Quantitative Real-time PCR has proven to be a powerful tool to accurately 13 estimate transgene copy number and exogenous gene expression in plants. 14 Compared to Southern and Northern blot analysis, accuracy, rapidity and low-cost 15 are the main advantages. However, this technique requires important preliminary 16 work for standardizing and optimizing the many parameters involved in the 17 analysis. Grapevine data on this topic are not presently available in the literature. 18 We have developed a method for properly performing quantitative assays in grapes 19 where the genes for the b-glucoronidase (GUS) under the control of the 35S 20 promoter and for the neomicin phosphotransferase (NPTII) were transferred. 21 Validation of our method was verified with Southern blot analysis comparison. The 22 optimal endogenous genes to be applied as referee on grape were exploited and nine23 cis-epoxycarotenoid dioxygenase2 (NCED2) resulted the most amenable. For 24 transgene copy number detection, the approach based on synthetic hybrid amplicons 25 proved to be promising.
EVALUATION OF EXOGENOUS DNA BY QUANTITATIVE REAL-TIME PCR IN TRANSGENIC GRAPE / Savazzini, F.; Dalla Costa, L.; Martinelli, L.. - In: ACTA HORTICULTURAE. - ISSN 0567-7572. - ELETTRONICO. - 689:689(2005), pp. 499-504. [10.17660/ActaHortic.2005.689.61]
EVALUATION OF EXOGENOUS DNA BY QUANTITATIVE REAL-TIME PCR IN TRANSGENIC GRAPE
SAVAZZINI, FEDERICA;MARTINELLI, LUCIA
2005
Abstract
Quantitative Real-time PCR has proven to be a powerful tool to accurately 13 estimate transgene copy number and exogenous gene expression in plants. 14 Compared to Southern and Northern blot analysis, accuracy, rapidity and low-cost 15 are the main advantages. However, this technique requires important preliminary 16 work for standardizing and optimizing the many parameters involved in the 17 analysis. Grapevine data on this topic are not presently available in the literature. 18 We have developed a method for properly performing quantitative assays in grapes 19 where the genes for the b-glucoronidase (GUS) under the control of the 35S 20 promoter and for the neomicin phosphotransferase (NPTII) were transferred. 21 Validation of our method was verified with Southern blot analysis comparison. The 22 optimal endogenous genes to be applied as referee on grape were exploited and nine23 cis-epoxycarotenoid dioxygenase2 (NCED2) resulted the most amenable. For 24 transgene copy number detection, the approach based on synthetic hybrid amplicons 25 proved to be promising.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
