Epoxide polymer surfaces

Abstract

Method and reagent composition for covalent attachment of target molecules, such as nucleic acids, onto the surface of a substrate. The reagent composition includes epoxide groups capable of covalently binding to the target molecule. Optionally, the composition can contain photoreactive groups for use in attaching the reagent composition to the surface. The reagent composition can be used to provide activated slides for use in preparing microarrays of nucleic acids.

Description

RELATED APPLICATIONS [0001] This application is a continuation-in-part of U.S. Ser. No. 09 / 227,913, filed Jan. 8, 1999, which is a continuation-in-part of U.S. Ser. No. 08 / 940,213 filed Sep. 30, 1997, now U.S. Pat. No. 5,858,653, the entire disclosures of which are incorporated herein by reference.TECHNICAL FIELD [0002] The present invention relates to reagents and support surfaces for immobilization of biomolecules, such as nucleic acids and proteins. BACKGROUND OF THE INVENTION [0003] The immobilization of deoxyribonucleic acid (DNA) onto support surfaces has become an important aspect in the development of DNA-based assay systems, including the development of microfabricated arrays for DNA analysis. See, for instance, “The Development of Microfabricated Arrays of DNA Sequencing and Analysis”, O'Donnell-Maloney et al., TIBTECH 14:401-407 (1996). Generally, such procedures are carried out on the surface of microwell plates, tubes, beads, microscope slides, silicon wafers or membran...

AI Technical Summary

Benefits of technology

[0017] The present application is particularly concerned with reagents having epoxide groups, in the manner described above, and provides further examples and advantages concerning the use of such epoxide-based reagents. Such advantages include, for instance, the ability to use the reagents to attach underivatized DNA, in addition to DNA derivatized to contain amine or other reactive groups, as described in parent application U.S. Ser. No. 09 / 227,913. Such advantages also include improved resistance to hydrolysis demonstrated by epoxides, as compared for instance, to NOS groups.

[0026] Applicants have found that polymers (and particularly hydrophilic polymers) containing epoxide groups, of the type described herein, have several advantages for DNA immobilization over previously used methods. These polymers, when coated onto silane-modified glass slides, for instance, provide an improved method for immobilizing underivatized DNA. Therefore, using these reagents, it is not necessary to modify the DNA with amines or other functional groups. Furthermore, the epoxide groups are significantly more stable to hydrolysis than are the amine-reactive ester groups. Compared with UV crosslinking of DNA onto polylysine or aminosilane, the coated surfaces of this invention are more convenient to use and tend to result in fewer undesirable side reactions, thereby resulting in less modification of the DNA.

Problems solved by technology

One disadvantage of this approach is that the UV crosslinking causes undesirable damage to the DNA that is not all useful for the immobilization.

Another disadvantages of this approach is that UV crosslinking tends to be limited to longer nucleic acids (e.g., over about 100-mers), as provided by cDNA's and PCR products (and in contrast to the shorter nucleic acids typically formed by synthesis and referred to as “oligonucleotides”).

It appears that the potential damage induced by UV radiation (e.g., the formation of thymine dimers) is simply too great, and / or the extent of immobilization is insufficient, to permit shorter nucleic acids to be used.

A population of longer nucleic acids, however, even when crosslinked by UV, will typically provide ample undamaged regions sufficient to permit accurate hybridization.

Only relatively few approaches to immobilizing DNA, to date, have found their way into commercial products.

Disadvantages of this approach are that it requires the extra step of adding the carbodiimide reagent as well as a five hour reaction time for immobilization of DNA, and it is limited to a single type of substrate material.

Although the product literature describes it as being useful for all common plastic surfaces used in the laboratory, it does have some limitations.

For example, Applicants were not able to demonstrate useful immobilization of DNA onto polypropylene using the manufacturer's instructions.

Furthermore, this product requires large amounts of DNA.

Since the DNA-BIND™ product is polystyrene based, it is of limited use for those applications that require elevated temperatures such as thermal cycling.

As expected, however, the reactive ester groups tend to be hydrolytically unstable, which limits the amount of time arrays can be printed without some loss of performance to approximately eight hours.

To date, however, there appears to be no description in the art, let alone commercial products, that provide an optimal combination of such properties as hydrolytic stability, ease of use, minimized DNA damage (due to exposure to crosslinking radiation), and the ability to immobilize underivatized nucleic acids and / or shorter nucleic acid segments.

In turn, there appear to be no products presently available, nor descriptions in the art, that provide or suggest the ability to use polymer-pendent epoxide groups adapted to immobilize either short or long nucleic acids, let alone in both derivatized and underivatized forms, and suitable for immobilization onto surfaces.

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