Acceleration of nucleic acid hybridization on DNA microarrays driven by pH tunable modifications.

A series of peptides containing histidine residues were designed as potential hybridization rate enhancers within a polymeric matrix of DNA microarrays. The polymeric matrix modified with these peptides showed strong attraction to DNA molecules under conditions of induction. DNA probes on the peptide-modified sites rapidly hybridized to their complementary targets with single base pair mismatch discrimination.

Oligonucleotides form a duplex with non-helical properties on a positively charged surface.

The double helix is known to form as a result of hybridization of complementary nucleic acid strands in aqueous solution. In the helix the negatively charged phosphate groups of each nucleic acid strand are distributed helically on the outside of the duplex and are available for interaction with cationic groups. Cation-coated glass surfaces are now widely used in biotechnology, especially for covalent attachment of cDNAs and oligonucleotides as surface-bound probes on microarrays. These cationic surfaces can bind the nucleic acid backbone electrostatically through the phosphate moiety. Here we describe a simple method to fabricate DNA microarrays based upon adsorptive rather than covalent attachment of oligonucleotides to a positively charged surface. We show that such adsorbed oligonucleotide probes form a densely packed monolayer, which retains capacity for base pair-specific hybridization with a solution state DNA target strand to form the duplex. However, both strand dissociation kinetics and the rate of DNase digestion suggest, on symmetry grounds, that the target DNA binds to such adsorbed oligonucleotides to form a highly asymmetrical and unwound duplex. Thus, it is suggested that, at least on a charged surface, a non-helical DNA duplex can be the preferred structural isomer under standard biochemical conditions.

Simultaneous multianalyte ELISA performed on a microarray platform.

BACKGROUND: A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Here we describe the construction and use of such a multiplex ELISA to measure prostate-specific antigen (PSA), alpha1-antichymotrypsin-bound PSA (PSA-ACT), and interleukin-6 (IL-6). METHODS: We silanized glass plates and printed the appropriate capture antibodies to allow for the construction of "sandwich" ELISA quantification assays. We examined specificity of the assay for appropriate antigen, assembled calibration curves, and obtained PSA concentrations for 14 human serum samples. We compared the serum PSA concentrations derived through the use of our array with values obtained independently using a standard ELISA method. RESULTS: R2 values generated by our microarray for the PSA and PSA-ACT calibration curves were 0.989 and 0.979, respectively. Analyte concentrations used for the construction of these curves were 0.31-20 microg of protein/L of diluent. IL-6 calibration curve concentrations were 4.9-300 ng of IL-6/L of diluent. The R2 value for the IL-6 calibration curve was 0.983. The 14 human serum samples screened by this micro-ELISA technique for PSA concentrations generated a regression equation (linear) with a slope of 0.83 +/- 0.10 and intercept of 0.74 +/- 0.70 (R2 = 0.88). CONCLUSIONS: Multiplexed ELISA arrays are a feasible option for analyte quantification in complex biologic samples.

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.

Nearly instantaneous, cation-independent, high selectivity nucleic acid hybridization to DNA microarrays.

Hybridization rate enhancement has been demonstrated for high molecular weight DNA target binding to a microarray. Microarrays were fabricated using biotin-modified oligonucleotides complexed with streptavidin (SA), which serves as an attachment to the underlying surface. It is shown that at low salt and pH 5, where SA develops a positive charge, duplex formation becomes at least 80-fold faster than seen under standard conditions, where SA is neutral or anionic. Duplex formation becomes independent of solution state cation concentration in the low pH state, under conditions where specificity remains high. The utility of such applied surface science is discussed.

DNA microarray technology for neonatal screening.

Modern molecular biology, owing much to the Human Genome Initiative, has elucidated many of the genetic mechanisms underlying heritable metabolic disease. While the use of molecular methods has flourished in research laboratories, complexity and cost have limited their utility in newborn screening. Newborn blood cards provide high quality DNA samples able to provide reliable support to highly multiplexed polymerase chain reactions (PCR). New manufacturing processes have reduced the cost of DNA microarray technology to the point where it is a practical tool for population screening. In a single assay, a DNA microarray facilitates the co-detection of amplification products diagnostic for several genetic diseases. High throughput is achieved with automation at every step, from DNA extraction to detection of hybrids. We suggest that it is both feasible and practical to develop a first-tier newborn screening protocol based upon multiplex PCR and analysis of amplification products using DNA microarrays. Initial data utilizing the model systems of sickle cell disease, alpha-1-antitrypsin deficiency and Factor V Leiden will be reported.

A microchip for quantitative detection of molecules utilizing luminescent and radioisotope reporter groups.

Through the marriage of microelectronics and molecular biology, a miniaturized device is presented for ultrasensitive detection of labeled molecules. The novelty of the approach is the direct integration of a charge-coupled device (CCD) and a probe-based assay. Specifically, a CCD detector serves as an active solid support that quantitatively detects and images the distribution of labeled target molecules near the spatially addressable pixels. The device exploits the inherent characteristics of microelectronics that accommodate highly parallel assays, ultrasensitive detection, high throughput, integrated data acquisition and computation. Hence, the technology presented offers substantial practical utility to both research and clinical diagnostic applications that require quantitative analysis of bound molecules. Specifically for probe-based assays such as reverse dot blots, hybridization of radiolabeled or fluorescently labeled, target DNA can be quantitatively assessed within seconds due to the high sensitivity and direct coupling employed.

Evaluation of the potent anti-hepatitis B virus agent (-) cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in a novel in vivo model.

A murine model was developed to investigate the in vivo activity of anti-hepatitis B virus (HBV) agents. Mice with subcutaneous tumors of HBV-producing 2.2.15 cells showed reductions in levels of HBV in serum and in intracellular levels of HBV when the mice were orally dosed with (-) cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (FTC). No effects on tumor size or alpha-fetoprotein levels were observed. FTC can selectively inhibit HBV replication at nontoxic doses.

Immunologic priming with recombinant hepatitis A virus capsid proteins produced in Escherichia coli.

Hepatitis A virus capsid proteins (VP0, VP3, and VP1) have been synthesized in Escherichia coli for use in antigenic and immunogenic analyses. Rabbits immunized with each of these individual recombinant capsid proteins developed a rapid neutralizing antibody response when subsequently challenged with a subimmunogenic dose of whole virus.