Application of DNA- and Protein-Based Detection Methods in Agricultural Biotechnology.

DNA- and protein-based detection methods are widely used tools for monitoring biotechnology-derived crops and their products globally. Agricultural biotechnology companies, food/feed suppliers and supply chains, diagnostic testing companies, and regulatory authorities heavily rely on these two technologies for product development, seed production, compliance, and contractual needs. The primary use of DNA- and protein-based detection methods is either to verify the presence or absence of genetically engineered (GE) materials or to quantify the amount of GE material present in a product. This review describes key parameters of DNA- and protein-based detection methods, and thorough assessment of their applications and their advantage and limitations in agricultural biotechnology are discussed in detail. The review highlights the principle and considerations of detection method selection, which will equip users to choose suitable technology and obtain reliable test results. The review also compares the compatibility of the two technologies in GE product testing using a case study.

Southern-by-Sequencing: A Robust Screening Approach for Molecular Characterization of Genetically Modified Crops.

Molecular characterization of events is an integral part of the advancement process during genetically modified (GM) crop product development. Assessment of these events is traditionally accomplished by polymerase chain reaction (PCR) and Southern blot analyses. Southern blot analysis can be time-consuming and comparatively expensive and does not provide sequence-level detail. We have developed a sequence-based application, Southern-by-Sequencing (SbS), utilizing sequence capture coupled with next-generation sequencing (NGS) technology to replace Southern blot analysis for event selection in a high-throughput molecular characterization environment. SbS is accomplished by hybridizing indexed and pooled whole-genome DNA libraries from GM plants to biotinylated probes designed to target the sequence of transformation plasmids used to generate events within the pool. This sequence capture process enriches the sequence data obtained for targeted regions of interest (transformation plasmid DNA). Taking advantage of the DNA adjacent to the targeted bases (referred to as next-to-target sequence) that accompanies the targeted transformation plasmid sequence, the data analysis detects plasmid-to-genome and plasmid-to-plasmid junctions introduced during insertion into the plant genome. Analysis of these junction sequences provides sequence-level information as to the following: the number of insertion loci including detection of unlinked, independently segregating, small DNA fragments; copy number; rearrangements, truncations, or deletions of the intended insertion DNA; and the presence of transformation plasmid backbone sequences. This molecular evidence from SbS analysis is used to characterize and select GM plants meeting optimal molecular characterization criteria. SbS technology has proven to be a robust event screening tool for use in a high-throughput molecular characterization environment.