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We have demonstrated that the dynamics of nucleic acid hybridization in microarrays depend on the physical structure of immobilized probes. We have immobilized oligonucleotide-3'-phosphates with and without stem-loop structure on epoxylated glass surface, followed by hybridization under different conditions, viz., hybridization buffer, pH condition, temperature and ionic strength. In a comparative study, we have established that array constructed using probes with stem-loop structure displayed approximately 2.2 times higher hybridization signals than the probes without it. The stem-loop DNA array format is simple and flexible in design and thus potentially useful in various DNA diagnostic tests.

A new immobilization chemistry for covalent attachment of phosphorylated oligonucleotides on epoxy-activated glass surface via opening of oxirane ring is described. The proposed strategy results in excellent immobilization efficiency, spot homogeneity, and morphology. The constructed microarray was successfully demonstrated for discrimination of nucleotide mismatches.

A facile and efficient method for direct immobilization of phosphorylated oligonucleotides on an epoxy-activated glass surface is described. The new immobilization strategy has been analyzed for its performance in DNA microarray under both microwave and thermal conditions. It reflects high immobilization efficiency ( approximately 23%), and signal-to-noise ratio ( approximately 98) and resulted in high hybridization efficiency ( approximately 36%) in comparison to those obtained with standard methods, viz., NTMTA ( approximately 9.76%) and epoxide-amine ( approximately 9.82%). The probes immobilized through the new strategy were found to be heat-stable, since the performance of microarray decreased by only approximately 7% after subjecting it to 20 PCR-like heat cycles, suggesting that the chemistry could be used in integrated PCR/microarray devices. The immobilization of probes following the proposed chemistry resulted in spots of superior quality in terms of spot morphology, spot homogeneity, and signal reproducibility. The constructed microarrays have been successfully used for the discrimination of nucleotide mismatches. In conclusion, these features make the new immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.

A synthesis of labeled oligonucleotides incorporating a new chemically cleavable linker (III) via a two-step method is described. The labeled oligomers obtained after cleavage and deprotection reactions [treatment with anhydrous tert-butylamine and dry methanol, 1:1 (v/v) for 12 h at room temperature, and lyophilization followed by subsequent reaction with aq NH4OH and methylamine (40%), 1:1 (v/v) for 5 min at 65 °C] were analyzed by RP-HPLC. A distinctive feature of this protocol is that free oligomers can be recovered from their labeled analogs under mild conditions (0.2 M NaOH containing 0.5 M NaCl over 30 min at room temperature) and are comparable to the corresponding standard oligonucleotides (HPLC).

Oligonucleotide-based arrays are increasingly becoming useful tools for the analysis of gene expression and single-nucleotide polymorphism. Here, we report a method that allows the direct immobilization of thiolated oligonucleotides onto an epoxy-activated glass surface via a stable thioether linkage under microwaves. The described chemistry efficiently immobilizes the probes via terminal thiol groups with uniform spot morphology. The thioether linkage could endure repeated PCR-like heat cycling with only 2.5% loss after 20 cycles, indicating that the chemistry can be used in integrated PCR/microarray devices. The highlighting feature of the proposed method is that the detection limit for the probe concentration can be reduced to 0.25 microM with 20-mer oligonucleotides. The efficiency of the projected method (approximately 33%) indicates its advantage over the existing standard methods, viz., NTMTA (approximately 9.8%), epoxide-amine (approximately 9.8%) and disulfide (approximately 1.7%). The constructed microarrays were validated through the detection of base mismatches and bacterial meningitis. These features make the projected strategy ideal for manufacturing oligonucleotide arrays and detection of mismatches and bacterial diseases.

Oligonucleotide microarray has become an important and powerful tool for various genomic analyses, where, unlike conventional methods, one can identify simultaneously a large number of targets in a given sample. Postsynthesis immobilization, the most widely used method, involves the noncovalent and covalent fixing of suitably modified oligonucleotides on the solid supports. Among the various functional groups aminoalkyl, hydroxyalkyl, mercaptoalkyl, aldehyde, epoxy, and carboxyl the most frequently used functional groups on the polymeric surfaces. Because glass and polypropylene, the most widely used materials, are nonporous in nature, the functional groups density on the surface remains very low. In order to know the exact concentration of a ligand to be immobilized, it is essential to estimate the accessible functional groups on these surfaces. For this purpose, sensitive methods are required to estimate exact density of available functional groups on the surfaces. Apart from physical methods, a number of sensitive chemical methods, by making use of high extinction coefficient of 4,4'-dimethoxytrityl cation (epsilon(498) = 70,000 L mol-1 cm-1), have been reported to estimate accessible functional groups on the glass based polymer supports. In this chapter, use of these reagents for spectrophotometric determination of functional group density on glass microslides and polypropylene film has been discussed.

A two-step general method for labeling of synthetic oligonucleotides is described. The protocol employs a cleavable universal linker, 5'-O-(4,4'-dimethoxytrityl)-3'-O-benzoyl-2'-O-(2-cyanoethyl-N,N-diisopropyl)-uridine phosphoramidite, to effect coupling to polymer-bound oligonucleotide chains. Sequentially, coupling with commercially available phosphoramidite reagent of an appropriate label (Biotin, HEX etc.) in an automated DNA synthesizer is carried out. The labeled oligomers, obtained after cleavage and deprotection reactions, are analyzed on RP-HPLC. A distinctive feature of this protocol is the recovery of free oligomers from their labeled analogs under mild conditions. The oligomers obtained are comparable to the corresponding standard oligonucleotides (HPLC).

A universal reagent 1- O -(4,4′-dimethoxytrityl)-6-aminohexanol (DTAH) is described for the estimation of surface-bound functionalities (epoxy, aldehyde, and carboxyl) required for preparation of oligonucleotide arrays (biochips). The method involves the reaction of universal reagent DTAH with surface-bound functionality under microwaves for 10 min, followed by washings to remove the excess reagent. In the subsequent step, a weighed amount of DTAH-treated surface is exposed to acid to liberate 4,4′-dimethoxytrityl cation, which is measured at 505 nm to determine the functional group loading on the surface.

A new efficient reusable universal polymer support for oligonucleotide synthesis, based on a non-ammoniacal cleavable linker, is described. Twenty six cycles of oligonucleotide synthesis have been carried out without compromising the quality of the fully deprotected oligonucleotides.