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COMBO-FISH:通过计算机选择的DNA寡核苷酸探针组合对未变性染色质靶标进行特异性标记。

COMBO-FISH: specific labeling of nondenatured chromatin targets by computer-selected DNA oligonucleotide probe combinations.

作者信息

Hausmann Michael, Winkler Ralph, Hildenbrand Georg, Finsterle Jutta, Weisel Andrea, Rapp Alexander, Schmitt Eberhard, Janz Siegfried, Cremer Christoph

机构信息

Kirchhoff-Institute of Physics, University of Heidelberg, Heidelberg.

出版信息

Biotechniques. 2003 Sep;35(3):564-70, 572-7. doi: 10.2144/03353rr03.

DOI:10.2144/03353rr03
PMID:14513562
Abstract

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 bp 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 homogenous 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.

摘要

在此,我们介绍组合寡核苷酸(COMBO)探针荧光原位杂交(FISH)的原理,这是一种用于基因组位点特异性标记的新方法。COMBO-FISH利用同型嘌呤/同型嘧啶寡核苷酸与完整的双链基因组DNA形成三链螺旋,无需像标准FISH方案中通常用于探针结合那样对靶序列进行预先变性。对人类基因组数据库的分析表明,长度超过14 bp的同型嘌呤/同型嘧啶序列在基因组中几乎均匀分布,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中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,该体积应包含150 - 200个同型嘌呤/同型嘧啶片段。利用DNA数据库信息,可以从这些片段中配置一组独特的、均匀标记的寡核苷酸探针,预期它们仅在一个250 kb的染色质结构域内共定位。由于显微镜的衍射极限分辨率,配置好的寡核苷酸探针集的荧光信号合并成一个典型的、几乎均匀的FISH斑点。我们使用一组32个同型嘧啶探针,在人类9号染色体的阿贝尔逊鼠白血病区域进行了实验,作为COMBO-FISH的一些首批原理验证。尽管目前的实验方案包含几个与活细胞条件不兼容的步骤,但该理论方法可能是朝着在活细胞的高分辨率荧光显微镜中进行特异性FISH这一长期但仍难以实现的目标迈出的首个方法学进展。

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