Illumina universal bead arrays.

This chapter describes an accurate, scalable, and flexible microarray technology. It includes a miniaturized array platform where each individual feature is quality controlled and a versatile assay that can be adapted for various genetic analyses, such as single nucleotide polymorphism genotyping, DNA methylation detection, and gene expression profiling. This chapter describes the concept of the BeadArray technology, two different Array of Arrays formats, the assay scheme and protocol, the performance of the system, and its use in large-scale genetic, epigenetic, and expression studies.

Whole-genome genotyping.

We have developed an array-based whole-genome genotyping (WGG) assay (Infinium) using our BeadChip platform that effectively enables unlimited multiplexing and unconstrained single nucleotide polymorphism (SNP) selection. A single tube whole-genome amplification reaction is used to amplify the genome, and loci of interest are captured by specific hybridization of amplified gDNA to 50-mer probe arrays. After target capture, SNPs are genotyped on the array by a primer extension reaction in the presence of hapten-labeled nucleotides. The resultant signal is amplified during staining and the array is read out on a high-resolution confocal scanner. We have employed our high-density BeadChips supporting up to 288,000 bead types to create an array that can query over 100,000 SNPs using the Infinium assay. In addition, we have developed an automated BeadChip processing platform using Tecan's GenePaint slide processing system. Hybridization, washing, array-based primer extension, and staining are performed directly in Tecan's capillary gap Te-Flow chambers. This automation process increases assay robustness and throughput greatly while enabling laboratory information management system control of sample tracking.

Binding stoichiometry of an RNA aptamer and its transcription factor target.

RNA molecules serve informational, structural, and catalytic roles in cells. RNA also offers an interesting raw material for the design or genetic selection of modifiers of gene expression. We have been interested in the possibility that natural and/or artificial RNA ligands might be identified for DNA-binding proteins. With these concepts in mind, our laboratory previously isolated a 31-nucleotide RNA aptamer that specifically binds to human transcription factor NF-kappaB. This RNA aptamer (alpha-p50) competitively inhibits DNA binding by NF-kappaB in vitro. The aptamer may target the DNA-binding groove formed by the junction of the two monomers of NF-kappaB, perhaps mimicking kappaB duplex DNA. This model predicts a binding stoichiometry of one RNA aptamer per NF-kappaB dimer. To test this hypothesis, two complementary biophysical methods were utilized. Both analytical ultracentrifugation and microelectrospray mass spectrometry suggest that 1 mol of alpha-p50 RNA binds per mole of NF-kappaB p50 homodimer. Such a result is consistent with the observed ability of the RNA aptamer to block the access of transcription factor NF-kappaB to its binding site on DNA and highlights the question of how an RNA stem-loop structurally mimics a DNA duplex. This work also demonstrates the successful application of mass spectrometry to characterize noncovalent RNA/protein interactions.

Rescue of an abasic hairpin ribozyme by cationic nucleobases: evidence for a novel mechanism of RNA catalysis.

The hairpin ribozyme catalyzes a reversible phosphodiester cleavage reaction. We examined the roles of conserved nucleobases in catalysis using an abasic ribozyme rescue strategy. Loss of the active site G8 nucleobase reduced the cleavage rate constant by 350-fold while loss of A9 and A10 nucleobases reduced activity less than 10-fold. Certain heterocyclic amines restored partial activity when provided in solution to the variant lacking G8. Heterocyclic amines that were capable of rescue shared the exocyclic amine and cyclic amide in common with the Watson-Crick hydrogen bonding face of guanine. In contrast to the shallow pH dependence of unmodified ribozyme activity, rescue activity increased sharply with decreasing pH. These results support a novel model for RNA catalysis in which a cationic nucleobase contributes electrostatic stabilization to negative charge developing in the transition state.