ABSTRACT

To evaluate crops generated by new breeding techniques, it is important to confirm the removal of recombinant DNAs (rDNAs) derived from foreign genes including unintentionally introduced short rDNA(s). We attempted to develop a sensitive detection method for such short rDNAs using Southern blot analysis and performed a model study targeting single-copy endogenous genes in plants. To increase the detection sensitivity, the general protocol for Southern blot analysis was modified. In the model study, we used endogenous-gene-targeting probes in which complementary sequences were serially replaced by dummy sequences, and detected complementary sequences as well as 30 bp. We further evaluated the sensitivity using short rDNAs derived from GM sequences as pseudoinsertions, and the results demonstrated that rDNA-insertions as small as 30 bp could be detected. The results suggested that unintentionally introduced rDNA-insertions were 30 bp or more in length could be detected by the Southern blot analysis.

摘要

为了评估通过新育种技术产生的农作物，重要的是确认去除了外源基因（包括无意引入的短rDNA）衍生的重组DNA（rDNA）。我们尝试使用Southern印迹分析开发针对此类短rDNA的灵敏检测方法，并进行了针对植物中单拷贝内源基因的模型研究。为了增加检测灵敏度，修改了用于Southern印迹分析的通用方案。在模型研究中，我们使用靶向内源基因的探针，其中互补序列被虚拟序列连续替换，并检测到互补序列以及30 bp。我们使用源自GM序列的短rDNA作为伪插入物进一步评估了敏感性，结果表明，可以检测到小至30 bp的rDNA插入。结果表明，通过Southern印迹分析可以检测到无意引入的rDNA插入长度为30 bp或更长。