RESUMEN
The quantification of GMOs by real-time PCR relies on an external calibrant. In this paper the suitability of two DNA calibrants, genomic DNA from plant leaves and plasmidic DNA, was investigated. The PCR efficiencies, the correlation coefficients of the calibration curves, and the ratios between PCR efficiencies of transgenic and endogenous sequences were compared for both calibrants using 59 data sets produced by 43 laboratories. There were no significant differences between plasmidic and genomic DNA except for the PCR efficiencies of the calibration curves for the transgene of the construct-specific real-time PCR method. In the GM system investigated, PCR efficiencies of plasmidic calibrants were slightly closer to the PCR efficiencies observed for the unknowns than those of the genomic DNA calibrant. Therefore, plasmidic DNA was the more suitable calibrant for the PCR measurements on genomic DNA extracted from MON 810 seeds. It is shown that plasmidic DNA is an appropriate choice for the calibration of measurements of MON 810 corn with respect to the DNA copy number ratio.
Asunto(s)
ADN de Plantas/análisis , Alimentos Modificados Genéticamente , Plantas Modificadas Genéticamente/genética , Zea mays/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Calibración , Endotoxinas/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas Hemolisinas/genética , Hojas de la Planta/genética , Reacción en Cadena de la Polimerasa , Semillas/genéticaRESUMEN
This paper is part of a set of three papers investigating metrological traceability of the quantification of DNA fragments as, for instance, used for quantification of genetic modifications. This paper evaluates the possible impact of several factors on results of real-time Polymerase Chain Reaction (PCR) measurements. It was found that the particle size of the powder samples does not have an influence, whereas the nature of the calibrant (plasmidic or genomic DNA) has a significant effect. Moreover, two real-time PCR detection methods (construct-specific and event-specific) for MON 810 corn were compared. The results obtained in a specifically designed interlaboratory study revealed a significant influence of the DNA extraction method on measurement results when the MON 810 construct-specific real-time PCR detection method was applied. Statistical analyses confirmed the importance of validating DNA extraction methods in conjunction with real-time PCR methods.
Asunto(s)
ADN de Plantas/análisis , Alimentos Modificados Genéticamente , Plantas Modificadas Genéticamente/genética , Reacción en Cadena de la Polimerasa/métodos , Zea mays/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Endotoxinas/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas Hemolisinas/genética , Semillas/genéticaRESUMEN
Thaumatin-like proteins (TLPs) were isolated and characterized from fruits and leaves of elderberry (Sambucus nigra) and their corresponding genes cloned. In addition, the developmental regulation and induction of the different TLPs was followed in some detail. Ripening berries accumulated a fruit-specific TLP during the final stages of maturation. This fruit-specific TLP had no antifungal activity and was devoid of beta-glucanase activity. Leaves constitutively expressed a TLP that closely resembled the fruit-specific homologue. Treatment with jasmonate methyl ester induced two additional TLPs in leaves but did not induce or enhance the expression of TLPs in immature berries. In contrast to jasmonate methyl ester, both ethephon and garlic extract induced the expression of a TLP in unripe berries that normally do not express any TLP. Sequence analysis and molecular modeling indicated that all elderberry thaumatin-like proteins share a high sequence similarity with group-5 pathogenesis-related proteins. However, the proteins encoded by the different sequences differed from each other in isoelectric point and the distribution of the charges on the surface of the molecule.