DNA extraction from bulked samples of canola seed and the use of multiplex PCR for detection of adventitious contamination with genetically modified seed

Abstract

A method for detecting adventitious contamination of genetically modified (GM) seed in a bulk sample of 100 traditional canola seeds is presented. This detection level fits sampling plans aimed at minimizing risk lo buyer and seller through the use of large sample sizes, in the area of 3000 seeds. It also provides opportunities for flexible approaches to testing schemes for different sampling plan needs. The extraction procedure can be completed for at least two 3000 seed samples in to 7.5 hour period by a single technician. The amplification by the polymerase chain reaction (PCR) and detection of amplicons by gel electrophoresis require a further 7 hours. This paper describes the general method for DNA extraction using fully defined components. The benefits and limitations of using a commercial kit are briefly discussed. The multiplex PCR developed detects the following transgenic canola varieties: glyphosate tolerant; bromoxynil tolerant; and all three events conferring glufosinate ammonium tolerance. The final PCR used to detect the presence of GM seed yields 6 bands ranging in size from 219-800 base pairs if all five events, plus the housekeeping gene, are detected in the sample.