Comparison Of Different Molecular Methods In Screening Genetically Modified Lentil

Abstract

Currently transgenic plants are grown in more than 20 countries with maize, soybean, canola and cotton being the most predominant crops. Inexperience in the outcomes of the technology and growing public concern necessitates proper detection and regulation of genetically modified organisms (GMOs) from farmland to market. Due to their high specifity and sensitivity, polymerase chain reaction (PCR) based systems are currently the method of choice in detection of genetic modifications. This study compares the efficiency of three different PCR based methods; reverse-transcription PCR (RT-PCR), real-time PCR (qPCR) and conventional PCR in reference with the transgene copy numbers assessed by Southern blot hybridization, in detection of genetic modification. In the study, first generation transgenic lentil (Lens culinaris M.) plants carrying beta-glucuronidase (gus) gene in control of CaMV-35S promoter and A.tumefaciens nos terminator was used. Conventional PCR was used in detection of gus gene signal and RT PCR was performed in detection of gene's expression. qPCR was used to detect expression signals of both 35S promoter and nos terminator. All of the methods were successful in producing amplification signals for each target gene. Although qPCR signal strengths were in consistency with the band intensities obtained by RT-PCR to some extent, outcomes of both PCR-based methods appeared to be independent from copy number of genes detected in Southern blot hybridization. Band intensities obtained by conventional PCR showed no particular correlation with any other PCR-based method. Inconsistency in copy number of gene and qPCR signal strength, even in pure DNA samples may have a contribution for the debates on the influence of various factors on qPCR and reliability of the method in genetic modification quantification.