Kinetics of hybridization on surface oligonucleotide microchips: theory, experiment, and comparison with hybridization on gel-based microchips.

The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5-1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5-20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2-4 times. These effects may be attributed to the better immobilization efficiency and to the higher thermodynamic association constants for duplex formation within gel pads.

Analysis of binding specificity of disulfide bonded dimeric lambda-Cro V55C protein with generic hexamer oligonucleotide microchip.

Binding specificity of mutant V55C disulfide bonded dimeric lambda-Cro protein (CroVC) to double-stranded DNA (dsDNA) was studied using generic hexamer oligonucleotide microchip. The curves of dissociation of hybridized DNA in the presence and absence of CroVC were converted into the effective discriminant constants to assess the relevant thermodynamic equilibrium binding constants for dsDNA-protein complexes. Then, tiling of longer oligonucleotides with shorter oligomers was used to search for sequence motifs with the highest binding specificity similarly to sequencing by hybridization. The comparison of the deduced sequences with the known natural operator half-sites demonstrated the principal ability to discern and reconstruct the major parts of 7-mer motifs corresponding to the strongest binding of CroVC subunits. Our results show the applicability of generic microchips to the analysis of binding specificity in the case of multi-subunit DNA-binding proteins.

Kinetics of hybridization on the oligonucleotide microchips with gel pads.

The kinetics of hybridization on the oligonucleotide microchip with gel pads is studied both theoretically and experimentally. The monitoring of kinetics was performed with the measurements of fluorescence intensity produced by the labeled target oligonucleotides. As is shown, the hybridization time depends on the stability of the formed duplexes, the concentrations of target and probe oligonucleotides, and the diffusion of target oligonucleotides in solution and gel pad. The initial stage of hybridization is determined by the flow of target oligonucleotides from solution, then, followed by the diffusive propagation with approximately constant concentration of oligonucleotides at the boundary of gel pad and, finally, by the exponential saturation. The theoretical predictions of hybridization kinetics reveal a good correspondence with the experimental results and may be used for the choice of the optimal hybridization conditions. The possible applications of kinetic hybridization curves to the discrimination problems and assessment of diffusion coefficients in gel pads are briefly discussed. Finally, we discuss the relationships between the binding kinetics and the general functioning of biomolecular microchips.

Comparison of complex DNA mixtures with generic oligonucleotide microchips.

The reproducibility of melting curves for repeated hybridizations of target DNA with generic oligonucleotide microchips is shown experimentally to depend on the character of matching between fragments of target DNA and immobilized oligonucleotides. The reproducibility of melting curves is higher for the perfect match duplexes and decreases as the number of mismatched pairs within duplexes increases. This effect was applied to the comparative analysis of complex DNA mixtures. We developed a scheme in which we can identify and discriminate between the probe oligonucleotides responsible for the distinctions between target DNA mixtures. A scheme is illustrated by comparing DNA mixtures corresponding to V-D-J genes connected with populations of mRNAs CDR3 TCR Vb (T-cell receptor beta complementarity determining region 3) from the thymus and pancreas of NOD mice. Our results demonstrate that generic microchips can be applied efficiently to the analysis of DNA mixtures.

Sequencing by hybridization with the generic 6-mer oligonucleotide microarray: an advanced scheme for data processing.

DNA sequencing by hybridization was carried out with a microarray of all 4(6) = 4,096 hexadeoxyribonucleotides (the generic microchip). The oligonucleotides immobilized in 100 x 100 x 20-microm polyacrylamide gel pads of the generic microchip were hybridized with fluorescently labeled ssDNA, providing perfect and mismatched duplexes. Melting curves were measured in parallel for all microchip duplexes with a fluorescence microscope equipped with CCD camera. This allowed us to discriminate the perfect duplexes formed by the oligonucleotides, which are complementary to the target DNA. The DNA sequence was reconstructed by overlapping the complementary oligonucleotide probes. We developed a data processing scheme to heighten the discrimination of perfect duplexes from mismatched ones. The procedure was united with a reconstruction of the DNA sequence. The scheme includes the proper definition of a discriminant signal, preprocessing, and the variational principle for the sequence indicator function. The effectiveness of the procedure was confirmed by sequencing, proofreading, and nucleotide polymorphism (mutation) analysis of 13 DNA fragments from 31 to 70 nucleotides long.

Study of correlations in DNA sequences.

We present a method for unified statistical analysis of short and long range correlations between various nucleotides in genomic DNA strands. The approach is based on the mutual study of Fourier structure factor spectra and pair correlation functions. The analysis of cross correlations in the different ranges of structural spectra permits identification of the main sources of correlations, namely, the coherent point mutations, coincident periodicities or large scale density variations. The technique for assessment of structural coupling between various genes in the genome is also described.