Oligonucleotide Design for Whole Genome Tiling Arrays

Oligonucleotides are short, single-stranded fragments of DNA or RNA, designed to readily bind with a unique part in the target sequence. They have many important applications including PCR (polymerase chain reaction) amplification, microarrays, or FISH (fluorescence in situ hybridization) probes. While traditional microarrays are commonly used for measuring gene expression levels by probing for sequences of known and predicted genes, high-density, whole genome tiling arrays probe intensively for sequences that are known to exist in a contiguous region. Current programs for designing oligonucleotides for tiling arrays are not able to produce results that are close to optimal since they allow oligonucleotides that are too similar with non-targets, thus enabling unwanted cross-hybridization. We present a new program, BOND-tile, that produces much better tiling arrays, as shown by extensive comparison with leading programs

Oligonucleotide that binds nuclear factor NF-kappa-B acts as a lymphoid-specific and inducible enhancer element

The immunoglobulin kappa light chain gene contains a lymphoid-specific enhancer that includes several short protein-binding sequences. The sequence that binds the nuclear factor NF-kappa B was tested for its ability to act independently as an enhancer element by inserting it into test plasmids containing the chloramphenicol acetyltransferase gene. When analyzed for activity by transient transfection into lymphoid and nonlymphoid cells, a single copy of the NF-kappa B binding site could act as a tissue-specific upstream activating element. Two copies (dimer) showed 10-fold higher activity than did one copy and could act as an enhancer element 2.5 kilobases downstream of the transcriptional start site. The enhancer activity of this sequence was correlated with the presence of the cognate binding protein, NF-kappa B. This sequence acted as an inducible enhancer under conditions that induce NF-kappa B binding activity. Thus, the NF-kappa B binding site acts by itself as a tissue-specific and inducible enhancer element, and two copies show cooperative interaction

Polymerase-endonuclease amplification reaction for large-scale enzymatic production of antisense oligonucleotide

Synthetic oligonucleotides are contaminated with highly homologous failure sequences. Oligonucleotide synthesis is difficult to scale up because it requires expensive equipments, hazardous chemicals, and tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR), for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI) cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves >100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation, so it has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs

A microfluidic oligonucleotide synthesizer

De novo gene and genome synthesis enables the design of any sequence without the requirement of a pre-existing template as in traditional genetic engineering methods. The ability to mass produce synthetic genes holds great potential for biological research, but widespread availability of de novo DNA constructs is currently hampered by their high cost. In this work, we describe a microfluidic platform for parallel solid phase synthesis of oligonucleotides that can greatly reduce the cost of gene synthesis by reducing reagent consumption (by 100-fold) while maintaining a 100 pmol synthesis scale so there is no need for amplification before assembly. Sixteen oligonucleotides were synthesized in parallel on this platform and then successfully used in a ligation-mediated assembly method to generate DNA constructs 200 bp in length

Effect of defects on thermal denaturation of DNA Oligomers

The effect of defects on the melting profile of short heterogeneous DNA chains are calculated using the Peyrard-Bishop Hamiltonian. The on-site potential on a defect site is represented by a potential which has only the short-range repulsion and the flat part without well of the Morse potential. The stacking energy between the two neigbouring pairs involving a defect site is also modified. The results are found to be in good agreement with the experiments.Comment: 11 pages including 5 postscript figure; To be appear in Phys. Rev.

Pd2 +-mediated base pairing in oligonucleotides

International audienceTwo short glycol nucleic acid (GNA) oligonucleotides, having either a terminal or an intrachain nucleobase replaced by the pyridine-2,6-dicarboxamide chelate of Pd2 +, have been synthesized and their hybridization properties studied by melting temperature measurements. In the termini of a double-stranded oligonucleotide, the Pd2 + chelates provided dramatic stabilization of the duplex relative to its metal-free counterpart, in all likelihood owing to formation of Pd2 +-mediated base pairs between pyridine-2,6-dicarboxamide and the opposing nucleobase. In contrast, no stabilization was observed when the Pd2 + chelate was placed in the middle of the chain. Furthermore, the results could not be reproduced by adding a Pd2 + salt in situ to the dilute oligonucleotide solutions but the palladated oligonucleotides had to be synthesized and purified prior to the hybridization studies. This behavior, presumably attributable to the relatively slow ligand-exchange reactions of Pd2 +, differs greatly from what is usually observed with more labile metal ions. The present results offer an explanation for the failure of previous attempts to incorporate Pd2 +-mediated base pairs into oligonucleotides