Holden Marcia J, Blasic Joseph R, Bussjaeger Louis, Kao Chuan, Shokere Luke A, Kendall Donald C, Freese Larry, Jenkins G Ronald
National Institute of Standards and Technology, Biotechnology Division, 100 Bureau Drive, MS-8312, Gaithersburg, MD 20899, USA.
J Agric Food Chem. 2003 Apr 23;51(9):2468-74. doi: 10.1021/jf0211130.
Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material requires pure, high-quality genomic DNA as template for subsequent amplification using the polymerase chain reaction (PCR). Six methodologies were evaluated for extracting DNA from ground corn kernels spiked with 0.1% (m/m) CBH351 (StarLink) corn. DNA preparations were evaluated for purity and fragment size. Extraction efficiency was determined. The alcohol dehydrogenase gene (adh1) and the CBH351 (cry9C, 35S promoter) genes in the genomic DNA were detected using PCR. DNA isolated by two of the methods proved unsuitable for performing PCR amplification. All other methods produced some DNA preparations that gave false negative PCR results. We observed that cornstarch, a primary component of corn kernels, was not an inhibitor of PCR, while acidic polysaccharides were. Our data suggest that amplification of an endogenous positive control gene, as an indicator for the absence of PCR inhibitors, is not always valid. This study points out aspects of DNA isolation that need to be considered when choosing a method for a particular plant/tissue type.
对植物材料中痕量生物技术衍生DNA进行灵敏且准确的检测，需要纯净、高质量的基因组DNA作为模板，以便后续使用聚合酶链反应（PCR）进行扩增。评估了六种从添加了0.1%（m/m）CBH351（星联）玉米的磨碎玉米粒中提取DNA的方法。对DNA制品的纯度和片段大小进行了评估。测定了提取效率。使用PCR检测基因组DNA中的乙醇脱氢酶基因（adh1）和CBH351（cry9C，35S启动子）基因。通过其中两种方法分离的DNA被证明不适用于进行PCR扩增。所有其他方法都产生了一些导致PCR结果为假阴性的DNA制品。我们观察到，玉米粒的主要成分玉米淀粉不是PCR的抑制剂，而酸性多糖是。我们的数据表明，扩增内源性阳性对照基因作为不存在PCR抑制剂的指标并不总是有效的。这项研究指出了在为特定植物/组织类型选择方法时需要考虑的DNA分离方面。
