrylamide solutions and polymerized directly into the gel matrix (Rehman et al.,
Nucleic Acids Research,
27, 649-655 (1999). This method still relies on acrylamide as the monomer. Depending on the choice of chemical functionality, similar problems in the stability of attachment, as with the above-mentioned methods, will also result.
Accordingly, the methods described in the prior art use post-modification of the matrix, or incorporation of a suitable co-monomer during the fabrication process. In addition to the disadvantages described above, toxic acrylamide monomer is used in manufacturing the arrays. The present invention seeks to overcome some of the aforesaid disadvantages of the prior art. In particular, the present invention provides biomolecules, solid supports, and hydrogels (particularly hydrogel arrays) comprising one or more reactive sites by which the biomolecules can be attached to the solid supports and hydrogels in a photochemical cycloaddition reaction. The invention further provides novel methods of preparing the hydrogels and solid supports with attached biomolecules that avoid the difficulties attendant the prior art. The present invention can be employed in an econo
Beuhler Allyson
Brush Charles Kimball
Johnson Travis
Lajos Robert Emil
McGowen John
Amersham Biosciences AB
Berman Susan W.
Ronning, Jr. Royal N.
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