Establishing an accurate and rapid method for copy number and zygosity determination can accelerate genetic engineering research process. In this study, droplet digital PCR (ddPCR), an emerging DNA absolute quantifica...Establishing an accurate and rapid method for copy number and zygosity determination can accelerate genetic engineering research process. In this study, droplet digital PCR (ddPCR), an emerging DNA absolute quantification technology, was used to identify single-copy homozygous transgenic lines from a batch of T0 transgenic rapeseed harboring 11 exogenous elements. Copy number of exogenous gene was evaluated in T0 generation based on calculated ratio between transgene and reference CruA gene, single-copy transformants were selected for selfing followed by subsequent zygosity analysis. Single-copy homozygous transgenic plants were successfully screened out in T1 generation by ddPCR.Segregation analysis with T2 seedlings verified that identification results of ddPCR were accurate and reliable. This study provides a novel rapid and accurate method for copy number and zygosity determination in transgenic rapeseed which overcomes disadvantages of traditional Southern analysis and recently developed real-time quantitative method.展开更多
文摘Establishing an accurate and rapid method for copy number and zygosity determination can accelerate genetic engineering research process. In this study, droplet digital PCR (ddPCR), an emerging DNA absolute quantification technology, was used to identify single-copy homozygous transgenic lines from a batch of T0 transgenic rapeseed harboring 11 exogenous elements. Copy number of exogenous gene was evaluated in T0 generation based on calculated ratio between transgene and reference CruA gene, single-copy transformants were selected for selfing followed by subsequent zygosity analysis. Single-copy homozygous transgenic plants were successfully screened out in T1 generation by ddPCR.Segregation analysis with T2 seedlings verified that identification results of ddPCR were accurate and reliable. This study provides a novel rapid and accurate method for copy number and zygosity determination in transgenic rapeseed which overcomes disadvantages of traditional Southern analysis and recently developed real-time quantitative method.
