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Quantitative real-time PCR with SYBR Green detection to assess gene duplication in insects: study of gene dosage in Drosophila melanogaster (Diptera) and in Ostrinia nubilalis (Lepidoptera)

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Quantitative real-time PCR with SYBR Green detection to assess gene duplication in insects: study of gene dosage in Drosophila melanogaster (Diptera) and in Ostrinia nubilalis (Lepidoptera)

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dc.contributor.author Bel Cortés, Yolanda es_ES
dc.contributor.author Ferré Manzanero, Juan es_ES
dc.contributor.author Escriche Soler, Baltasar es_ES
dc.date.accessioned 2015-06-22T09:50:06Z
dc.date.available 2015-06-22T09:50:06Z
dc.date.issued 2011 es_ES
dc.identifier.citation BMC Research Notes Vol. 4 pp. 84-84 es_ES
dc.identifier.uri http://hdl.handle.net/10550/44665
dc.description.abstract BackgroundThe accurate determination of the number of copies of a gene in the genome (gene dosage) is essential for a number of genetic analyses. Quantitative real time PCR (qPCR) with TaqMan detection has shown advantages over traditional Southern-blot and FISH techniques, however the high costs of the required labeled probes is an important limitation of this method. qPCR with SYBR Green I detection is a simple and inexpensive alternative, but it has never been applied to the determination of the copy number of low copy number genes in organisms with high allelic variability (as some insects), where a very small margin of error is essential.FindingsWe have tested the suitability of the qPCR with SYBR Green I detection methodology for the detection of low copy number genes in two insects: the genetically well characterized Drosophila melanogaster (Diptera) and the poor genetically characterized Ostrinia nubilalis (Lepidoptera). The system was applied to determine the copy number of: (1) the O. nubilalis cadherin gene, involved in the mode of action of Bacillus thuringiensis toxins, which showed indirect evidence of duplication, and (2) the D. melanogaster BarH1 and BarH2 genes, located within the Bar region of the X chromosome, to clearly determine whether they both are covered by the tandem duplication in the classical Bar (B1) mutant. Our results showed that the O. nubilalis cadherin gene is an autosomal single copy gene and that BarH1, but not BarH2, is duplicated in the Drosophila B1 mutant.ConclusionsThis work shows that qPCR with SYBR Green I detection can be specific and accurate enough to distinguish between one and two gene copies per haploid genome of genes with high allelic variability. The technique is sensitive enough to give reliable results with a minimum amount of sample (DNA from individual thoraxes) and to detect gene duplications in tandem. es_ES
dc.title Quantitative real-time PCR with SYBR Green detection to assess gene duplication in insects: study of gene dosage in Drosophila melanogaster (Diptera) and in Ostrinia nubilalis (Lepidoptera) es_ES
dc.type journal article es_ES
dc.identifier.doi 10.1186/1756-0500-4-84 es_ES
dc.identifier.idgrec 070866 es_ES

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