Two meters in ten micrometers? Let’s have a closer look

Visualizing specific DNA loci in their natural context – the cell’s nucleus – is fundamental to understanding how two meters of DNA can be packed in a space six orders of magnitude smaller. With this goal in mind, we have developed a simple but powerful quantitative method to rapidly and cost effectively engineer probes for visualizing virtually any locus in the human and mouse genome as well as entire chromosomes at high definition in single cells, using fluorescence in situ hybridization.

HFSP Long-Term Fellows Magda Bienko and Shalev Itzkovitz and colleagues
authored on Thu, 24 January 2013

DNA fluorescence in situ hybridization (DNA FISH) is a powerful technique to visualize DNA loci and even entire chromosomes at single-cell level. Despite great technological advancements, however, it has been difficult to rapidly engineer FISH probes that would allow for high-resolution quantitative visualization of dozens of DNA loci simultaneously in individual cells. This is fundamental to gain insights into the laws that govern genome organization – be it positioning of individual chromosomes in the nucleus, inter- or intra-chromosomal interactions or local chromatin folding. How these aspects of DNA architecture change during development, differentiation or pathological processes such as cancer is also of great interest. Finally, learning how DNA packaging inside the cell’s nucleus is achieved will bring us closer to understanding the relationship between genome organization and gene expression.


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Figure: HD FISH workflow is very simple: after identifying a region of interest in the genome database,corresponding primer pairs are found in the HD FISH primer database.Primers are synthesized in 96-well plate format, and used in real-time PCR to create a pool of specific amplicons (each 200-bp long). Amplicons are then labeled with so-called Universal Linkage System (ULSÔ), which adds fluorescent dyes to guanine residues at a frequency of 1-3 dyes every 100 bases. A probe can consist of small number of amplicons (~20-50) – if one is interested in achieving high resolution – or large number of amplicons (~100-500) – when one wants to visualize a locus with high precision. The 96-well plate format allows for flexible usage of primers in any combination, while the post-PCR labeling strategy allows for a combinatorial use of dyes. After hybridizing a probe or a pool of probes to fixed cells, one can visualize either a single locus or many loci at a time.

To meet these challenges, we have developed a genome-scale PCR-based method for high-definition DNA FISH (HD FISH). We started by creating a long-awaited database of PCR primer pairs for both human and mouse genomes, which produce amplicons complimentary to unique sequences in the genome. After being fluorescently labeled with a dye of choice, these PCR products serve as HD FISH probes. The fact that probes prepared in such a way are highly specific allowed us to drastically minimize the size of the FISH signal, bringing the optical resolution of the technique to its limit. In addition, the ease, high speed of preparation, and cost-effectiveness of this approach enabled us to create a rich library of probes for various applications. For instance, by designing about twenty equally spaced probes along human chromosome 1 and 17, we were able to visualize these chromosomes as clusters of discrete spots, where individual probes served as reference points to detect much larger objects in 3D. In fact, sampling of chromosome territories using our approach proved not only to be more cost-effective, much less laborious and more time-efficient, but also quantitatively superior to the conventional approach that uses so-called paint probes. Importantly, the format in which HD FISH probes are created allows for combinatorial labeling, swapping of dyes, and flexible adjustment of probe size, making HD FISH extremely versatile. Finally, we were able to combine HD FISH with single-molecule RNA FISH, and simultaneously visualized a gene locus and its messenger RNA product in individual cells.

Our method opens up a new broad landscape of possibilities to quantitatively study genome organization as well as the relationship between chromosome architecture and gene expression, providing at the same time a user-friendly and cost-effective solution for cytogenetic diagnostics.


A versatile genome-scale PCR-based pipeline for high-definition DNA FISH. Bienko M, Crosetto N, Teytelman L, Klemm S, Itzkovitz S, van Oudenaarden A. Nat Methods.2012 Dec. doi: 10.1038/nmeth.2306.

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