Kirchhoff-Institute for Physics, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany; E-Mails:
Int J Mol Sci. 2010 Oct 21;11(10):4094-105. doi: 10.3390/ijms11104094.
With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15-30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)-synthetic DNA analogues with a neutral backbone-which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488(®) showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10-20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.
随着基因组数据库的完备，开发出一种新型的荧光原位杂交（FISH）技术——COMBO-FISH（组合寡核苷酸 FISH）成为可能。与其他 FISH 技术不同，COMBO-FISH 采用生物信息学方法进行探针集设计。通过计算机基因组数据库搜索，选择几个典型长度为 15-30 个核苷酸的寡核苷酸片段，使得所有片段都在给定的基因组目标处唯一共定位。这里应用的探针是肽核酸（PNA）——一种具有中性骨架的合成 DNA 类似物，它们是在高纯度条件下合成的。对于在着丝粒 9 中重复高亮显示的探针，测试了用不同染料标记的 PNA，其中 Alexa 488（®）显示出可恢复的光漂白（在暗态和亮态之间闪烁），这是 SPDM（光谱精度距离/位置确定显微镜）的应用前提，SPDM 是一种新的高分辨率荧光定位显微镜技术。尽管在 SPDM 条件下标记的细胞核的 COMBO-FISH 有时会显示荧光背景，但通过信号强度和检测到的寡核苷酸片段的定位精度（在 10-20nm 范围内），可以清楚地区分特定的基因座。结果表明，具有闪烁染料的 COMBO-FISH 探针非常适合 SPDM，这将为基因组的分子纳米结构分析开辟新的前景。
