Scholler P, Heber S, Hoheisel J D
Molecular-Genetic Genome Analysis Group, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Electrophoresis. 1998 Apr;19(4):504-8. doi: 10.1002/elps.1150190409.
Large-scale hybridization-based genome mapping projects, such as the production of sequence-ready physical clone maps, call for robust and cheap DNA labeling techniques that are amenable to automation. We routinely use a high-throughput protocol based on fluorescence detection. DNA probes are labeled via polymerase chain reaction (PCR) amplification with primers that are digoxigenin-modified at their 5' ends. Alternatively, digoxigenin-labeled dUTP is incorporated in a random hexamer priming reaction. Hybridization takes place in small volumes by sandwiching the probe between filters and plastic sheets. A fluorescence signal is produced by the activity of alkaline phosphatase attached to an anti-digoxigenin antibody upon the addition of AttoPhos substrate. Signals are directly detected with a charge-coupled device (CCD) camera and scored by an image data analysis system. DNA filters can be reused at least 40 times without loss of data quality. Significant advantages compared to radioactive techniques are the reduced health risk, enabling highly parallel processing; the production of spot signals uniform in size and intensity, which is essential for efficient image analysis; and a cost reduction of about 70%.
大规模基于杂交的基因组图谱绘制项目，例如制备可用于测序的物理克隆图谱，需要可靠且廉价的DNA标记技术，并且这些技术要适合自动化操作。我们通常使用基于荧光检测的高通量方案。DNA探针通过聚合酶链反应（PCR）扩增进行标记，所用引物在其5'端用洋地黄毒苷修饰。或者，在随机六聚体引物反应中掺入洋地黄毒苷标记的dUTP。杂交在小体积中进行，通过将探针夹在滤膜和塑料片之间实现。加入AttoPhos底物后，与抗洋地黄毒苷抗体相连的碱性磷酸酶的活性产生荧光信号。信号由电荷耦合器件（CCD）相机直接检测，并由图像数据分析系统进行评分。DNA滤膜可重复使用至少40次而不损失数据质量。与放射性技术相比，其显著优势在于降低了健康风险，可实现高度并行处理；产生大小和强度均匀的斑点信号，这对于高效图像分析至关重要；成本降低约70%。
