Microgels and apparatus for PAGE of nucleic acids in one or two dimensions

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked FilesWe describe a system for horizontal 1D or 2D PAGE comprising an apparatus and microgels. There is no buffer outside the gel, making handling and sample loading easy. Specially designed electrodes on all four sides allow 2D electrophoresis without gel rotation. Electrophoresis is completed within 20 min and sensitivity is in the subnanogram range. The system is temperature controlled for speed, denaturation of nucleic acid molecules and maintaining molecules single-stranded. The system allows characterization of structure, conformation and damage in complex nucleic acid preparations. Besides quick 1D PAGE, 2D applications include characterization of efficiency of complex molecular procedures, checking quality of biosamples and detecting DNA damage in cells and body fluids. The system should also run protein gels.Scottish Government Icelandic Technology Development Fund grant

Sequence specificity of single-stranded DNA-binding proteins: a novel DNA microarray approach

We have developed a novel DNA microarray-based approach for identification of the sequence-specificity of single-stranded nucleic-acid-binding proteins (SNABPs). For verification, we have shown that the major cold shock protein (CspB) from Bacillus subtilis binds with high affinity to pyrimidine-rich sequences, with a binding preference for the consensus sequence, 5′-GTCTTTG/T-3′. The sequence was modelled onto the known structure of CspB and a cytosine-binding pocket was identified, which explains the strong preference for a cytosine base at position 3. This microarray method offers a rapid high-throughput approach for determining the specificity and strength of ss DNA–protein interactions. Further screening of this newly emerging family of transcription factors will help provide an insight into their cellular function

Factors involved in 3' splice site selection in eukaryotic pre-messenger RNA.

The work presented in this thesis is an investigation of factors involved in 3' splice site selection. To try to determine the intrinsic strengths of 3' splice site sequences, a cis-competition assay system was used. This system was based on the large intervening sequence (IVS-2) of the rabbit beta-globin gene. Synthetic 3' splice site sequence oligonucleotides were inserted into the EcoRl restriction site, forty nine nucleotides downstream of the authentic rabbit beta-globin IVS-2 3' splice site. The oligonucleotides conformed to the established 3' splice site consensus sequence and allowed for variations within this sequence. The authentic site served as a constant reference site against which the strengths of the synthetic sites could be measured. When spliced in HeLa cells in vivo, all constructs tested were seen to choose the authentic 3' splice site over the synthetic 3' splice site under test. A series of mutageneses was carried out to try to decrease the intrinsic strength of the authentic site and/or improve the environment of the synthetic site such that the overall strengths of the two sites might be balanced. An AG→CG mutation at the authentic 3' splice site caused the synthetic 3' splice site to be activated as a cryptic site in vivo and in vitro. In this case lariat formation was mapped to an artificially created branch point within exon 3. Splicing component binding to both 3' splice sites was investigated by looking at protection of the RNA from oligonucleotide directed cleavage by RNase H. Initial protection of both 3' splice sites was independent of the final choice of site. However, branch point protection was dependent on the 3' splice site chosen. Components bound to the authentic 3' splice site could be immunoprecipitated whether that site was chosen or not. The synthetic 3' splice site was poorly precipitated even when it was chosen. This data tends to suggest that the synthetic 3' splice site directs inefficient complex assembly, and that at least partial complex assembly occurs at a 3' splice site which has been inactivated by an AG→CG mutation. Preliminary work was carried out to develop a method for the analysis of splicing component binding to either or both 3' splice sites of material within fully and partially assembled splicing complexes (spliceosomes) isolated by sucrose gradient sedimentation

Impact of different ChIP-Seq protocols on DNA integrity and quality of bioinformatics analysis results.

To access publisher's full text version of this article click on the hyperlink at the bottom of the pageDifferent ChIP-Seq protocols may have a significant impact on the final outcome in terms of quality, number and distribution of called peaks. Sample DNA undergoes a long procedure before the final sequencing step, and damaged DNA can result in excessive mismatches in the alignment with reference genome. In this letter, we present the effect of well-defined modifications (timing of formaldehyde crosslink reversal, brand of the sonicator) of standard ChIP-Seq protocol on parallel samples derived from the same cell line correlating the initial DNA quality control metrics to the final bioinformatics analysis results.Associazione Italiana Ricerca sul Cancro Italian Ministry of Education, University, and Research PRIN 2008NY72SJ FIRB RBFR087JMZ FIRB RBFR10URHP Icelandic Research Council 110473-061