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FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.

Giorgi D, Farina A, Grosso V, Gennaro A, Ceoloni C, Lucretti S - PLoS ONE (2013)

Bottom Line: All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity.The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement.It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

View Article: PubMed Central - PubMed

Affiliation: ENEA-Italian National Agency for New Technologies, Energy and Sustainable Economic Development, CASACCIA Research Center, Rome, Italy.

ABSTRACT
The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

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Biparametric dot plot analysis of pasta wheat cv Creso chromosomes.The fluorescence intensity emissions from chromosomes stained with DAPI (DNA content) and labeled by FISHIS with GAA-FITC are joint together into a bi-parametric dot plot where each dot represents a single particle (blue: DNA stained by DAPI; green: (GAA)7-FITC labeling). Similar particles with a similar fluorescence emission are clustered and can then be enclosed into a sorting region for flow sorting and single-type chromosome isolation (colored regions). Panels showing the chromosome content from each relevant dot plot sorting region display various purity levels. The sorting purity is presented as a percentage of the main sorted chromosome in respect to the total number of the sorted population. Chromosome region distribution is directly proportional to the whole intensity of the fluorescence hybridization pattern. Different colored regions R1–R5 were used to assess the MESF (Molecules of Equivalent Soluble Fluorescein) values (Figure S5). The (GAA)7 oligonucleotides hybridize less with the A-genome chromosomes than the B-genome ones. As expected, the A-genome chromosomes are found within regions R1–R3 and the B-genome chromosomes are enclosed in regions R4 and R5. Bar  = 10 µm.
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pone-0057994-g002: Biparametric dot plot analysis of pasta wheat cv Creso chromosomes.The fluorescence intensity emissions from chromosomes stained with DAPI (DNA content) and labeled by FISHIS with GAA-FITC are joint together into a bi-parametric dot plot where each dot represents a single particle (blue: DNA stained by DAPI; green: (GAA)7-FITC labeling). Similar particles with a similar fluorescence emission are clustered and can then be enclosed into a sorting region for flow sorting and single-type chromosome isolation (colored regions). Panels showing the chromosome content from each relevant dot plot sorting region display various purity levels. The sorting purity is presented as a percentage of the main sorted chromosome in respect to the total number of the sorted population. Chromosome region distribution is directly proportional to the whole intensity of the fluorescence hybridization pattern. Different colored regions R1–R5 were used to assess the MESF (Molecules of Equivalent Soluble Fluorescein) values (Figure S5). The (GAA)7 oligonucleotides hybridize less with the A-genome chromosomes than the B-genome ones. As expected, the A-genome chromosomes are found within regions R1–R3 and the B-genome chromosomes are enclosed in regions R4 and R5. Bar  = 10 µm.

Mentions: All chromosome analysis and sorting were performed on a dual laser FACS Vantage SE flow cytometer (BD Bioscience, San Jose, CA). Flow cytometric analysis were carried out after calibration with the PeakFlow Standard Particles (ST: cod. P14825 for UV alignment and cod. P14827 for 488 nm excitation, LifeTechnology, Carlsbad, CA). For DNA denaturation and the FCM analysis by Acridine Orange (AO) metachromasia, the argon laser (Innova Coherent 90/5UV) was tuned at 488 nm with a power output of 200 mW. Green fluorescence emanating from the AO stained dsDNA was collected through a band-pass filter (BP) at 530/20 nm and a dichroic mirror at 560 nm. A long pass filter at 640 nm captured the red AO-stained ssDNA fluorescence. For the FISHIS analysis, the first argon ion laser was tuned to multiline UV (wavelength  = 353–361 nm) at 200 mW output, to excite the DAPI stained chromosomes and to generate the trigger system signal. The DAPI fluorescence emission was collected through a BP filter at 420/30 nm. The second argon ion laser (Innova Coherent 305c) was tuned at 488 nm (FITC labeling; FL3 filter BP  = 530/20 nm) or at 514 nm (Cy3 labeling; FL4  =  LP580 nm) with 400 mW power output. The FACS Vantage SE was equipped with a 70 µm flow tip, running at 27psi with a sheath fluid of 50 mM NaCl. The sample throughput was set to 400particles/sec as injected by the step motor-driven 1 ml syringe. Sorting was at a 29.7 KHz drop drive frequency, with a sorting rate of 5–20/sec in dual-sorting mode. Sorted chromosomes were collected either on glass slides for immediate identification, or in DNA Eppendorf LoBind tubes (Eppendorf, Germany) in ddH2O for further processing. Flow cytometric data were collected and analyzed using the software package CellQuest Pro v4.01 (BD Bioscience, San Jose, CA). As already described [9], the primary analysis gate was set on a dual parameter dot plot comprising Forward Scatter (FSC) versus FL1H (H =  signal height, DAPI fluorescence) to discriminate chromosomes from debris and chromosome aggregates. Sorting windows (Figure 2) were drawn on the fluorescence dot plot of FL1A (A =  signal area; DAPI fluorescence) versus FL3H (FITC fluorescence) or FL4H (Cy3 fluorescence). Sorting purity was evaluated on glass slides under a fluorescence microscope by counting one hundred FISHIS chromosomes three times per sort run, and cataloging chromosome types according to their hybridization patterns [14].


FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy.

Giorgi D, Farina A, Grosso V, Gennaro A, Ceoloni C, Lucretti S - PLoS ONE (2013)

Biparametric dot plot analysis of pasta wheat cv Creso chromosomes.The fluorescence intensity emissions from chromosomes stained with DAPI (DNA content) and labeled by FISHIS with GAA-FITC are joint together into a bi-parametric dot plot where each dot represents a single particle (blue: DNA stained by DAPI; green: (GAA)7-FITC labeling). Similar particles with a similar fluorescence emission are clustered and can then be enclosed into a sorting region for flow sorting and single-type chromosome isolation (colored regions). Panels showing the chromosome content from each relevant dot plot sorting region display various purity levels. The sorting purity is presented as a percentage of the main sorted chromosome in respect to the total number of the sorted population. Chromosome region distribution is directly proportional to the whole intensity of the fluorescence hybridization pattern. Different colored regions R1–R5 were used to assess the MESF (Molecules of Equivalent Soluble Fluorescein) values (Figure S5). The (GAA)7 oligonucleotides hybridize less with the A-genome chromosomes than the B-genome ones. As expected, the A-genome chromosomes are found within regions R1–R3 and the B-genome chromosomes are enclosed in regions R4 and R5. Bar  = 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585268&req=5

pone-0057994-g002: Biparametric dot plot analysis of pasta wheat cv Creso chromosomes.The fluorescence intensity emissions from chromosomes stained with DAPI (DNA content) and labeled by FISHIS with GAA-FITC are joint together into a bi-parametric dot plot where each dot represents a single particle (blue: DNA stained by DAPI; green: (GAA)7-FITC labeling). Similar particles with a similar fluorescence emission are clustered and can then be enclosed into a sorting region for flow sorting and single-type chromosome isolation (colored regions). Panels showing the chromosome content from each relevant dot plot sorting region display various purity levels. The sorting purity is presented as a percentage of the main sorted chromosome in respect to the total number of the sorted population. Chromosome region distribution is directly proportional to the whole intensity of the fluorescence hybridization pattern. Different colored regions R1–R5 were used to assess the MESF (Molecules of Equivalent Soluble Fluorescein) values (Figure S5). The (GAA)7 oligonucleotides hybridize less with the A-genome chromosomes than the B-genome ones. As expected, the A-genome chromosomes are found within regions R1–R3 and the B-genome chromosomes are enclosed in regions R4 and R5. Bar  = 10 µm.
Mentions: All chromosome analysis and sorting were performed on a dual laser FACS Vantage SE flow cytometer (BD Bioscience, San Jose, CA). Flow cytometric analysis were carried out after calibration with the PeakFlow Standard Particles (ST: cod. P14825 for UV alignment and cod. P14827 for 488 nm excitation, LifeTechnology, Carlsbad, CA). For DNA denaturation and the FCM analysis by Acridine Orange (AO) metachromasia, the argon laser (Innova Coherent 90/5UV) was tuned at 488 nm with a power output of 200 mW. Green fluorescence emanating from the AO stained dsDNA was collected through a band-pass filter (BP) at 530/20 nm and a dichroic mirror at 560 nm. A long pass filter at 640 nm captured the red AO-stained ssDNA fluorescence. For the FISHIS analysis, the first argon ion laser was tuned to multiline UV (wavelength  = 353–361 nm) at 200 mW output, to excite the DAPI stained chromosomes and to generate the trigger system signal. The DAPI fluorescence emission was collected through a BP filter at 420/30 nm. The second argon ion laser (Innova Coherent 305c) was tuned at 488 nm (FITC labeling; FL3 filter BP  = 530/20 nm) or at 514 nm (Cy3 labeling; FL4  =  LP580 nm) with 400 mW power output. The FACS Vantage SE was equipped with a 70 µm flow tip, running at 27psi with a sheath fluid of 50 mM NaCl. The sample throughput was set to 400particles/sec as injected by the step motor-driven 1 ml syringe. Sorting was at a 29.7 KHz drop drive frequency, with a sorting rate of 5–20/sec in dual-sorting mode. Sorted chromosomes were collected either on glass slides for immediate identification, or in DNA Eppendorf LoBind tubes (Eppendorf, Germany) in ddH2O for further processing. Flow cytometric data were collected and analyzed using the software package CellQuest Pro v4.01 (BD Bioscience, San Jose, CA). As already described [9], the primary analysis gate was set on a dual parameter dot plot comprising Forward Scatter (FSC) versus FL1H (H =  signal height, DAPI fluorescence) to discriminate chromosomes from debris and chromosome aggregates. Sorting windows (Figure 2) were drawn on the fluorescence dot plot of FL1A (A =  signal area; DAPI fluorescence) versus FL3H (FITC fluorescence) or FL4H (Cy3 fluorescence). Sorting purity was evaluated on glass slides under a fluorescence microscope by counting one hundred FISHIS chromosomes three times per sort run, and cataloging chromosome types according to their hybridization patterns [14].

Bottom Line: All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity.The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement.It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

View Article: PubMed Central - PubMed

Affiliation: ENEA-Italian National Agency for New Technologies, Energy and Sustainable Economic Development, CASACCIA Research Center, Rome, Italy.

ABSTRACT
The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

Show MeSH
Related in: MedlinePlus