Zhu Z, Chao J, Yu H, Waggoner A S
Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, PA 15213.
Nucleic Acids Res. 1994 Aug 25;22(16):3418-22. doi: 10.1093/nar/22.16.3418.
Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR.
通过缺口平移法和聚合酶链反应（PCR），利用连接有荧光标记Cy3且带有不同长度连接臂的dUTP，制备了直接标记的荧光DNA探针。在通过PCR制备探针的过程中，我们发现连接臂长度会影响Cy3-dUTP的掺入效率、标记探针的产量以及与染色体靶序列杂交的标记探针的信号强度。对于缺口平移法和PCR而言，当连接臂长度增加时，掺入水平和杂交荧光信号会同时增加。在最佳条件下，PCR产生的标记探针标记密度更高，然而，PCR标记探针的产量会随着标记线性密度的增加而降低。在最佳条件下，使用连接臂最长的Cy3修饰dUTP，通过PCR有可能以合理的产量标记靶DNA上高达28%的可能取代位点，通过缺口平移法可标记18%。提出了一种机制，该机制涉及聚合酶、模板上的花菁标记位点与延伸链以及修饰的dUTP底物之间的空间相互作用，以解释标记效率与PCR合成DNA探针产量之间的负相关关系。
