Himmelspach M, Gruber F, Antoine G, Falkner F G, Dorner F, Hämmerle T
Biomedical Research Center, Immuno AG, Orth/Donau, Austria.
Anal Biochem. 1996 Nov 15;242(2):240-7. doi: 10.1006/abio.1996.0459.
An assay was developed to specifically quantitate concentrations as low as 50 fg/ml genomic DNA based on the amplification of repetitive sequences. Reliable results were obtained by using internal standard molecules which were coextracted, coamplified, and coanalyzed with the nucleic acids of interest. Amplification was performed by the polymerase chain reaction in the presence of fluorescent dye-labeled primers followed by quantitation of fluorescence derived from the reaction products after separation by PAGE. Based on the known amount of added internal standard molecules and the intensities of the fluorescence of the reaction products, the primary results of the assay were obtained as copies per milliliter of sample. These were converted into mass units of DNA by applying an experimentally determined conversion factor. Chicken DNA has been used as an example for genomic DNA, and the sequences amplified were CR1 repetitive elements. This type of assay may be applied in cases where a sensitive and precise quantitation of genomic DNA is required, such as in the quality control of biological products.
基于重复序列的扩增，开发了一种检测方法，用于特异性定量低至50 fg/ml的基因组DNA浓度。通过使用与感兴趣的核酸共提取、共扩增和共分析的内标分子，获得了可靠的结果。在荧光染料标记引物存在的情况下，通过聚合酶链反应进行扩增，然后通过PAGE分离后对反应产物产生的荧光进行定量。根据添加的内标分子的已知量和反应产物荧光的强度，获得检测的初步结果为每毫升样品的拷贝数。通过应用实验确定的转换因子，将这些结果转换为DNA的质量单位。鸡DNA已被用作基因组DNA的示例，扩增的序列是CR1重复元件。这种检测方法可应用于需要对基因组DNA进行灵敏和精确定量的情况，例如生物制品的质量控制。
