Detection and identification of multiple genetically modified events using DNA insert fingerprinting

Abstract

Current screening and event-specific polymerase chain reaction (PCR) assays for the detection and identification of genetically modified organisms (GMOs) in samples of unknown composition or for the detection of non-regulated GMOs have limitations, and alternative approaches are required. A transgenic DNA fingerprinting methodology using restriction enzyme digestion, adaptor ligation, and nested PCR was developed where individual GMOs are distinguished by the characteristic fingerprint pattern of the fragments generated. The inter-laboratory reproducibility of the amplified fragment sizes using different capillary electrophoresis platforms was compared, and reproducible patterns were obtained with an average difference in fragment size of 2.4 bp. DNA insert fingerprints for 12 different maize events, including two maize hybrids and one soy event, were generated that reflected the composition of the transgenic DNA constructs. Once produced, the fingerprint profiles were added to a database which can be readily exchanged and shared between laboratories. This approach should facilitate the process of GMO identification and characterization.

The Development and Standardization of Testing Methods for Genetically Modified Organisms and their Derived ProductsF

“…However, the evidence for the presence of unauthorized GMOs can only be indirectly inferred from the matrix approach because of the non‐specificity of the screening method. To overcome this weakness, various approaches have been developed as alternative methods to identify GM events, including the use of differential quantitative PCR (Cankar et al 2008), DNA insert fingerprinting (Raymond et al 2010) or anchor‐PCR GM fingerprinting method (Ruttink et al 2010), microarray‐based method (Tengs et al 2007; Prins et al 2008) etc.…”

Four new SYBR®Green qPCR screening methods for the detection of Roundup Ready®, LibertyLink®, and CryIAb traits in genetically modified products

“…Several multiplex qPCR formats are available reducing the number of analyses and facilitating high throughput but requiring multi-channel detection devices and often including costly detection probes for at least some of the targets [8][9][10]. In other cases, the applied chemistries are quite complex or are to be combined with other technologies that are less appropriate for routine applications [11][12][13][14][15][16]. Several of these approaches involve the use of PCR with multiple targets and consecutive detection and identification of the amplification products using microarrays [11][12][13].…”

“…Apart from requiring additional costly array analysis equipment, these approaches are often prone to variable quality of the array chips making them less suitable for routine or enforcement purposes [17]. Raymond et al [14] and Nadal et al [15] combined PCR with capillary electrophoresis in a very effective way, but requiring costly investment in additional equipment. Finally, an effective novel approach based on so-called padlock-probes [18] was developed, which could in the future represent an elegant solution to cover the broad diversity of GMO [16].…”

Event-Specific Qualitative and Quantitative Detection in Transgenic Soybean OsDREB3 Based on the 5′ Flanking Sequence

“…In past years, the event-specific detection method was validated for GM wheat, GM corn, GM soybean, GM rice, and so on. Raymond et al (2010) developed transgenic DNA fingerprinting methodology, and the individual GMOs were distinguished by the characteristic fingerprint pattern of the fragments generated. This approach should facilitate the process of GMO identification and characterization.…”