Quantitative Detection of Transgenic and Endogenous Dna Sequences in Seeds After Automated Dna Preparation

Abstract

In applied transgenic research as well as in agriculture there is an increasing need for high-throughput analyses of plants for genotypic selection or to identify the purity of seed stocks, e.g. for transgenic contaminations or the identification of pathogens. We developed and optimised conditions for the isolation of DNA from single seeds using an automated high-throughput protocol. Our results show that the system provided is capable of isolating DNA from any tested seed source. Furthermore, seeds remain capable of germinating during the homogenisation procedure. Quantification of endogenous and transgenic sequences by Real-Time PCR revealed that the prepared DNA is suitable in quality and quantity for multiple PCR analyses with both SYBR Green and hybridisation probe detection. The described method will be a useful tool for routine analyses like screening of large populations or the specific detection of genetically modified organisms (GMO).

Assessment of DNA extraction methods for PCR testing of discontinued or unapproved biotech events in single seeds of canola, flax and soybean

“…In general, soybean seeds used for food such as Tofu are larger than soybean seeds used for feed; thus the former could result in more DNA yield than the latter. In our case, the soybean DNA yield was much higher than that reported by Peterhansel et al (2004).…”

“…Thus, the DNA yield of intact soybean seed for relatively high-throughput extraction method was likely underestimated. According to Peterhansel et al (2004), a mean DNA yield of 500 AE 74 ng was obtained from single crushed soybean seeds. In general, soybean seeds used for food such as Tofu are larger than soybean seeds used for feed; thus the former could result in more DNA yield than the latter.…”

Evaluation and Validation of Reference Genes for Normalization of Quantitative Real-Time PCR Based Gene Expression Studies in Peanut

“…Similarly, ATPsyn (ATP synthase) gene that is involved in the synthesis of adenosine triphosphate (ATP) has so far not been reported as stably expressing internal gene, with only one report where it was used as an endogenous gene in soybean [43]. Since ATPsyn was not stable across the experimental conditions, it is not recommended as a reference gene for gene expression studies in peanut.…”

“…The candidate reference genes were selected through bibliographic reviews of studies in crop plants such as peanut ( LEC [42]; ADH3 [37]), soybean ( ATPsyn [43]; ACT11 , ELF1B , CYP2 and G6PD [29]), and cotton ( UBC1 ) [44]) followed by an in silico analysis using the BLAST tools of the NCBI database [45]. For instance, a previously selected EST in soybean was submitted to the BLASTN tool to obtain EST orthologous in peanut (Table 1).…”