Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-18
2004-02-17
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S022100, C536S023100, C536S024300, C536S025300
Reexamination Certificate
active
06692912
ABSTRACT:
BACKGROUND OF THE INVENTION
Various methods have been developed to analyze nucleic acid molecules present in experimental or diagnostic samples. Many of these techniques are assays wherein the sample is placed in contact with a solid support. The solid support contains nucleic acid molecules which have been immobilized by covalent or noncovalent attachment. Immobilization of a nucleic acid molecule to a spatially defined position on a solid support can be used in many ways. These uses include: hybridization assays which are able to identify an individual nucleic acid of interest present in an experimental or diagnostic sample containing multiple unique nucleic acids (Southern,
Trends in Genetics
12:110-115 (1996)); hybridization assays which are able to identify genes which have a mutation such that the gene present in the experimental or diagnostic sample differs from that of the wild-type gene (Southern, WO 89/10977 (1989)); and in polymerase extension assays where the immobilized nucleic acids serve as primers for DNA synthesis by a DNA polymerase enzyme following hybridization to complementary target nucleic acids that may be present in the sample (Shumaker et al.,
Hum. Mut
. 7:346-354 (1996); Syvanen et al.,
Am. J. Hum. Genet
. 52:46-59 (1993)).
Presently, there are a number of known methods for covalently coupling a nucleic acid to a solid support for use in an experimental or diagnostic assay . These can be divided into two categories: 1) those in which preformed nucleic acids are coupled to the support; and 2) those in which the nucleic acids are synthesized in situ on the support.
In the first approach, the nucleic acids are deposited on the support either by hand or by automated liquid handling equipment (Lamture et al.,
Nucleic Acids Research
22:2121-2125 (1994); Yershov et al.,
Proc. Natl. Acad. Sci. USA
93:4913-4918 (1996)). To effect covalent attachment of the nucleic acids to the support, either the support, the nucleic acids, or both, are chemically activated prior to deposition. Alternatively, the nucleic acids can be deposited on the support and nonspecifically immobilized by physical means such as heat or irradiation with ultraviolet light (Life Science Research Product Catalog, BioRad Laboratories, Richmond, Calif., pg.269-273 (1996); Meinkoth and Wahl,
Analytical Biochemistry
138:267-284 (1984)). In general, chemically mediated coupling is preferred since specific, well defined attachments can be accomplished, thereby minimizing the risk of unwanted artifacts from the immobilization process.
In the second approach, oligonucleotides are synthesized directly on the support using chemical methods based on those used for solid phase nucleic acid synthesis (Southern et al.,
Nucleic Acids Research
22:1368-1373 (1994)). Recently, specialized apparatus and photolithographic methods have been introduced which allow the synthesis of many different oligonucleotides at discrete, well-defined positions on planar glass or silica supports (Pease et al.,
Proc. Natl Acad. Sci. USA
91:5022-5026 (1994)). In general, these methods are most useful for applications which require many hundreds or thousands of different immobilized nucleic acids, such as sequencing by hybridization.
Yet another method presently in use to couple a nucleic acid molecule to a solid support involves the formation of an electroconducting conjugated polymerized layer (Livache et al.,
Nucleic Acids Research
22:2915-2921 (1994)). This polymerized layer is formed by copolymerization of a mixture containing pyrrole monomers and oligonucleotides covalently linked to a pyrrole monomer. The copolymerization reaction initiates following application of an electrical charge through the electrode which has been placed into the mixture containing the copolymerizable components. The dimensions of the polymerized layer which coats the surface of the electrode can be varied by adjusting the surface area of the electrode which is placed into the mixture.
Each of the methods disclosed above have specific limitations. For instance, the polymerized layer which coats the surface of an electrode can not be formed on a solid support which is not able to transmit an electrical charge into the mixture containing the copolymerizable monomer units. Most of the other disclosed methods are also limited to solid supports of a particular type. In addition, several of these methods require special types of equipment, and involve a degree of technical difficulty which may make it difficult to covalently link a nucleic acid molecule to a solid support in a reproducible manner.
SUMMARY OF THE INVENTION
The invention relates in one aspect to a polymerizable complex comprising a nucleic acid molecule which is derivatized by attachment to a first polymerizable ethylene-containing monomer unit which, under appropriate conditions, is capable of copolymerization with a second polymerizable ethylene-containing monomer unit. The copolymerization produces a polymerized layer which contains the covalently linked nucleic acid molecule. The nucleic acid molecule is attached to the first polymerizable ethylene-containing monomer unit either directly or through a chemical linker group.
In another aspect, the present invention relates to a &bgr;-cyanoethyl phosphoramidite reagent comprising:
wherein X contains either a polymerizable ethylene-containing monomer unit, or a chemically protected version of same which can be deprotected using appropriate techniques.
In a further aspect, the present invention relates to a &bgr;-cyanoethyl phosphoramidite reagent comprising:
wherein X contains either a polymerizable ethylene-containing monomer unit, or a chemically protected version of same which can be deprotected using appropriate techniques, and DMT is a 4,4′-dimethoxytrity group.
The invention relates in another aspect to a support for oligonucleotide synthesis having the general structure:
wherein S is a solid support material and L is a linker group. The linker group is cleavable by appropriate means at the end of oligonucleotide synthesis to release the completed oligonucleotide product. X contains either a polymerizable ethylene-containing monomer unit or a chemically protected version of same, which can be deprotected using appropriate techniques, and DMT is a 4,4′-dimethoxytrityl group.
In another aspect, the present invention relates to a support for oligonucleotide synthesis having the general structure:
wherein S is a support material, L is a linker group, and X contains either a polymerizable ethylene-containing monomer unit, or a chemically protected version of same which can be deprotected using appropriate techniques, and DMT is a 4,4′-dimethoxytrity group.
In a further aspect of the present invention, the first and second polymerizable ethylene-containing monomer units are selected from the group consisting of monosubstituted ethylenes of general structure CH
2
═CHX, or unsymmetrically (1,1-) disubstituted ethylenes of the general structure CH
2
═CXY. In preferred embodiments of the invention, the first polymerizable ethylene-containing monomer unit is a derivative of acrylamide, methacrylamide, acrylic acid, or methacrylic acid.
The present invention further relates to a polymerized composition which is produced by a method comprising incubating a polymerizable complex and at least one second polymerizable ethylene-containing monomer unit under conditions appropriate for polymerization. The first and second polymerizable ethylene groups are selected from the group consisting of monosubstituted ethylenes of general structure CH2═CHX, or unsymmetrically (1,1-) disubstituted ethylenes of the general structure CH2═CXY.
In another aspect, the present invention relates to a method for producing a polymerized composition by incubating a polymerizable complex and at least one second polymerizable ethylene-containing monomer unit under conditions appropriate for polymerization. The first and second polymerizable ethylene groups are selected from the group consisting of monosubstituted ethy
Adams Christopher P.
Boles T. Christian
Kron Stephen J.
Matrix Technologies Corporation
Riley Jezia
Wood Herron & Evans L.L.P.
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