The incredible shrinking world of DNA microarrays

The efficacy of microarrays in examining gene expression, gene and genome structure, protein-DNA interactions, whole-genome similarities and differences, microRNA expression, methylation (and more) is no longer in question. It is a fast-developing, cutting edge technology that has grown up along with massive sequence databases and is likely to become part of everyday patient care. Many advances have recently expanded the power and utility of microarrays; among them is our development of a new array tiling technique that dramatically increases the scope of coverage of an oligonucleotide tiling array without substantially increasing its cost.     

Exploring the sequence space of a DNA aptamer using microarrays

The relationship between sequence and binding properties of an aptamer for immunoglobulin E (IgE) was investigated using custom DNA microarrays. Single, double and some triple mutations of the aptamer sequence were created to evaluate the importance of specific base composition on aptamer binding. The majority of the positions in the aptamer sequence were found to be immutable, with changes at these positions resulting in more than a 100-fold decrease in binding affinity. Improvements in binding were observed by altering the stem region of the aptamer, suggesting that it plays a significant role in binding. Results obtained for the various mutations were used to estimate the information content and the probability of finding a functional aptamer sequence by selection from a random library. For the IgE-binding aptamer, this probability is on the order of 10⁻¹⁰ to 10⁻⁹. Results obtained for the double and triple mutations also show that there are no compensatory mutations within the space defined by those mutations. Apparently, at least for this particular aptamer, the functional sequence space can be represented as a rugged landscape with sharp peaks defined by highly constrained base compositions. This makes the rational optimization of aptamer sequences using step-wise mutagenesis approaches very challenging.     

Facilitating RNA structure prediction with microarrays

Determining RNA secondary structure is important for understanding structure-function relationships and identifying potential drug targets. This paper reports the use of microarrays with heptamer 2'-O-methyl oligoribonucleotides to probe the secondary structure of an RNA and thereby improve the prediction of that secondary structure. When experimental constraints from hybridization results are added to a free-energy minimization algorithm, the prediction of the secondary structure of Escherichia coli 5S rRNA improves from 27 to 92% of the known canonical base pairs. Optimization of buffer conditions for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding and improve discrimination between AU and GU pairs are also described. The results suggest that probing RNA with oligonucleotide microarrays can facilitate determination of secondary structure.     

The expanding world of DNA and RNA

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Production of bulk amounts of universal RNA for DNA microarrays

In DNA microarray technology, repeatability and reliability are very important to compare multiple RNA samplesfrom different experiments. The application of common or universal RNA as a standard control equalizes the differences in hybridization parameters and array variations. For this purpose, high-quality reference RNA is necessary in bulk amounts. A novel approach was developed to get milligrams of sense or antisense RNA, starting from micrograms of pooled total RNA from different cell lines, tissues, or organisms. This method is inexpensive and allows further labeling procedures using poly(dT) or random oligomers as primers. In addition, amplified, sense reference RNA is suitable for standard labeling protocols, while the antisense reference RNA can be used with antisense RNA from the linear sample amplification method. Here we produced universal RNA for human, rat, and alfalfa and demonstrated the quality using specific cDNA microarrays.     

DNA microarrays with PAMAM dendritic linker systems

The DNA microarray-based analysis of single nucleotide polymorphisms (SNPs) is important for the correlation of genetic variations and individual phenotypes, and for locating disease-causing genes. To facilitate the development of surfaces suitable for immobilization of oligonucleotides, we report here a novel method for the surface immobilization of DNA using pre-fabricated polyamidoamine (PAMAM) starburst dendrimers as mediator moieties. Dendrimers containing 64 primary amino groups in their outer sphere are covalently attached to silylated glass supports and, subsequently, the dendritic macromolecules are modified with glutaric anhydride and activated with N-hydroxysuccinimide. As a result of the dendritic PAMAM linker system the surfaces reveal both a very high immobilization efficiency for amino-modified DNA-oligomers, and also a remarkable high stability during repeated regeneration and re-using cycles. The performance of dendrimer-based DNA microarrays in the discrimination of SNPs is demonstrated.     

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.     

DNA microarrays: raising the profile

Expression profiling using DNA microarrays is starting to come of age. The past year has seen significant advances in the number, scope and quality of studies that incorporate expression profiling experiments. Attention is starting to move on from making DNA microarrays to appropriate experimental design and sophisticated data analysis techniques.