DNA aptamers against the Lup an 1 food allergen.

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. Pe...

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Autores principales:	Pedro Nadal, Alessandro Pinto, Marketa Svobodova, Nuria Canela, Ciara K O'Sullivan
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ebac8da05efc4340b6e3afef4a2e863a
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spelling	oai:doaj.org-article:ebac8da05efc4340b6e3afef4a2e863a2021-11-18T07:21:55ZDNA aptamers against the Lup an 1 food allergen.1932-620310.1371/journal.pone.0035253https://doaj.org/article/ebac8da05efc4340b6e3afef4a2e863a2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22529997/?tool=EBIhttps://doaj.org/toc/1932-6203Using 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.Pedro NadalAlessandro PintoMarketa SvobodovaNuria CanelaCiara K O'SullivanCiara K O'SullivanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 4, p e35253 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Pedro Nadal Alessandro Pinto Marketa Svobodova Nuria Canela Ciara K O'Sullivan Ciara K O'Sullivan DNA aptamers against the Lup an 1 food allergen.
description	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.
format	article
author	Pedro Nadal Alessandro Pinto Marketa Svobodova Nuria Canela Ciara K O'Sullivan Ciara K O'Sullivan
author_facet	Pedro Nadal Alessandro Pinto Marketa Svobodova Nuria Canela Ciara K O'Sullivan Ciara K O'Sullivan
author_sort	Pedro Nadal
title	DNA aptamers against the Lup an 1 food allergen.
title_short	DNA aptamers against the Lup an 1 food allergen.
title_full	DNA aptamers against the Lup an 1 food allergen.
title_fullStr	DNA aptamers against the Lup an 1 food allergen.
title_full_unstemmed	DNA aptamers against the Lup an 1 food allergen.
title_sort	dna aptamers against the lup an 1 food allergen.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/ebac8da05efc4340b6e3afef4a2e863a
work_keys_str_mv	AT pedronadal dnaaptamersagainstthelupan1foodallergen AT alessandropinto dnaaptamersagainstthelupan1foodallergen AT marketasvobodova dnaaptamersagainstthelupan1foodallergen AT nuriacanela dnaaptamersagainstthelupan1foodallergen AT ciarakosullivan dnaaptamersagainstthelupan1foodallergen AT ciarakosullivan dnaaptamersagainstthelupan1foodallergen
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DNA aptamers against the Lup an 1 food allergen.

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