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Development of a Standard, Validated Procedure for the Isolation of Transgene Flanking Regions in GM Crops and Detailed Analysis of Transgene Insertion

Institutions
John Innes Centre, Norwich, UK
Start date
2006
End date
2009
Objective
This project will involve the refinement and validation of a rapid polymerase chain reaction (PCR) based method for characterisation of junction sequences in a range of genetically modified (GM) crops.

A new methodology will be compared to the existing technology to see if it offers any advantages in throughput, reproducibility and cost.

The project will provide standard validated methods for the analysis of junction sequences in a range of GM crops, containing transgene insertions of different complexity.

The methods developed will be validated by the analysis of a standard set of GM samples at two different sites. The project will also provide valuable information on the patterns of transgene insertion in a range of GM crops.

More information
The isolation and analysis of transgene flanking regions forms a key component of the molecular analysis and safety assessment of GM plants.

Until recently the characterisation of the insertion point for crop plants has been difficult and time consuming.

However, work carried out under the G02 programme and other recently published work has resulted in discovery of a rapid PCR based method, which can be applied to the identification of junction sequences.

The characterisation of the insertion point of the introduced transgenic trait will provide information as to whether any functional genes are disrupted and whether any new open reading frames (ORFs) are created resulting in the synthesis of novel proteins.

If so, further work can be carried out to determine whether these ORFs may code for potential allergens or toxins.

The project will allow refinement and validation of this method, advancing the state-of-the art technology in this field. The standard validated methods developed could form part of the risk assessment procedure for a large range of GM crops and different types of transgene insertion.

Results and findings:
During the project it emerged that two commercially available kits were appropriate for the isolation of transgene junction regions. The DNA Walking SpeedUpTM Kit II (manufactured by Seegene) was found to be suitable for use in all GM crop types tested where the number of transgene insertions was low. This kit produced reliable and rapid results from small amounts of DNA. The APAgeneTM GOLD Walking Kit (manufactured by BIO S&T) was found to be appropriate for the analysis of potato lines with both simple and more complex transgene insertions and gave reliable results.

As a result of the work carried out in this project, it is now possible to recommend standard operating procedures (SOPs) using the Seegene kit for the molecular analysis and safety assessment of a wide range of GM plant material, including potato, barley, maize, pea, wheat and Brassica. For more complex material (especially in potato), the SOP using the APAgeneTM GOLD kits, together with additional analysis steps, is appropriate.

The results obtained during the project identified some DNA rearrangements at the transgene insertion site in older experimental GM lines not intended to be grown commercially. Rearrangements were less frequent in GM lines produced more recently. Additional analysis is required to identify the reason for such rearrangements and in some cases to determine the origin of the additional sequences and potential consequences of such rearrangements.

The current guidelines for the molecular characterisation of GM crops specify that the junctions of the transgene and the plant genome should be identified and the DNA sequence determined to ensure that there are no rearrangements or other unintended effects at the site of insertion. The methods developed in this project will speed up this process and allow rapid screening and selection of candidate lines for commercialisation that lack such rearrangements or other unintended effects.

Find more about this project and other FSA food safety-related projects at the Food Standards Agency Research webpage.

Funding Source
Food Standards Agency
Project number
G03026
Categories
Risk Assessment, Management, and Communication
Sanitation and Quality Standards
Commodities
Grains, Beans, Legumes
Produce