Researchers have expanded the capabilities of a diagnostic tool to capture the first high-resolution, 3D images of the shortest non-repetitive genomic sequence in a human or mouse nuclear genome.
Fluorescent in situ hybridisation (FISH) is a molecular diagnostic technique for spotting abnormalities in genes or chromosomes. First developed in the early 1980s, it includes the use of fluorescent probes that bind to specific parts of the chromosome to detect the presence or absence of DNA sequences.
Now, writing in the journal eLife mainly from two labs in Shenzhen and Tsinghua University, China, researchers have pushed the ability of FISH to image a specific segment of the genome in super resolution. They developed a simple FISH method that uses molecular beacon (MB) probes (MB-FISH) and stochastic optical reconstruction microscopy (STORM) to label and reveal 3D shapes of a non-repetitive endogenous genome sequence as short as 2.5 kilobases. This is similar to being able to pinpoint an individual in a crowded room of more than a million people.
“The conventional FISH method was constrained by various factors, including sequence and optical resolution,” says co-corresponding author Professor Michael Zhang, from Tsinghua University and the University of Texas Dallas.
In 2015, a new method, which combined oligopaint probe-based FISH (Oligopaint-FISH) with STORM, enabled super-resolution imaging of non-repetitive genomic regions as short as 4.9 kilobases. “Although this technique is very powerful for studying chromatin domain structures, we wanted to further increase the sequence resolution and develop a simpler method without the tedious procedure of generating probes. With this method, it is possible to target gene enhancers and promoters precisely and visualize their interactions, or to detect small DNA sequence alternations in cancer and other diseases,” says Zhang.
To develop the method, first and co-corresponding author Dr. Yanxiang Ni from Shenzhen University applied the MB concept in labelling short genomic targets. The MB design largely diminishes non-specific probe fluorescence by forming a hairpin structure and quenching an unbound or off-target probe’s fluorophore.
"The actual results are much better than what I expected,” says Dr. Ni. “The MB design not only significantly reduces the fluorescence of unbound and off-target probes but also protects the probes from non-specifically associating with other genomic regions.
“Our results highlight MB-FISH and 3D-STORM’s capability of visualizing the shortest non-repetitive genomic sequence in human and mouse genomes alike, and we are now using the tool to study enhancer-promoter interaction further,” she adds. “We are continually improving the method and believe its potential in studying 3D genome structure and function is yet to be unleashed.”
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eLife is a unique collaboration between the funders and practitioners of research to improve the way important research is selected, presented and shared. eLife publishes outstanding works across the life sciences and biomedicine — from basic biological research to applied, translational and clinical studies. All papers are selected by active scientists in the research community. Decisions and responses are agreed by the reviewers and consolidated by the Reviewing Editor into a single, clear set of instructions for authors, removing the need for laborious cycles of revision and allowing authors to publish their findings quickly. eLife is supported by the Howard Hughes Medical Institute, the Max Planck Society and the Wellcome Trust. Learn more at elifesciences.org.
