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©2005 Integrated DNA Technologies. All rights reserved. 1
Strategies for Attaching Oligonucleotides to Solid Supports
Eric J. Devor, Ph.D.
1,2
and Mark A. Behlke, M.D., Ph.D.
1,3
 Molecular Genetics
1
, Bioinformatics
2
, and Biophysics
3
 Integrated DNA TechnologiesIntroduction
While most molecular reactions involving synthetic oligonucleotides, notably PCR and DNA sequencing, are carried out in liquid phase, a rapidly growing number of important applications utilize synthetic oligonucleotides attached to solid supports. Ingeneral, the purpose for using a solid support-attached oligonucleotide is to capturesomething, hold on to it, enrich it, and identify and/or purify it. The major class of solidsupport-attached oligonucleotides, DNA oligonucleotide microarrays, provide a powerfultool for analysis of gene expression. The most generally accessible approach for  producing oligonucleotide microarrays is to synthesize the individual oligonucleotidesseparately prior to immobilization on the solid surface. In this case the oligonucleotide ismodified with a functional group that allows attachment to a reactive group on a solidsurface. Another important class is the micro-bead-attached oligonucleotide.Here, the chemistries available for solid support attachment are presented. Specialreference to micro-beads is presented as
Supplemental Material
. Modificationsavailable at IDT include for oligonucleotide attachment are:
•
Amine-oligos covalently linked to an activated carboxylate group or succinimidylester.
•
SH-oligos covalently linked via an alkylating reagent such as an iodoacetamide or maleimide.
•
Acrydite-oligos covalently linked through a thioether.
•
Biotin-oligos captured by immobilized Streptavidin
Oligonucleotide Microarrays
In general, amino, thiol, and Acrydite
TM
modifications have been routinely used toconstruct oligonucleotide arrays. Construction of arrays involves a number of parameterseach of which must be optimized for efficient and effective experimental design. Oneimportant parameter is the method of choice for attachment of the syntheticoligonucleotides. Some of the issues to be considered in choosing an appropriate supportand the associated attachment chemistry include: the level of scattering and fluorescence background inherent in the support material and added chemical groups; the chemicalstability and complexity of the construct; the amenability to chemical modification or 
 
©2005 Integrated DNA Technologies. All rights reserved. 2
derivatization; surface area; loading capacity and the degree of non-specific binding of the final product (Guo et al., 1994).Substrates for arrays are usually silicon chips or glass microscope slides, with slides being the most common medium. Glass is a readily available and inexpensive supportmedium, possessing a relatively homogeneous chemical surface whose properties have been well studied and which is amenable to chemical modification using very versatileand well developed silanization chemistry (Guo et al., 1994). Although silianizedoligonucleotides can be covalently linked to an unmodified glass surface, most arrayattachment protocols involve chemically modifying the glass surface to facilitateattachment of the oligo (Kumar et al., 2000).
Surface Modification
Preparation of the two dimensional array surface usually involves treatment with anamino silane reagent to the glass or silicon surface resulting in a uniform layer of primaryamines or epoxides (Figure 1) (Guo et al., 1994;Lamture, et al., 1994). Thesemodifications meet several criteria (Maskos and Southern, 1992); the linkages arechemically stable, sufficiently long to eliminate undesired steric interference from thesupport, and hydrophilic enough to be freely soluble in aqueous solution and not producenon-specific binding to the support (Guo et al., 1994). Once these modifications haveactivated the surface, the efficiency of attaching the oligonucleotides depends largely onthe chemistry used and how the oligonucleotide targets are modified (Lindroos et al.,2001). Oligonucleotides modified with an NH
2
group can be immobilized onto epoxysilane-derivatized (Lamture et al., 1994) or isothiocyanate coated glass slides (Guo et al.,1994). Succinylated oligonucleotides can be coupled to aminophenyl- or aminopropyl-derivitized glass slides by peptide bonds (Joos, et al.1997), and disulfide-modifiedoligonucleotides can be immobilized onto a mercaptosilanized glass support by athiol/disulfide exchange reactions (Rogers et al., 1999) or through chemical cross linkers.
Fig. 1. Surface modification. A. Amino modification with 3-aminopropyltrimethoxysilane. B. Epoxidemodification with 3’ glycidoxy propyltrimethoxysilane.
 
©2005 Integrated DNA Technologies. All rights reserved. 3
The surface density of the oligonucleotide probe is expected to be an important parameter of any array. A low surface coverage will yield a correspondingly lowhybridization signal, and decrease the hybridization rate.Conversely, high surface densities may result in steric interference between thecovalently immobilized oligonucleotides, impeding access to the target DNA strand (Guoet al., 1994). Guo et. al. (Guo et al., 1994) found an optimal surface coverage at which amaximum amount of a complementary PCR product is hybridized. In a series of hybridization studies using PCR products as probes these authors determined the optimalsurface coverage at which a maximum amount of the complementary PCR product ishybridized. For a 157 base probe, the optimum occurred at about 5.0 mM oligonucleotideconcentration, which corresponded to a surface density of 500 Å2/molecule (Guo et al.,1994). The optimum for a longer 347 nt fragments was found to be approximately 30%lower, probably reflecting the greater steric interference of the longer fragment.The planar surface structure of glass slides or silicon chips can limit the loadingcapacity of oligonucleotides. This limitation has been addressed by applying acrylamidegels to glass slides to construct a three dimensional surface, greatly increasing the surfacearea per spot (Mirzabekov, 1994; Proudnikov et al., 1998). The increased surface area permits relatively large amounts of DNA to be linked to the surface resulting in strongsignal intensities and a good dynamic range. This effect is partly offset by a higher  background produced mainly by the same structural characteristic - the large surface area per spot - responsible for the high loading capacity (Beier and Hoheisel, 1999).
Amino-modified Oligonucleotides
A primary amine can be used to covalently attach a variety of products to anoligonucleotide, including fluorescent dyes (Connolly and Rider, 1985; Zuckerman et al.,1987), biotin (Sproat et al, 1987), alkaline phosphatase (Li et al., 1987), EDTA (Dreyer and Dervan 1985), or to a solid surface. An amino modifier can be place at the 5'-end, 3'-end or internally using and amino-dC or amino-dT modified base.Attaching an amino modified oligonucleotide to a surface or another molecule requires anacylating reagent that forms carboxamides, sulfonamides, ureas or thioureas uponreaction with the amine moiety. The kinetics of the reaction depends on the reactivity andconcentration of both the acylating reagent and the amine. Buffers that contain freeamines such as Tris and glycine must be avoided when using any amine-reactive reagent.The most significant factors affecting an amine's reactivity are its class and its basicity.Oligonucleotides with an amino modification have a free aliphatic amine that ismoderately basic and reactive with most acylating reagents. However, the concentrationof the free base form of aliphatic amines below pH 8 is very low; thus, the kinetics of acylation reactions of amines by isothiocyanates, succinimidyl esters and other reagentsare strongly pH dependent. A pH of 8.5-9.5 is usually optimal for oligonucleotideconjugation. Aromatic amines, present within each base, are very weak bases and thus arenot protonated at pH 7 or above. Most attachment chemistries currently in use for amino
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