Manufacturing DNA microarrays from unpurified PCR products

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.     

Determination of hepatitis B virus DNA in serum by molecular hybridization

Hepatitis B virus (HBV) DNA was detected by direct spotting of alkali-denatured serum on a nitrocellulose filter and molecular hybridization with cloned HBV DNA as the probe. Measurement of the autoradiographic signals as the intensity of hybridization allowed the quantitation of HBV DNA content in serum specimens in reference to cloned HBV DNA. Direct spotting of denatured serum was approximately three times as sensitive as the conventional method in which proteinase-treated serum was extracted with phenol-chloroform. The intensity of hybridization with 25 specimens of HB virion concentrates correlated well with DNA polymerase activity (r = 0.89, P less than 0.01).     

'FloraArray' for screening of specific DNA probes representing the characteristics of a certain microbial community

To investigate uncharacterized microbial communities, a custom DNA microarray named 'FloraArray' was developed for screening specific probes that would represent the characteristics of a microbial community. The array was prepared by spotting 2000 plasmid DNAs from a genomic shotgun library of a sludge sample on a DNA microarray. By comparative hybridization of the array with two different samples of genomic DNA, one from the activated sludge and the other from a nonactivated sludge sample of an anaerobic ammonium oxidation (anammox) bacterial community, specific spots were visualized as a definite fluctuating profile in an MA (differential intensity ratio vs. spot intensity) plot. About 300 spots of the array accounted for the candidate probes to represent anammox reaction of the activated sludge. After sequence analysis of the probes and examination of the results of blastn searches against the reported anammox reference sequence, complete matches were found for 161 probes (58.3%) and >90% matches were found for 242 probes (87.1%). These results demonstrate that 'FloraArray' could be a useful tool for screening specific DNA molecules of unknown microbial communities.     

Manufacturing DNA microarrays of high spot homogeneity and reduced background signal

Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-L-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N:-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.     

Impact of surface chemistry and blocking strategies on DNA microarrays

The surfaces and immobilization chemistries of DNA microarrays are the foundation for high quality gene expression data. Four surface modification chemistries, poly-L-lysine (PLL), 3-glycidoxypropyltrimethoxysilane (GPS), DAB-AM-poly(propyleminime hexadecaamine) dendrimer (DAB) and 3-aminopropyltrimethoxysilane (APS), were evaluated using cDNA and oligonucleotide sub-arrays. Two un-silanized glass surfaces, RCA-cleaned and immersed in Tris-EDTA buffer were also studied. DNA on amine-modified surfaces was fixed by UV (90 mJ/cm(2)), while DNA on GPS-modified surfaces was immobilized by covalent coupling. Arrays were blocked with either succinic anhydride (SA), bovine serum albumin (BSA) or left unblocked prior to hybridization with labeled PCR product. Quality factors evaluated were surface affinity for cDNA versus oligonucleotides, spot and background intensity, spotting concentration and blocking chemistry. Contact angle measurements and atomic force microscopy were preformed to characterize surface wettability and morphology. The GPS surface exhibited the lowest background intensity regardless of blocking method. Blocking the arrays did not affect raw spot intensity, but affected background intensity on amine surfaces, BSA blocking being the lowest. Oligonucleotides and cDNA on unblocked GPS-modified slides gave the best signal (spot-to-background intensity ratio). Under the conditions evaluated, the unblocked GPS surface along with amine covalent coupling was the most appropriate for both cDNA and oligonucleotide microarrays.     

Kinetics of target site localization of a protein on DNA: a stochastic approach

It is widely recognized that the cleaving rate of a restriction enzyme on target DNA sequences is several orders-of-magnitude faster than the maximal one calculated from the diffusion-limited theory. It was therefore commonly assumed that the target site interaction of a restriction enzyme with DNA has to occur via two steps: one-dimensional diffusion along a DNA segment, and long-range jumps coming from association-dissociation events. We propose here a stochastic model for this reaction which comprises a series of one-dimensional diffusions of a restriction enzyme on nonspecific DNA sequences interrupted by three-dimensional excursions in the solution until the target sequence is reached. This model provides an optimal finding strategy which explains the fast association rate. Modeling the excursions by uncorrelated random jumps, we recover the expression of the mean time required for target site association to occur given by Berg et al. in 1981, and we explicitly give several physical quantities describing the stochastic pathway of the enzyme. For competitive target sites we calculate two quantities: processivity and preference. By comparing these theoretical expressions to recent experimental data obtained for EcoRV-DNA interaction, we quantify: 1), the mean residence time per binding event of EcoRV on DNA for a representative one-dimensional diffusion coefficient; 2), the average lengths of DNA scanned during the one-dimensional diffusion (during one binding event and during the overall process); and 3), the mean time and the mean number of visits needed to go from one target site to the other. Further, we evaluate the dynamics of DNA cleavage with regard to the probability for the restriction enzyme to perform another one-dimensional diffusion on the same DNA substrate following a three-dimensional excursion.     

Physicochemical perspectives on DNA microarray and biosensor technologies

Detection and sequence-identification of nucleic acid molecules is often performed by binding, or hybridization, of specimen "target" strands to immobilized, complementary "probe" strands. A familiar example is provided by DNA microarrays used to carry out thousands of solid-phase hybridization reactions simultaneously to determine gene expression patterns or to identify genotypes. The underlying molecular process, namely sequence-specific recognition between complementary probe and target molecules, is fairly well understood in bulk solution. However, this knowledge proves insufficient to adequately understand solid-phase hybridization. For example, equilibrium binding constants for solid-phase hybridization can differ by many orders of magnitude relative to solution values. Kinetics of probe-target binding are affected. Surface interactions, electrostatics and polymer phenomena manifest themselves in ways not experienced by hybridizing strands in bulk solution. The emerging fundamental understanding provides important insights into application of DNA microarray and biosensor technologies.     

Comparative modeling and analysis of microfluidic and conventional DNA microarrays

A theoretical analysis was developed to predict molecular hybridization rates for microarrays where samples flow through microfluidic channels and for conventional microarrays where samples remain stationary during hybridization. The theory was validated by using a multiplexed microfluidic microarray where eight samples were hybridized simultaneously against eight probes using 60-mer DNA strands. Mass transfer coefficients ranged over three orders of magnitude where either kinetic reaction rates or molecular diffusion rates controlled overall hybridization rates. Probes were printed using microfluidic channels and also conventional spotting techniques. Consistent with the theoretical model, the microfluidic microarray demonstrated the ability to print DNA probes in less than 1 min and to detect 10-pM target concentrations with hybridization times in less than 5 min.     

Integrated capillary fluorescence DNA biosensor

Covalent attachment of dsDNA molecules inside a glass capillary without the need for hybridization is described. It is shown that the glass capillary has a surface density of 2.5 x 10(13) molecules/cm(2) with specific binding capacity of 62.5%. The resulting substrate was used to develop a biosensor for determining fluorescent organic analytes and metal binding with DNA. The biosensor combines highly specific immobilization chemistry with a capillary-geometry flow cell arrangement. The results show that fluorescent dyes are retained in the dsDNA-modified surface and that exposure to concentrations of nickel and lead ions resulted in a recoverable, highly reproducible diminishment of the fluorescence intensity.     

A dimensionless number analysis of the hybridization process in diffusion- and convection-driven DNA microarray systems

The present theoretical analysis aims at providing a general understanding of the combined effect the many different process variables have on the hybridization rate in diffusion- and convection-driven DNA microarray systems. It is shown that all process variables can be grouped into only four different dimensionless numbers (the Damkohler number Da, the dimensionless association constant kappa(A), the dimensionless initial concentration C'(0) and a geometrical ratio alpha). These four numbers have a straightforward physical meaning and only contain easily measurable parameters. Reducing the solution space from 7D to 4D, the dimensionless number representation greatly facilitates the insight in the conditions leading to the occurrence of diffusion-limited hybridization rates in both diffusion- and convection-driven DNA microarray systems. This in turn simplifies their design and the interpretation of the experimental results that are obtained with these systems.     

Creating highly dense and uniform protein and DNA microarrays through photolithography and plasma modification of glass substrates

We demonstrate a method to create high density protein microarrays with excellent spot uniformity using photolithography and plasma processing on low cost commercially available microscope glass slides. Protein deposition and fluorescence signal evaluation on these substrates are performed by standard arrayers and scanners. To this end, spots of commercial photoresists (AZ5214, SU8 and Ormocomp(?)) were defined through lithography on glass substrates followed by short SF(6) plasma treatment and selective protein adsorption on these spots with respect to glass (spot to background fluorescence signal ratios 30:1 to 40:1) was demonstrated using model protein binding assays. Among the photoresists tested, Ormocomp was selected since it provided the highest protein binding capacity. No ageing of Ormocomp/glass substrates in terms of protein binding capacity was observed for at least two months. Besides to protein microarrays, DNA microarrays were also developed by spotting streptavidin-biotinylated oligonucleotide conjugates corresponding to wild- and mutant-type sequences of four deleterious BRCA1 gene mutations. For all of the examined mutations, higher specific hybridization signals (1.5-4 times) and improved discrimination ratios between wild- and mutant-type sequences as well as higher spot uniformity and repeatability were demonstrated on Ormocomp/glass substrates with intra- and inter-spot CVs of 8.0% and 4.5%, respectively, compared to commercial polystyrene (intra- and inter-spot CVs 36% and 18%) and epoxy-coated glass (intra- and inter-spot CVs 26% and 20%) slides. Thus, the proposed substrates can be readily applied to protein and DNA microarrays fabrication and, moreover, the described method for selective protein adsorption can be advantageously implemented in various analytical microdevices for multi-analyte detection.     

Protein determination by ponceau S using digital color image analysis of protein spots on nitrocellulose membranes

A procedure was developed to estimate protein concentrations using color image analysis of protein spots stained with ponceau S. The method involved spotting a constant volume (2 microl) of the protein solutions on nitrocellulose paper, staining with acidic ponceau S, destaining, and air drying the paper. The image of the nitrocellulose paper was grabbed using a digital color scanner and thresholded with an optimal value to mark the area of the spot. The intensity of the color in the spot was measured in an arbitrary unit of intensity termed as inverse integrated gray value. This value showed a discernible increase with protein concentrations from 0.1 to 50 microg protein per spot (0.05-25 mg/ml). The method is simple and convenient compared to the conventional spectrophotometric procedures and allows several samples to be analyzed simultaneously. It can also be used to estimate protein concentration in the spots stained with Coomassie brilliant blue or other dyes.     

Systematic spatial bias in DNA microarray hybridization is caused by probe spot position-dependent variability in lateral diffusion

Background

The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained.

Methodology/Principal Findings

This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias.

Conclusions

Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization.     

Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides.

A new method of DNA sequencing by hybridization using a microchip containing a set of immobilized oligonucleotides is being developed. A theoretical analysis is presented of the kinetics of DNA hybridization with deoxynucleotide molecules chemically tethered in a polyacrylamide gel layer. The analysis has shown that long-term evolution of the spatial distribution and of the amount of DNA bound in a hybridization cell is governed by "retarded diffusion," i.e., diffusion of the DNA interrupted by repeated association and dissociation with immobile oligonucleotide molecules. Retarded diffusion determines the characteristic time of establishing a final equilibrium state in a cell, i.e., the state with the maximum quantity and a uniform distribution of bound DNA. In the case of cells with the most stable, perfect duplexes, the characteristic time of retarded diffusion (which is proportional to the equilibrium binding constant and to the concentration of binding sites) can be longer than the duration of the real hybridization procedure. This conclusion is indirectly confirmed by the observation of nonuniform fluorescence of labeled DNA in perfect-match hybridization cells (brighter at the edges). For optimal discrimination of perfect duplexes from duplexes with mismatches the hybridization process should be brought to equilibrium under low-temperature nonsaturation conditions for all cells. The kinetic differences between perfect and nonperfect duplexes in the gel allow further improvement in the discrimination through additional washing at low temperature after hybridization.     

Long oligonucleotide arrays on nylon for large-scale gene expression analysis

To date, most studies of multigenic expression patterns by long DNA array have used DNA fragments as probes. These probes are usually obtained as PCR products, and this represents a time-consuming and error-prone approach, requiring strict quality control. The present study examines the use of 40- and 70-mer synthetic oligonucleotides as probes for DNA array analysis with radioactive labeled targets. Design, spotting onto nylon filters, and hybridization conditions were determined and optimized. In this approach, the sensitivity and the specificity of the hybridization appear comparable to the conventional long DNA probes assay, permitting the analysis of small samples of approximately 1 microg total RNA. The long oligonucleotide array thus provides a very convenient method for the analysis of gene expression patterns in biological specimens and in clinical research.     

Ultrathin functional star PEG coatings for DNA microarrays

In this study, star PEG coatings on glass substrates have been used as support material for oligonucleotide microarrays. These coatings are prepared from solutions of six armed star shaped prepolymers that carry reactive isocyanate endgroups. As described earlier, such films prevent the adsorption of proteins and the adhesion of cells but can easily be functionalized for specific biological recognition. Here we used the high functionality of these coatings for the covalent immobilization of amino terminated 20mer oligonucleotides, both by microcontact printing and spotting techniques. The permanent immobilization of fluorescently labeled DNA as well as hybridization of 20mer oligonucleotides have been monitored by fluorescence microscopy. The hybridization efficiency as determined by fluorescence intensity varied from 30% to 80% depending on the way of layer preparation. The direct spotting without additional activation and blocking steps of the surface demonstrates the potential of star PEG coatings as ultrathin surface modification for microarrays.     

DNA intermediates and telomere addition during genome reorganization in Euplotes crassus

Three gene-sized molecules cloned intact from macronuclear DNA served as hybridization probes to study excision of these molecules from chromosomes and their processing during macronuclear development in the hypotrich Euplotes crassus. These molecules occur in integrated forms within polytene chromosomal DNA during macronuclear developmental. After transection of the polytene chromosomes, the three molecules occur in intermediate forms. One of the three molecules first appeared in a large intermediate that was subsequently replaced by a second intermediate, approximately 140 bp larger than the final molecule. The other two macronuclear molecules were detected only in intermediates approximately 140 bp larger than the mature form. These penultimate intermediates are larger by virtue of oversized telomeres, which are pared to yield the mature gene-sized molecules.