Simultaneous Confirmatory Analysis of Different Transgenic Maize (Zea mays) Lines Using Multiplex Polymerase Chain Reaction−Restriction Analysis and Capillary Gel Electrophoresis with Laser Induced Fluorescence Detection

2008 ◽  
Vol 56 (18) ◽  
pp. 8280-8286 ◽  
Author(s):  
Virginia García-Cañas ◽  
Alejandro Cifuentes
Keyword(s):  
Polymerase Chain Reaction ◽  
Gel Electrophoresis ◽  
Multiplex Polymerase Chain Reaction ◽  
Restriction Analysis ◽  
Laser Induced Fluorescence ◽  
Chain Reaction ◽  
Capillary Gel Electrophoresis ◽  
Laser Induced Fluorescence Detection ◽  
Confirmatory Analysis ◽  
Polymerase Chain

scholarly journals Multiplex Polymerase Chain Reaction-Capillary Gel Electrophoresis: A Promising Tool for GMO ScreeningAssay for Simultaneous Detection of Five Genetically Modified Cotton Events and Species

2009 ◽  
Vol 92 (3) ◽  
pp. 765-772 ◽  
Author(s):  
Anna Nadal ◽  
Teresa Esteve ◽  
Maria Pla
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Gel Electrophoresis ◽  
Multiplex Polymerase Chain Reaction ◽  
Genetically Modified ◽  
Simultaneous Detection ◽  
Chain Reaction ◽  
Capillary Gel Electrophoresis ◽  
Cotton Species ◽  
Polymerase Chain

Abstract A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7 of false classification rate), with limit of detection values of 0.1 for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary.

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