Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-08-28
2002-12-10
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S174000, C435S283100, C435S287200, C435S287900, C536S022100, C536S023100, C536S024300, C536S025300, C422S050000, C422S067000, C422S068100, C530S300000
Reexamination Certificate
active
06492118
ABSTRACT:
BACKGROUND OF THE INVENTION
A variety of assay systems utilize ligands, e.g., nucleic acids, immobilized on the surface of a solid support. Effective immobilization of the nucleic acids is difficult, both because a range of materials are used to form the solid supports utilized in these assays, and because individual assays have special requirements. Therefore, although a number of attachment mechanisms have been developed, none are universally acceptable and most exhibit notable deficiencies. Among other drawbacks, present methods tend to require large amounts of nucleic acids, have high background noise levels or lack versatility (Duran et al. U.S. Pat. No. 5,858,653 issued Jan. 12, 1999).
The reproducible production of solid supports containing immobilized nucleic acids can also be problematic. For example, a convenient method of attachment utilizes nucleic acids with acrylamide functional groups which can be copolymerized to polyacrylamide gel matrices by free radical polymerization. However, oxidation can affect the copolymerization process resulting in variability in the results achieved using different supports, even when prepared using the same materials. Moreover, long-term stability of supports containing immobilized ligands has been difficult to achieve, often limiting the period of use to shortly after preparation.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of a novel and convenient method of immobilizing a ligand, e.g., a nucleic acid, on a solid support. The method utilizes a covalent bond formed between a thiol group immobilized on the solid support and an acrylamide functional group contained on the nucleic acid to immobilize the nucleic acid to the support. In a particular embodiment, the covalent bond formed is a sulfide, a thioether, bond. The solid support can contain a polymer layer.
The method and the supports it produces are advantageous in several respects. The method utilizes reagents which are both readily available and compatible with the types of analysis conducted with solid supports. Because the materials can be used in aqueous solutions, the need for special skills and sophisticated chemical apparatus are minimized. In addition, because the materials and the supports they form are quite stable, the reproducibility from support to support which has previously proved so difficult to achieve can be realized. This stability also permits the components forming the bond to be combined at different times. For example, because solid supports containing the latent thiol groups of the invention are extremely stable, they can be produced under consistent conditions for use at a later time. Prior to analysis, the latent thiol groups can be activated and contacted with the acrylamide modified nucleic acids to form a support containing immobilized nucleic acids. In a particular embodiment, the thiol groups are activated by contact with a reducing agent.
In one embodiment, the invention is directed to a method of immobilizing an affinity ligand on a solid support comprising providing a solid support comprising an immobilized thiol group, contacting the thiol group with a nucleic acid comprising an acrylamide functional group, and forming a covalent bond between the two groups, thereby immobilizing the ligand on the solid support.
In a particular embodiment, the ligand is a nucleic acid, a modified nucleic acid or a nucleic acid analog. The solid support can comprise a plurality of thiol groups. A plurality of ligands can be immobilized on the solid support. In alternate embodiments, the solid support is formed from glass, silica, ceramic, plastic or metal compounds. The solid support can comprises two or more spatially distinct regions, each region comprising a plurality of immobilized nucleic acids. The solid support can further comprise a polymer layer. In a particular embodiment, the solid support can comprise a microarray. The thiol groups can comprise disulfide groups.
In another embodiment, the invention is directed to a method of immobilizing an affinity ligand on a solid support comprising the steps of providing a solid support comprising immobilized latent thiol groups, activating the latent thiol groups, and reacting the activated thiol groups with an affinity ligand having at least one acrylamide functional group, thereby immobilizing an affinity ligand on a solid support.
In a particular embodiment, the ligand is selected from the group consisting of a nucleic acid, a modified nucleic acid and a nucleic acid analog. The steps of activating the latent thiol groups and reacting the activated thiol groups can occur essentially simultaneously. In alternate embodiments, the solid support is formed from glass, ceramic, plastic and metal. The solid support can comprise two or more spatially distinct regions, each region comprising a plurality of immobilized nucleic acids. The solid support can further comprises a polymer layer. The solid support can comprise a microarray.
In another aspect, the invention is directed to the product formed by the method of forming a solid support described above.
In another embodiment, the invention is directed to a method of immobilizing an affinity ligand on microarray comprising the steps of providing a solid support comprising immobilized latent thiol groups, activating the latent thiol groups, and reacting the activated thiol groups with an affinity ligand having at least one &agr;,&bgr; unsaturated carbonyl functional group, thereby immobilizing an affinity ligand on a solid support. In a particular embodiment, the ligand is selected from the group consisting of a nucleic acid, a modified nucleic acid and a nucleic acid analog. The steps of activating the latent thiol groups and reacting the activated thiol groups can occur essentially simultaneously.
In another embodiment, the invention is directed to a method of immobilizing an affinity ligand on a microarray comprising the steps of providing a solid support comprising immobilized latent thiol groups, activating the latent thiol groups, and reacting the activated thiol groups with an affinity ligand having at least one &agr;,&bgr; unsaturated carbonyl functional group, thereby immobilizing an affinity ligand on a solid support. In a particular embodiment, the ligand is a nucleic acid, a modified nucleic acid or a nucleic acid analog. The steps of activating the latent thiol groups and reacting the activated thiol groups can occur essentially simultaneously.
The method can additionally include contacting a glass solid support with a silane compound to form a solid support having an unsaturated aliphatic surface. The silane compound can be represented by Structural Formula I:
In Structural Formula I, X is a halogen, and R
1
, R
2
and R
3
are each, independently, a halogen, an alkyl group, an alkenyl group or a group having at least one &agr;,&bgr;-unsaturated carbonyl, provided that at least one of R
1
, R
2
or R
3
is an alkenyl group or a group having at least one &agr;,&bgr;-unsaturated carbonyl. The unsaturated aliphatic surface is then contacted with a polymerization solution containing free radical initiator, a disulfide bisacrylamide, and optionally containing an acrylamide to form a solid support comprising immobilized latent thiol groups. Disulfide bisacrylamides can be represented by Structural Formula IIA:
In Structural Formula IIA, n and m are each, independently, a positive integer.
The latent thiol groups can be activated by contacting the solid support with a disulfide reducing agent. When it is desirable to have a crosslinked gel having immobilized thiol groups, the polymerization solution can additionally include alkylene bisacrylamide.
In an alternative embodiment, the unsaturated aliphatic surface is then contacted with a polymerization solution containing free radical initiator, a compound having a &agr;,&bgr;-unsaturated carbonyl and a protected thiol group, and optionally containing an acrylamide to form a solid support comprising immobilized latent thiol groups. The compound having an (&agr;,
Abrams Ezra S.
Mielewczyk Slawomir
Patterson Brian C.
Zhang Tianhong
Forman B J
Jones W. Gary
Matrix Technologies Corporation
Wood Herron & Evans LLP
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