COMBO-FISH: specific labeling of nondenatured chromatin targets by computer-selected DNA oligonucleotide probe combinations
COMBO-FISH: specific labeling of nondenatured chromatin targets by computer-selected DNA oligonucleotide probe combinations
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DOI：
10.2144/03353rr03
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发表时间：
2003-09-01
期刊：
影响因子：
2.7
通讯作者：
Cremer, C
中科院分区：
文献类型：
--
作者：
Hausmann, M;Winkler, R;Cremer, C
在这里，我们提出了荧光原位杂交（FISH）与组合寡核苷酸（COMBO）探针的原理，作为基因组位点特异性标记的新方法。COMBO-FISH利用同嘌呤/同嘧啶寡核苷酸与完整的双工基因组DNA形成三螺旋，而不需要在标准FISH方案中通常用于探针结合的目标序列的事先变性。对人类基因组数据库的分析表明，长度超过14倍的同嘌呤/同嘧啶序列几乎均匀地分布在整个基因组中，它们占整个基因组的1%至2%。由于配备高数值孔径透镜的共聚焦激光扫描显微镜的观察体积通常对应于正常哺乳动物细胞核中约250-kb的染色质结构域，因此该体积应包含150-200个同嘌呤/同嘧啶延伸。利用DNA数据库信息，人们可以从这些延伸中配置一组不同的、均匀标记的寡核苷酸探针，这些探针预计将在250 kb的染色质结构域中独家共定位。由于显微镜的衍射极限分辨率，配置的寡核苷酸探针组的荧光信号合并成一个典型的，几乎均匀的FISH点。使用一组32个同型嘧啶探针，我们在人类9号染色体的Abelson小鼠白血病区域进行了实验，作为COMBO-FISH的一些最初的原理证明。虽然实验方案目前包含了与活细胞条件不相容的几个步骤，但理论上的方法可能是第一个方法上的进步，朝着在高分辨率荧光显微镜下对重要细胞进行特异性FISH的长期但仍然难以实现的目标迈进。
Here we present the principle of fluorescence in situ hybridization (FISH) with combinatorial oligonucleotide (COMBO) probes as a new approach for the specific labeling of genomic sites. COMBO-FISH takes advantage of homopurine/homopyrimidine oligonucleotides that form triple helices with intact duplex genomic DNA, without the need for prior denaturation of the target sequence that is usually applied for probe binding in standard FISH protocols. An analysis of human genome databases has shown that homopurine/homopyrimidine sequences longer than 14 by are nearly homogeneously distributed over the genome, and they represent from 1% to 2% of the entire genome. Because the observation volume in a confocal laser-scanning microscope equipped with a high numerical aperture lens typically corresponds to an approximate 250-kb chromatin domain in a normal mammalian cell nucleus, this volume should contain 150-200 homopurine/homopyrimidine stretches. Using DNA database information, one can configure a set of distinct, uniformly labeled oligonucleotide probes from, these stretches that is expected to exclusively co-localize within a 250-kb chromatin domain. Due to the diffraction-limited resolution of a microscope, the fluorescence signals of the configured oligonucleotide probe set merge into a typical, nearly homogeneous FISH spot. Using a set of 32 homopyrimidine probes, we performed experiments in the Abelson murine leukemia region of human chromosome 9 as some of the very first proofs-of-principle of COMBO-FISH. Although the experimental protocol currently contains several steps that are incompatible with living cell conditions, the theoretical approach may be the first methodological advance toward the long-term but still elusive goal of carrying out specific FISH in high-resolution fluorescence microscopy of vital cells.