LT-RADE: An Efficient User-Friendly Genome Walking Method Applied to the Molecular Characterization of the Insertion Site of Genetically Modified Maize MON810 and Rice LLRICE62

Information on the insertion site and characterization of the transgene(s) in genetically modified organisms (GMO) is very important for safety assessment and identification of a GMO. The generation of such information, especially when evidence suggests the presence of unauthorized GMO on the market, requires efficient and in the later situation also rapid approaches. Here we report on the optimization of a restriction independent method named 'Rapid Amplification of genomic DNA Ends' (RADE). The method was developed using maize event MON810 genomic DNA as a model system, testing a standard Taq polymerase or a blend of polymerases (standard Taq and proofreading Tgo polymerases (LT-RADE). Both methods produce an initial single strand DNA, followed by nested PCR steps and yield easy-to-isolate DNA fragments for further manipulation. We showed that the application of the Taq/Tgo polymerase blend significantly increased the size of the obtained PCR products. Using LT-RADE we could successfully isolate the flanking regions of the transgenic insert of the GM maize event MON810 and confirmed the existing data on the adjacent regions of the insert. In addition, we applied the same methodology to report on the DNA sequences surrounding the insert of GM rice event LLRICE62.

SPALINSKAS Rapolas;  VAN DEN BULCKE Marc;  VAN DEN EEDE Guy;  MILCAMPS Anne; 

2013-04-15

SPRINGER

JRC68722

1936-9751,   

https://publications.jrc.ec.europa.eu/repository/handle/JRC68722,   

10.1007/s12161-012-9438-y,