Fluorescence in situ hybridization (FISH) is a powerful assay that can visualize the position of DNA and RNA molecules in individual cells. Here, I describe the development of a method that utilizes complex oligonucleotide (oligo) libraries as a renewable source of FISH probes, which we term ‘Oligopaints’. Our novel FISH platform includes a reliable and robust protocol for the bulk production of fluorescently labeled, strand-specific, single-stranded DNA (ssDNA) probe sets and a bioinformatic pipeline able to identify optimal target sequences for in situ hybridization on a genome-wide scale. A key advantage of Oligopaints is that it permits the researcher to precisely define the genomic sequence contained within each probe molecule, specify the placement of fluorophores, and engineer ssDNA overhangs to which activities can be targeted. We harness this control to make two significant technological advances in FISH- based imaging. In one, Oligopaint probes are programmed to carry 5’ ssDNA overhangs that enable stochastic super-resolution microscopy via two methodologies, STORM and DNA- PAINT. We have used these probes to produce <25 nm resolution images of developmentally regulated chromatin in Drosophila and mouse, which are to our knowledge the first images at this resolution of single-copy chromosomal regions produced by FISH. In the second, we utilize single nucleotide polymorphism (SNP) data to generate FISH probes that can for the first time visually distinguish single-copy regions of the maternal and paternal homologous chromosomes, thus allowing the examination of parent-of-origin dependent effects on chromosome positioning and gene expression in individual cells.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/14226040 |
| Date | 01 March 2016 |
| Creators | Beliveau, Brian J. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation, text |
| Format | application/pdf |
| Rights | open |
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