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本文以转基因玉米NK603为例,介绍了一种检测目标基因纯和体的PCR方法。该方法利用4个引物的多重PCR反应,这4个PCR引物分别来自于目标基因的5’端(5’TP)和3’端(3’TP)DNA序列,目标基因5’端一侧的玉米基因组DNA序列(5’GP),以及目标基因3’端一侧的玉米基因组DNA序列(3’GP)。视玉米个体有无目标基因以及目标基因的杂合/纯和状态,PCR扩增可得到三种不同的结果:如果被检测个体无目标基因,5’GP与3’GP间的玉米基因组DNA将被扩增,PCR只产生一条条带;如果被检测个体是目标基因的纯合体,5’GP与5’TP及3’TP与3’GP间的DNA将被扩增,PCR则产生两条条带;如果被检测个体是目标基因的杂合体,5’GP与3’GP、5’GP与5’TP以及3’TP与3’GP间的DNA将都被扩增,PCR则产生三条条带。三条不同长短的PCR产物条带在琼脂糖凝胶上清晰可分,易于辨别。和费时昂贵的定量PCR相比,该方法简单、快速、结果准确,在目标基因位点玉米基因组DNA序列已知的前提下,该方法可扩展到诸如Bt11、Event176、GA21、MON810、MON863 和 TC1507 等任何转基因玉米的回交转育程序中。
Taking transgenic corn NK603 as an example, this paper introduced a PCR method to detect the pure and the body of the target gene. The method utilizes multiplex PCR of four primers from the 5 ’end (5’TP) and 3’ end (3’TP) DNA sequences of the target gene and the 5 ’end The maize genomic DNA sequence (5’GP), and the maize genomic DNA sequence (3’GP) on the 3’-end side of the target gene. Depending on the presence or absence of a target gene in a corn individual and the heterozygosity / purity and status of the target gene, three different results can be obtained by PCR amplification: If the test individual has no target gene, the maize genomic DNA between 5’GP and 3’GP Is amplified, PCR produces only one band; if the tested individual is a homozygote of the target gene, DNA between 5’GP and 5’TP and between 3’TP and 3’GP will be amplified and PCR will produce two If the test individual is a heterozygote of the target gene, both the 5 ’GP and 3’ GP, the 5 ’GP and 5’ TP, and the DNA between 3 ’TP and 3’ GP will be amplified and PCR will generate three Bands. Three different lengths of PCR product bands are clearly distinguishable on an agarose gel. This method is simple, rapid and accurate in comparison with the time-consuming and expensive quantitative PCR. The method can be extended to other DNA sequences such as Bt11, Event176, GA21, MON810, MON863 and TC1507 Etc. Any genetically modified maize backcrossing program.