Abstract
Chromosome visualization is essential for chromosome analysis and genetic diagnostics. Here, we developed a click chemistry approach for multicolor imaging of chromosomal DNA instead of the traditional dye method. We first demonstrated that the commercially available reagents allow for the multicolor staining of chromosomes. We then prepared two pro-fluorophore moieties that served as light-up reporters to stain chromosomal DNA based on click reaction and visualized the clear chromosomes in multicolor. We applied this strategy in fluorescence in situ hybridization (FISH) and identified, with high sensitivity and specificity, telomere DNA at the end of the chromosome. We further extended this approach to observe several basic stages of cell division. We found that the click reaction enables direct visualization of the chromosome behavior in cell division. These results suggest that the technique can be broadly used for imaging chromosomes and may serve as a new approach for chromosome analysis and genetic diagnostics.
Highlights
The study of human chromosomes provides valuable insight into processes of clinical diagnosis for many genetic disorders and analysis of chromosome architecture[1,2,3]
Observing the chromosome in the desired color is possible by changing the fluorescent dye molecules that react with the DNA and achieving an overlay mode, suggesting that the click reaction strategy provides a useful tool for imaging chromosomes
The click reaction connects the chromosomal DNA and commercial dye molecules and allows for the multicolor staining of chromosomes
Summary
The study of human chromosomes provides valuable insight into processes of clinical diagnosis for many genetic disorders and analysis of chromosome architecture[1,2,3]. The existing approaches of chromosome imaging tend to work effectively, but depend upon the binding of the fluorescent dyes with the chromosomal DNA This condition raises a series of questions regarding the affinity of dyes to chromosomal DNA, the number of differently colored fluorescent dyes, and the photostability of the dyes[4,5,6,7]. The traditional dye molecules used in the study of chromosomal behavior do not form covalent bonds with chromosomal DNA; it is difficult to monitor chromosome dynamics To overcome these limitations and further enable the potential of chromosome imaging to be fully exploited in both research and diagnostic laboratories, we have developed a chemistry-based strategy for imaging chromosomal DNA in multicolor in place of the traditional dye pairing. After its metabolic incorporation into DNA, EdU can be detected with fluorescent azides by click reaction This method was used to label DNA in cell level by microscopic analysis[39]. We successfully found an unusual nucleic-acid structure formed by DNA and RNA50
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