Specific and nonspecific hybridization of oligonucleotide probes on microarrays

Gene expression analysis by means of microarrays is based on the sequence-specific binding of RNA to DNA oligonucleotide probes and its measurement using fluorescent labels. The binding of RNA fragments involving sequences other than the intended target is problematic because it adds a chemical background to the signal, which is not related to the expression degree of the target gene. The article presents a molecular signature of specific and nonspecific hybridization with potential consequences for gene expression analysis. We analyzed the signal intensities of perfect match (PM) and mismatch (MM) probes of GeneChip microarrays to specify the effect of specific and nonspecific hybridization. We found that these events give rise to different relations between the PM and MM intensities as function of the middle base of the PM, namely a triplet-like (C > G approximately T > A > 0) and a duplet-like (C approximately T > 0 > G approximately A) pattern of the PM-MM log-intensity difference upon binding of specific and nonspecific RNA fragments, respectively. The systematic behavior of the intensity difference can be rationalized on the level of basepairings of DNA/RNA oligonucleotide duplexes in the middle of the probe sequence. Nonspecific binding is characterized by the reversal of the central Watson-Crick (WC) pairing for each PM/MM probe pair, whereas specific binding refers to the combination of a WC and a self-complementary (SC) pairing in PM and MM probes, respectively. The Gibbs free energy contribution of WC pairs to duplex stability is asymmetric for purines and pyrimidines of the PM and decreases according to C > G approximately T > A. SC pairings on the average only weakly contribute to duplex stability. The intensity of complementary MM introduces a systematic source of variation which decreases the precision of expression measures based on the MM intensities.     

Fabrication of DNA microarrays using unmodified oligonucleotide probes

Microarrays printed on glass slides are often constructed by covalently linking oligonucleotide probes to a derivatized surface. These procedures typically require relatively expensive amine- or thiol-modified oligonucleotide probes that add considerable expense to larger arrays. We describe a system by which unmodified oligonucleotide probes are bound to either nonderivatized or epoxy-silane-derivatized glass slides. Biotinylated PCR products are heat denatured, hybridized to the arrays, and detected using an enzymatic amplification system. Unmodified probes appear to detach from the slide surface at high pH (> 10.0), suggesting that hydrogen bonding plays a significant role in probe attachment. Regardless of surface preparation, high temperature (up to 65 degrees C) and low ionic strength (deionized water) do not disturb probe attachment; hence, the fabrication method described here is suitable for a wide range of hybridization stringencies and conditions. We illustrate kinetics of room temperature hybridizations for probes attached to nonderivatized slides, and we demonstrate that unmodified probes produce hybridization signals equal to amine-modified, covalently bound probes. Our method provides a cost-effective alternative to conventional attachment strategies that is particularly suitable for genotyping PCR products with nucleic acid microarrays.     

DNA microarrays based on noncovalent oligonucleotide attachment and hybridization in two dimensions

Short oligonucleotide probes have been linked to a solid support by simple electrostatic adsorption onto a positively charged surface film. Attachment was obtained by microfluidic application of unmodified oligonucleotides in distilled water onto amino-silanized glass. It has been demonstrated that an extremely stable monolayer of oligonucleotide is obtained by this method, at a density of about 10(11) molecules/mm(2), which approaches the limit expected for a two-dimensional closest-packed array. Application of oligonucleotide by adsorption is followed by capping with acetic anhydride in the vapor phase, and then capping with succinic anhydride in solution to form a surface with weak negative charge. The capping method has been successfully employed for microarray fabrication and for the analysis of single nucleotide polymorphisms in the k-ras gene. The data reveal that, subsequent to capping, the adsorptive association of oligonucleotide to the surface yields a probe layer which is capable of single nucleotide base mismatch discrimination and high apparent binding affinity.     

Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays

The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

Tests of rRNA hybridization to microarrays suggest that hybridization characteristics of oligonucleotide probes for species discrimination cannot be predicted

Hybridization of rRNAs to microarrays is a promising approach for prokaryotic and eukaryotic species identification. Typically, the amount of bound target is measured by fluorescent intensity and it is assumed that the signal intensity is directly related to the target concentration. Using thirteen different eukaryotic LSU rRNA target sequences and 7693 short perfect match oligonucleotide probes, we have assessed current approaches for predicting signal intensities by comparing Gibbs free energy (ΔG°) calculations to experimental results. Our evaluation revealed a poor statistical relationship between predicted and actual intensities. Although signal intensities for a given target varied up to 70-fold, none of the predictors were able to fully explain this variation. Also, no combination of different free energy terms, as assessed by principal component and neural network analyses, provided a reliable predictor of hybridization efficiency. We also examined the effects of single-base pair mismatch (MM) (all possible types and positions) on signal intensities of duplexes. We found that the MM effects differ from those that were predicted from solution-based hybridizations. These results recommend against the application of probe design software tools that use thermodynamic parameters to assess probe quality for species identification. Our results imply that the thermodynamic properties of oligonucleotide hybridization are by far not yet understood.     

Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ

Summary A fast, reproducible and non-hazardous technique for non-isotopic DNA fingerprinting is presented. The method is based on digoxigenated oligonucleotides, which are specific for simple repetitive DNA sequences. The use of digoxigenin/ anti-digoxigenin detection avoids many drawbacks inherent in e.g. the biotin/streptavidin system which often causes a poor signal-to-background ratio. Synthesis and purification of digoxigenated oligonucleotides and their use in filter hybridization are described in detail. Hybridization patterns obtained with four different radioactively labeled oligonucleotides have been compared with those of the respective digoxigenated probes. When slightly less stringent hybridization conditions are applied for digoxigenated oligonucleotides than for those labeled with ³²P, the signal intensities are satisfying but additional minor bands occur as a result of the reduced strigency. With one explainable exception, these bands increase the information content of the fingerprint. In addition, hybridization of the digoxigenated (CAC)₅ probe has been performed in situ with human metaphase chromosomes. The hybridization patterns in many mitoses resemble R-bands.     

Kinetics of oligonucleotide hybridization to DNA probe arrays on high-capacity porous silica substrates

We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of approximately 23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3-microm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3-microm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22 degrees C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values (k(a), k(d), and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22 degrees C is described well by the predictive equations for morphology, whereas the behavior at 45 degrees C deviates from expectations and suggests that more complex phenomena may be occurring in that temperature regime.     

The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes

Oligonucleotide probes labeled with fluorescent dyes are used in a variety of in situ applications to detect specific DNA or RNA molecules. It has been described that probe fluorescence might be quenched upon hybridization in a sequence specific way. Here, a set of 17 oligonuleotides labeled with 6-carboxyfluorescein was used to examine the relevance of nucleotide specific quenching for fluorescence in situ hybridization (FISH) to whole fixed bacterial cells. Probes quenched upon hybridization to a guanine-rich region of purified RNA in solution were not quenched upon FISH. Among other factors the high protein concentration within cells may prevent quenching of probe fluorescence in situ.     

Improved hybridization assays employing tailed oligonucleotide probes: a direct comparison with 5'-end-labeled oligonucleotide probes and nick-translated plasmid probes

Terminal deoxynucleotidyl transferase was used to add labeled dAMP residues to the 3' end of oligonucleotide probes that hybridize to the 5' end of the neomycin phosphotransferase II gene. Southern hybridization conditions were described in which the sensitivity per unit of exposure time was about 30-fold greater for the tailed probe as compared to the 5'-end-labeled probe. The tailed oligonucleotide probe had the sensitivity per unit of exposure time comparable to that of a nick-translated probe of high specific activity: in 3 h of autoradiographic exposure both easily detected an amount of target equivalent to a single-copy gene in 10 micrograms of human DNA. The thermal dissociation profiles of 5'-end-labeled and tailed oligonucleotide probes were virtually identical and the tailed oligonucleotide probe was as allele specific as the 5'-end-labeled oligonucleotide probe. The useful lifetime of a 32P-tailed probe was about 1-2 weeks. Finally, by adding 50 35S-labeled nucleotides to the 3' end, we prepared a stable oligonucleotide probe with a sensitivity per unit of exposure time comparable to that of the unstable 5'-32P-labeled oligonucleotide probe.     

The effect of surface probe density on DNA hybridization

The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.     

DNA microarrays with stem-loop DNA probes: preparation and applications

We have developed DNA microarrays containing stem-loop DNA probes with short single-stranded overhangs immobilized on a Packard HydroGel chip, a 3-dimensional porous gel substrate. Microarrays were fabricated by immobilizing self-complementary single-stranded oligonucleotides, which adopt a partially duplex structure upon denaturing and re-annealing. Hybridization of single-stranded DNA targets to such arrays is enhanced by contiguous stacking interactions with stem-loop probes and is highly sequence specific. Subsequent enzymatic ligation of the targets to the probes followed by stringent washing further enhances the mismatched base discrimination. We demonstrate here that these microarrays provide excellent specificity with signal-to-background ratios of from 10- to 300-fold. In a comparative study, we demonstrated that HydroGel arrays display 10-30 times higher hybridization signals than some solid surface DNA microarrays. Using Sanger sequencing reactions, we have also developed a method for preparing nested 3'-deletion sets from a target and evaluated the use of stem-loop DNA arrays for detecting p53 mutations in the deletion set. The stem-loop DNA array format is simple, robust and flexible in design, thus it is potentially useful in various DNA diagnostic tests.     

Optical detection of DNA hybridization based on fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles

A novel system for the detection of DNA hybridization in a homogeneous format is developed. This method is based on fluorescence quenching by gold nanoparticles used as both nanoscaffolds for the immobilization of capture sequences and nanoquenchers of fluorophores attached to detection sequences. The oligonucleotide-functionalized gold nanoparticles are synthesized by derivatizing the colloidal gold solution with 5'-thiolated 12-base oligonucleotides. Introduction of sequence-specific target DNAs (24 bases) into the mixture containing dye-tagged detection sequences and oligonucleotide-functionalized gold nanoparticles results in the quenching of carboxytetramethylrhodamine-labeled DNA fluorescence because DNA hybridization occurs and brings fluorophores into close proximity with oligonucleotide-functionalized gold nanoparticles. The quenching efficiency of fluorescence increases with the target DNA concentration and provides a quantitative measurement of sequence-specific DNA in sample. A linearity is obtained within the range from 1.4 to 92 nM. The target sequence is detected down to 2 nM. This new system not only overcomes many of the drawbacks inherent in radioisotopic measurement or enzyme-linked assay but also avoids the requirement for the stem-loop structure compared with conventional molecular beacons. Furthermore, the background signal that is defined as fluorescence quenching arising from electrostatic attraction between positively charged fluorophores and negatively charged gold nanoparticles is comparatively low due to electrostatic repulsion between negatively charged oligonucleotides. In addition, this is a homogeneous assay that can offer the potential to be monitored in real time, be amenable to automation, eliminate washing steps, and reduce the risk of contamination.     

An oligonucleotide probe to assay lysis and DNA hybridization of a diverse set of bacteria

A computer bank of 16 S rRNA bacterial sequences was searched to determine a consensus sequence expected to hybridize with DNA from a wide variety of bacteria. An oligonucleotide probe, named a panprobe, containing this sequence was used to assay the degree of lysis of bacterial colonies on filter paper heated in a microwave oven and subsequently treated with NaOH. As determined by colony hybridization with the panprobe, lysis was achieved for 51 of 59 different species of bacteria tested. DNA, isolated from the eight bacteria not detected by colony hybridization, did hybridize with the panprobe in slot blot hybridizations.     

Oligo Design: a computer program for development of probes for oligonucleotide microarrays

Oligonucleotide microarrays have demonstrated potential for the analysis of gene expression, genotyping, and mutational analysis. Our work focuses primarily on the detection and identification of bacteria based on known short sequences of DNA. Oligo Design, the software described here, automates several design aspects that enable the improved selection of oligonucleotides for use with microarrays for these applications. Two major features of the program are: (i) a tiling algorithm for the design of short overlapping temperature-matched oligonucleotides of variable length, which are useful for the analysis of single nucleotide polymorphisms and (ii) a set of tools for the analysis of multiple alignments of gene families and related short DNA sequences, which allow for the identification of conserved DNA sequences for PCR primer selection and variable DNA sequences for the selection of unique probes for identification. Note that the program does not address the full genome perspective but, instead, is focused on the genetic analysis of short segments of DNA. The program is Internet-enabled and includes a built-in browser and the automated ability to download sequences from GenBank by specifying the GI number. The program also includes several utilities, including audio recital of a DNA sequence (useful for verifying sequences against a written document), a random sequence generator that provides insight into the relationship between melting temperature and GC content, and a PCR calculator.