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Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes.

Beliveau BJ, Boettiger AN, Avendaño MS, Jungmann R, McCole RB, Joyce EF, Kim-Kiselak C, Bantignies F, Fonseka CY, Erceg J, Hannan MA, Hoang HG, Colognori D, Lee JT, Shih WM, Yin P, Zhuang X, Wu CT - Nat Commun (2015)

Bottom Line: We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems.Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals.These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

No MeSH data available.


Related in: MedlinePlus

Secondary oligos are specific and efficient.(a) One synthesis strategy for Oligopaints, in which complex ssDNA libraries consisting of a stretch of genomic sequence (black lines) on the order of tens of bases flanked by non-genomic regions (coloured lines) containing primer sequences are amplified, labelled and then processed in any of a variety of ways to produce ssDNA probes that carry non-genomic sequences at one (shown) or both (not shown) ends (adapted from ref. 7; also see Supplementary Figs 2 and 3 for more details on MainStreet incorporation and placement strategies). The primer sequence can constitute the entirety, or just a portion, of the non-genomic region, called MainStreet, which will remain single-stranded when Oligopaint probes are hybridized to their target. (b) A binding site for a secondary (2°) oligo probe can be introduced to MainStreet by PCR amplification with a primer that carries the binding site. Here, the secondary oligo carries a single, 5′ fluorophore that matches the fluorophore present on the Oligopaint (primary) probe, but in practice the number, identity and placement of fluorophores on the secondary oligo can vary. Also see Supplementary Figs 2 and 3. (c) Grayscale and multicolour images from a two-colour co-localization experiment in diploid human WI-38 cells. DNA is stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Images are maximum Z projections from a laser scanning confocal microscope. (d) Two-colour co-localization experiments in diploid Drosophila clone 8 cells and WI-38 cells. The genomic target, span of the target, number of nuclei examined (n), per cent of nuclei (% Labelling) that had at least one signal from the primary (1°, Oligopaint) probe and at least one signal from the secondary oligo and per cent primary signals that have an overlapping secondary signal (% Co-localization) are given for each experiment.
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f1: Secondary oligos are specific and efficient.(a) One synthesis strategy for Oligopaints, in which complex ssDNA libraries consisting of a stretch of genomic sequence (black lines) on the order of tens of bases flanked by non-genomic regions (coloured lines) containing primer sequences are amplified, labelled and then processed in any of a variety of ways to produce ssDNA probes that carry non-genomic sequences at one (shown) or both (not shown) ends (adapted from ref. 7; also see Supplementary Figs 2 and 3 for more details on MainStreet incorporation and placement strategies). The primer sequence can constitute the entirety, or just a portion, of the non-genomic region, called MainStreet, which will remain single-stranded when Oligopaint probes are hybridized to their target. (b) A binding site for a secondary (2°) oligo probe can be introduced to MainStreet by PCR amplification with a primer that carries the binding site. Here, the secondary oligo carries a single, 5′ fluorophore that matches the fluorophore present on the Oligopaint (primary) probe, but in practice the number, identity and placement of fluorophores on the secondary oligo can vary. Also see Supplementary Figs 2 and 3. (c) Grayscale and multicolour images from a two-colour co-localization experiment in diploid human WI-38 cells. DNA is stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Images are maximum Z projections from a laser scanning confocal microscope. (d) Two-colour co-localization experiments in diploid Drosophila clone 8 cells and WI-38 cells. The genomic target, span of the target, number of nuclei examined (n), per cent of nuclei (% Labelling) that had at least one signal from the primary (1°, Oligopaint) probe and at least one signal from the secondary oligo and per cent primary signals that have an overlapping secondary signal (% Co-localization) are given for each experiment.

Mentions: Here we report two strategies for in situ single-cell imaging, one that facilitates two forms of single-molecule super-resolution microscopy and another that utilizes SNPs to visually distinguish homologous chromosomal regions. Both make use of Oligopaints, which are highly efficient, renewable, strand-specific fluorescence in situ hybridization (FISH) probes derived from complex single-stranded DNA (ssDNA) libraries in which each oligo carries a short stretch of homology to the genome (Fig. 1a). In contrast to classical FISH probes, which are produced from segments of purified genomic DNA amplified in bacterial vectors or PCR reactions, Oligopaints belong to a new generation of probes that are derived entirely from synthetic DNA oligonucleotides (oligos)567. Such probes have their sequences chosen bioinformatically; thus, they can be designed to target any organism whose genome has been sequenced, engineered to avoid repetitive elements, and selected to have specific hybridization properties. Our studies take advantage of two features of Oligopaints: the inclusion of non-genomic sequences, which enable super-resolution imaging, and a programmable insert of genomic homology, which makes it possible for Oligopaints to bind specifically at SNPs.


Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes.

Beliveau BJ, Boettiger AN, Avendaño MS, Jungmann R, McCole RB, Joyce EF, Kim-Kiselak C, Bantignies F, Fonseka CY, Erceg J, Hannan MA, Hoang HG, Colognori D, Lee JT, Shih WM, Yin P, Zhuang X, Wu CT - Nat Commun (2015)

Secondary oligos are specific and efficient.(a) One synthesis strategy for Oligopaints, in which complex ssDNA libraries consisting of a stretch of genomic sequence (black lines) on the order of tens of bases flanked by non-genomic regions (coloured lines) containing primer sequences are amplified, labelled and then processed in any of a variety of ways to produce ssDNA probes that carry non-genomic sequences at one (shown) or both (not shown) ends (adapted from ref. 7; also see Supplementary Figs 2 and 3 for more details on MainStreet incorporation and placement strategies). The primer sequence can constitute the entirety, or just a portion, of the non-genomic region, called MainStreet, which will remain single-stranded when Oligopaint probes are hybridized to their target. (b) A binding site for a secondary (2°) oligo probe can be introduced to MainStreet by PCR amplification with a primer that carries the binding site. Here, the secondary oligo carries a single, 5′ fluorophore that matches the fluorophore present on the Oligopaint (primary) probe, but in practice the number, identity and placement of fluorophores on the secondary oligo can vary. Also see Supplementary Figs 2 and 3. (c) Grayscale and multicolour images from a two-colour co-localization experiment in diploid human WI-38 cells. DNA is stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Images are maximum Z projections from a laser scanning confocal microscope. (d) Two-colour co-localization experiments in diploid Drosophila clone 8 cells and WI-38 cells. The genomic target, span of the target, number of nuclei examined (n), per cent of nuclei (% Labelling) that had at least one signal from the primary (1°, Oligopaint) probe and at least one signal from the secondary oligo and per cent primary signals that have an overlapping secondary signal (% Co-localization) are given for each experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430122&req=5

f1: Secondary oligos are specific and efficient.(a) One synthesis strategy for Oligopaints, in which complex ssDNA libraries consisting of a stretch of genomic sequence (black lines) on the order of tens of bases flanked by non-genomic regions (coloured lines) containing primer sequences are amplified, labelled and then processed in any of a variety of ways to produce ssDNA probes that carry non-genomic sequences at one (shown) or both (not shown) ends (adapted from ref. 7; also see Supplementary Figs 2 and 3 for more details on MainStreet incorporation and placement strategies). The primer sequence can constitute the entirety, or just a portion, of the non-genomic region, called MainStreet, which will remain single-stranded when Oligopaint probes are hybridized to their target. (b) A binding site for a secondary (2°) oligo probe can be introduced to MainStreet by PCR amplification with a primer that carries the binding site. Here, the secondary oligo carries a single, 5′ fluorophore that matches the fluorophore present on the Oligopaint (primary) probe, but in practice the number, identity and placement of fluorophores on the secondary oligo can vary. Also see Supplementary Figs 2 and 3. (c) Grayscale and multicolour images from a two-colour co-localization experiment in diploid human WI-38 cells. DNA is stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Images are maximum Z projections from a laser scanning confocal microscope. (d) Two-colour co-localization experiments in diploid Drosophila clone 8 cells and WI-38 cells. The genomic target, span of the target, number of nuclei examined (n), per cent of nuclei (% Labelling) that had at least one signal from the primary (1°, Oligopaint) probe and at least one signal from the secondary oligo and per cent primary signals that have an overlapping secondary signal (% Co-localization) are given for each experiment.
Mentions: Here we report two strategies for in situ single-cell imaging, one that facilitates two forms of single-molecule super-resolution microscopy and another that utilizes SNPs to visually distinguish homologous chromosomal regions. Both make use of Oligopaints, which are highly efficient, renewable, strand-specific fluorescence in situ hybridization (FISH) probes derived from complex single-stranded DNA (ssDNA) libraries in which each oligo carries a short stretch of homology to the genome (Fig. 1a). In contrast to classical FISH probes, which are produced from segments of purified genomic DNA amplified in bacterial vectors or PCR reactions, Oligopaints belong to a new generation of probes that are derived entirely from synthetic DNA oligonucleotides (oligos)567. Such probes have their sequences chosen bioinformatically; thus, they can be designed to target any organism whose genome has been sequenced, engineered to avoid repetitive elements, and selected to have specific hybridization properties. Our studies take advantage of two features of Oligopaints: the inclusion of non-genomic sequences, which enable super-resolution imaging, and a programmable insert of genomic homology, which makes it possible for Oligopaints to bind specifically at SNPs.

Bottom Line: We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems.Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals.These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.

No MeSH data available.


Related in: MedlinePlus