Functional analysis and identification of cis-regulatory elements of human chromosome 21 gene promoters.

Given the inherent limitations of in silico studies relying solely on DNA sequence analysis, the functional characterization of mammalian promoters and associated cis-regulatory elements requires experimental support, which demands cloning and analysis of putative promoter regions. Focusing on human chromosome 21, we cloned 182 gene promoters of 2500 bp in length and conducted reporter gene assays on transfected-cell arrays. We found 56 promoters that were active in HEK293 cells, while another 49 promoters could be activated by treatment of cells with Trichostatin A or depletion of serum.

LNA-modified oligodeoxynucleotide hybridization with DNA microarrays printed on nanoporous membrane slides.

We report a robust method for the detection of hybridization events using a microarray-based assay on a nanoporous membrane platform. The technique is characterized by a hybridization time of only 1 hour and uses Cy5-labeled, 7-mer oligodeoxynucleotide probes modified with locked nucleic acid (LNA) nucleotides. We show that the volume of the DNA spotted onto a nanomembrane can be reduced to approximately 4 nL with detectable signal intensity.

Liquid-based hybridization assay with real-time detection in miniaturized array platforms.

An assay for the fluorescent detection of short oligonucleotide probe hybridization in miniaturized high-density array platforms is presented. It combines hybridization in solution with real-time fluorescent detection, which involves measurement of fluorescence increase by means of an induced fluorescence resonance energy transfer. The feasibility of this approach using DNA or RNA as a target, and short DNA- as well as LNA (locked nucleic acid)-modified oligonucleotides as probes is shown.

Multiplexed hybridizations of positively charge-tagged peptide nucleic acids detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Peptide nucleic acid (PNA) is a novel class of DNA analogues in which the entire sugar-phosphate backbone is replaced by a pseudopeptide counterpart. Owing to its neutral character and the consequent lack of electrostatic repulsion, PNA exhibits very stable heteroduplex formation with complementary nucleic acid that is essentially ionic strength independent and enables hybridization under minimum salt conditions. This feature as well as its superior ion stability and easy ionization compared to DNA renders PNA very attractive for hybridization-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) applications.