Nucleic acid hybridization is a fundamental technique in molecular biology. Nucleic acid hybridization assays have been used extensively in molecular biology to establish the sequence similarity of populations of nucleic acids. Hybridization is simply the annealing or pairing of single stranded nucleic acid molecules (DNA or RNA) to form double strands. The most common technique employing hybridization is the Southern blot hybridization technique, in which a set of unknown target DNA molecules is immobilized on a membrane and a solution containing labeled DNA probe molecules is used to bathe the membrane under conditions where complementary molecules will anneal (Southern, E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517 (1975)). In an analogous technique called Northern blot hybridization (Alwine J. C. et al. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. 74:5350-5354 (1977); Alwine, J. C. et al. Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. Methods Enzymol. 68:220-242 (1979)), RNA molecules immobilized on membranes are the targets. The labeled probe DNA used in the liquid phase can be as short as 10 to 20 nucleotides. The probes are usually labeled with radioisotopes, although other reporter groups, e.g. fluorescein, biotin, etc., can be used.
Reverse blot hybridization employs the opposite approach. Instead of immobilizing unknown DNAs, a set of well defined DNA probes are immobilized on a solid surface and the unknown labeled DNA is present in the liquid phase. Theoretically, a high density array containing a large number of probes can be used for reverse hybridizations with a single target molecule. By decoding the hybridization pattern of the unknown DNA to positions of known sequence on the solid phase array, sequence information from several positions of the unknown target DNA can be obtained. While the idea of sequencing by hybridization (SBH) has generated much excitement, the use of reverse hybridization assays to detect known DNA sequences and their alterations is a more practical application at present.
Several methods for the constructing biomolecule arrays of sufficiently high density for sequencing applications are currently under development. Arrays of peptides and oligonucleotides have been created using photolithographic techniques. (Fodor, S. P. A., et al., Light-Directed Spatially Addressable Parallel Chemical Synthesis, Science 251:767-773 (1991); Pease, A. C., et al., Light-generated oligonucleotide arrays for rapid DNA sequence analysis, Proc. Natl. Acad. Sci. USA 91:5022-5026 (1994)) Biomolecules are attached to reactive groups on the surface of a solid support, which can be selectively blocked or deblocked through the use of photolabile protecting groups. Alternatively, a physical mask may be used and the desired chemical reactions carried out on the unmasked portion of the support. (Southern, E. M. et al. Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: Evaluation using experimental models. Genomics 13:1008-1017 (1992)) A third alternative is a printer-like device, which can deposit an array of drops on the matrix. (U.S. Pat. No. 5,474,796) Despite these promising early developments, existing or suggested methods do not reliably produce the very large high density arrays needed for sequencing applications in a rapid and reproducible manner.
There are two fundamental ways of immobilizing oligonucleotides at specific sites on solid supports: the oligonucleotides may be synthesized on the solid phase in their respective positions, i.e., in situ, or they may be synthesized apart from the solid support and attached later. The former method has been successfully achieved in several different ways. The first reverse hybridization arrays were made using glass modified with an aliphatic poly(ether) linker as a solid support (Southern, E. M. et al. 1992). More recently, polypropylene was used as a support for the in situ synthesis of oligonucleotides (U.S. Pat. No. 5,554,501).
There are also various methods available for immobilizing pre-synthesized biomolecules onto solid supports. Such methods include: simple adsorption, ultra violet cross linking or covalent attachment. In adsorption and ultra violet crosslinking, the attachment of molecules onto the surface of the support is by random process. Moreover, the specific sites can become inaccessible to binding with complementary sequences. In a covalently coupled system, the attachment of the functionalized or activated oligonucleotide to the surface of the polymeric support is at specific sites.
In general, the attachment of standard oligonucleotides to unmodified glass or plastic surfaces is inefficient. For this reason, many investigators trying to immobilize oligonucleotides modify them with molecules that promote adsorption or enable attachment to the support. Oligonucleotides modified with bovine serum albumin adsorb passively to microtiter plates designed to bind protein molecules (Southern, E. M. International Patent Application PCT GB 89/00460 (1988)). Biotinylated oligonucleotides bind tightly to plates or beads that are coated with avidin or streptavidin. Oligonucleotides with polythimidylate tails have been photochemically crosslinked to nylon (Bains, W., et al., A novel method for nucleic acid sequence determination. J. Theoret. Biol. 135:303-307 (1988)). More recently, oligonucleotides with terminal amino (Drmanac, R., et al. Sequencing of megabase plus DNA by hybridization: Theory of the method. Genomics 4:114-128 (1989), Lysov et al. Determination of the DNA nucleotide sequence by hybridization with oligonucleotides. A new method. Proc USSR Acad. Sci 303:1508-1511 (1988)) or methyluridine (Khrapko, K. R., et al., An oligonucleotide hybridization approach to DNA sequencing. FEBS Lett. 256:118-122 (1989)) groups have been covalently crosslinked to compatible reactive groups on multi-well plate surfaces.
Another approach is to modify the solid support with a suitable functional group and/or linker. For example, there are numerous reports of DNA becoming covalently bound to polystyrene supports, which carry different active groups on their surfaces, e.g., hydroxyl, carboxyl, amine, aldehyde, hydrazine, epoxide, bromoacetyl, maleimide and thiol groups (U.S. Pat. No. 5,474,895; Lund, V., et al., Assessment of methods for covalent binding of nucleic acids to magnetic beads, DynabeadsJ, and the characteristics of the bound nucleic acids in hybridization reactions, Nucleic Acids Research 16:10861-10880 (1988)) or having a spacer arm ending with an active group (Rasmussen, S. R., et al., Covalent Immobilization of DNA onto Polystyrene Microwells: The Molecules Are Only Bound at the 5' End, Anal. Biochem. 198:138-142 (1991)). However, these methods generally entail trade offs between high coupling yields and non-specific binding of nucleic acids during subsequent hybridization procedures.
Immobilizing pre-synthesized oligonucleotides and in situ synthesis have different advantages for array construction. Synthesis in situ does not involve the handling of thousands of independent oligonucleotides, each of which must be produced on a scale that far exceeds what is required for the array. In contrast, the ability to freely arrange the members of an array after oligonucleotide synthesis is only possible with pre-synthesized oligonucleotides.
Thus, a need exists for immobilizing procedures that permit greater flexibility in constructing arrays of pre-synthesized oligonucleotides on suitable solid supports. Preferably, the attachment procedures are amenable to automation using repeatable steps in order to facilitate their use in the clinical laboratory.