Single-stranded DNA (ssDNA) generation is a crucial step in several molecular biology applications, such as sequencing or DNA chip and microarray technology. Molecules of ssDNA also play a key role in the selection of ssDNA aptamers through Systematic Evolution of Ligands by EXponential enrichment (SELEX). With particular interest for this application, herein we present a comparative study of the most used methods for generation of ssDNA used in SELEX, such as asymmetric PCR, enzyme digestion and magnetic separation with streptavidin beads. In addition, we evaluate a new technique that combines asymmetric PCR and enzyme digestion with the aim to achieve the maximum efficiency in ssDNA generation. The methods studied were compared in terms of quality of ssDNA using electrophoretic analysis and generated ssDNA yields were quantitatively measured using an Enzyme-Linked OligoNucleotide Assay (ELONA).

Lupin has recently been added to the list of allergens requiring mandatory advisory labeling on foodstuffs sold in the European Union, and since December 2008, all products containing even trace amounts of lupin must be labeled correctly. Lupin globulins consist of two major globulins called α-conglutin (11S and "legumin-like") and β-conglutin (7S and "vicilin-like") and another additional two globulins, γ-conglutin and δ-conglutin, which are present in lower amounts. We report on a methodology to facilitate the extraction of each of these proteins using centrifugation and isolation by anion-exchange chromatography followed by size-exclusion chromatography. The isolated subunits were characterized using reducing and non-reducing polyacrylamide gel electrophoresis, western blotting, and peptide mass fingerprinting, all of which revealed that the individual protein subunits are highly pure and can be used as immunogens for the production of antibodies specific for each of the conglutin fractions, as well as standards, and the extraction protocol can be used for the selective extraction of each of the subunits from foodstuffs, thus facilitating a highly accurate determination of the lupin concentration. Furthermore, the subunits can be used to elucidate information regarding the toxicity of each of the subunits, by looking at their interaction with the IgE antibodies found in the serum of individuals allergic to lupin, providing critical information for the definition of the requirements of analytical assays for the detection of lupin in foodstuffs.

Changes at proteomic level in cardiovascular tissues may partially account for the underlying mechanisms of VOO phenols cardiovascular protection being the SEC effects higher than free HT.

Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs.