Single-step multicolor fluorescence In situ hybridization using semiconductor quantum dot-DNA conjugates

We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences using novel quantum dot-based fluorescence in situ hybridization (FISH) probes (QD-FISH). Short DNA oligonucleotides were attached on QDs and used in a single hybridization/detection step of target sites in situ. QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting of dense chromatin domains with minimal steric hindrances. We further demonstrated that QD's broad absorption spectra allowed different colored probes specific for distinct subnuclear genetic sequences to be simultaneously excited with a single excitation wavelength and imaged free of chromatic aberrations in a single exposure. Thus, these results demonstrate that QD-FISH probes are very effective in multicolor FISH applications. This work also documents new possibilities of using QD-FISH probes detection down to the single molecule level.     

Direct labelling of BAC-DNA by rolling-circle amplification

Efficient amplification and labelling of probes are crucial for successful sequence detection by fluorescent in situ hybridization (FISH). In particular, chromosome painting to visualize chromosome segments or entire chromosomes by FISH requires large amounts of probes derived from extended templates. There are a number of techniques for probe labelling. The most widespread is nick translation, based on the replicational incorporation of modified nucleotides. Here we demonstrate successful rolling-circle amplification (RCA) of very low amounts of long circular template sequences (single bacterial artificial chromosomes (BACs) or pools of BACs). The amplicons were suitable for labelling by nick translation and subsequent FISH. A novel achievement is the use of RCA for simultaneous amplification and labelling of single BACs or BAC pools in a labour- and cost-effective manner.     

Spectral karyotyping analysis of human and mouse chromosomes

Classical banding methods provide basic information about the identities and structures of chromosomes on the basis of their unique banding patterns. Spectral karyotyping (SKY), and the related multiplex fluorescence in situ hybridization (M-FISH), are chromosome-specific multicolor FISH techniques that augment cytogenetic evaluations of malignant disease by providing additional information and improved characterization of aberrant chromosomes that contain DNA sequences not identifiable using conventional banding methods. SKY is based on cohybridization of combinatorially labeled chromosome-painting probes with unique fluorochrome signatures onto human or mouse metaphase chromosome preparations. Image acquisition and analysis use a specialized imaging system, combining Sagnac interferometer and CCD camera images to reconstruct spectral information at each pixel. Here we present a protocol for SKY analysis using commercially available SkyPaint probes, including procedures for metaphase chromosome preparation, slide pretreatment and probe hybridization and detection. SKY analysis requires approximately 6 d.     

FISH landmarks for barley chromosomes (Hordeum vulgare L.).

Barley metaphase chromosomes (2n = 14) can be identified by fluorescence in situ hybridization (FISH) and digital imaging microscopy using heterologous 18S rDNA and 5S rDNA probe sequences. When these sequences are used together, FISH landmark signals were seen so that all 7 chromosomes were uniquely identified and unambiguously oriented. The chromosomal location of the landmark signals was determined by FISH to a barley trisomic series using the 18S and 5S probes labeled with different fluorophores. The utility of these FISH landmarks for barley physical mapping was also demonstrated when an Amy-2 cDNA clone and a BAC clone were hybridized with the FISH landmark probes.     

Characterization and application of soybean YACs to molecular cytogenetics

Yeast artificial chromosomes (YACs) are widely used in the physical analysis of complex genomes. In addition to their value in chromosome walking for map-based cloning, YACs represent excellent probes for chromosome mapping using fluorescence in situ hybridization (FISH). We have screened such a library for low-copy-number clones by hybridization to total genomic DNA. Four clones were chosen for chromosome tagging based upon their low or moderate signal. By using degenerate oligonucleotide-primed PCR (DOP-PCR), we were able to use relatively small amounts of soybean YAC DNA, isolated directly by preparative pulsed-field gel electrophoresis, as FISH probes for both metaphase chromosome spreads and interphase nuclei. FISH chromosomal analysis using the three of the clones as probes resulted in relatively simple hybridization patterns consistent with a single homologous locus or two homoeologous loci. The fourth YAC probe resulted in a diffuse hybridization pattern with signal on all metaphase chromosomes. We conclude that YACs represent a valuable source of probes for chromosomal analysis in soybean.     

Double telomeric signals on single chromatids revealed by FISH and PRINS

FISH probes for all human telomeres and specific telomeric probes that hybridize to unique sequences on individual chromosomes have been used to characterize the telomeric hybridization pattern of human peripheral blood lymphocytes and bone-marrow cells in interphase and metaphase chromosomes. We have identified the existence of double hybridization signals on chromatids both with the (TTAGGG)n telomere repeat arrays and on non chromosome-specific subtelomeric regions as well as on chromosome-specific sequences located several kilobases from the end of chromosomes. Preliminary results using cosmid or YAC probes that hybridize to regions rich in GC sequences also revealed double fluorescent spots on a single chromatid. Double spots were detected by PRINS on terminal and interstitial telomeric sequences in avian cells. The significance of this phenomenon is discussed based on some models of chromatid and DNA organization such as uninemy, looped chromatid organization and quartet DNA structures. The occurrence of double spots should be taken into consideration for the clinical cytogenetic diagnosis of duplications.     

Characterization of double minute chromosomes’ DNA content in a human high grade astrocytoma cell line by using comparative genomic hybridization and fluorescence in situ hybridization

The presence of double minute chromosomes (dmin) in cancer cells is known to be correlated with gene amplifications. In human high grade astrocytomas or glioblastomas, about 50% of cytogenetically characterized cases display dmin. G5 is a cell line which has been established from a human glioblastoma containing multiple dmin. In order to identify the DNA content of these dmin, three techniques were successively used: conventional cytogenetic analysis, comparative genomic hybridization (CGH), and fluorescent in situ hybridization (FISH). The karyotype of G5 cells showed numerical chromosome changes (hypertriploidy), several marker chromosomes, and multiple dmin. CGH experiments detected two strong DNA amplification areas located in 9p21-22 and 9p24, as well as an underrepresentation of chromosomes 6, 10, 11, 13, 14, and 18q. By using FISH with a chromosome 9-specific painting probe to metaphase chromosomes of the G5 cell line, dmin were shown to contain DNA sequences originating from chromosome 9. This study demonstrates the usefulness of a combination of classical karyotyping, CGH, and FISH to identify the chromosomal origin of amplified DNA sequences in dmin. Received: 30 October 1994 / Revised: 25 February 1996     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

Genome sequence comparison between Chinese hamster ovary (CHO) DG44 cells and mouse using end sequences of CHO BAC clones based on BAC-FISH results

Chinese hamster ovary (CHO) cells have frequently been used in biotechnology as a mammalian host cell platform for expressing genes of interest. Previously, we constructed a detailed physical chromosomal map of the CHO DG44 cell line by fluorescence in situ hybridization (FISH) imaging using 303 bacterial artificial chromosome (BAC) clones as hybridization probes (BAC-FISH). BAC-FISH results revealed that the two longest chromosomes were completely paired. However, other chromosomes featured partial deletions or rearrangements. In this study, we determined the end sequences of 303 BAC clones (BAC end sequences), which were used for BAC-FISH probes. Among 606 BAC-end sequences (BESs) (forward and reverse ends), 558 could be determined. We performed a comparison between all determined BESs and mouse genome sequences using NCBI BLAST. Among these 558 BESs, 465 showed high homology to mouse chromosomal sequences. We analyzed the locations of these BACs in chromosomes of the CHO DG44 cell line using a physical chromosomal map. From the obtained results, we investigated the regional similarities among CHO chromosomes (A–T) and mouse chromosomes (1–19 and sex) about 217 BESs (46.7% of 465 high homologous BESs). Twenty-three specific narrow regions in 13 chromosomes of the CHO DG44 cell line showed high homology to mouse chromosomes, but most of other regions did not show significant correlations with the mouse genome. These results contribute to accurate alignments of chromosomes of Chinese hamster and its genome sequence, analysis of chromosomal instability in CHO cells, and the development of target locations for gene and/or genome editing techniques.     

Chromosomal distribution of endogenous Jaagsiekte sheep retrovirus proviral sequences in the sheep genome

A family of endogenous retroviruses (enJSRV) closely related to Jaagsiekte sheep retrovirus (JSRV) is ubiquitous in domestic and wild sheep and goats. Southern blot hybridization studies indicate that there is little active replication or movement of the enJSRV proviruses in these species. Two approaches were used to investigate the distribution of proviral loci in the sheep genome. Fluorescence in situ hybridization (FISH) to metaphase chromosome spreads using viral DNA probes was used to detect loci on chromosomes. Hybridization signals were reproducibly detected on seven sheep chromosomes and eight goat chromosomes in seven cell lines. In addition, a panel of 30 sheep-hamster hybrid cell lines, each of which carries one or more sheep chromosomes and which collectively contain the whole sheep genome, was examined for enJSRV sequences. DNA from each of the lines was used as a template for PCR with JSRV gag-specific primers. A PCR product was amplified from 27 of the hybrid lines, indicating that JSRV gag sequences are found on at least 15 of the 28 sheep chromosomes, including those identified by FISH. Thus, enJSRV proviruses are essentially randomly distributed among the chromosomes of sheep and goats. FISH and/or Southern blot hybridization on DNA from several of the sheep-hamster hybrid cell lines suggests that loci containing multiple copies of enJSRV are present on chromosomes 6 and 9. The origin and functional significance of these arrays is not known.     

Multicolor fluorescence in situ hybridization with combinatorial labeling probes enables a detailed karyotype analysis of Larix principis-rupprechtii

The chromosomes (2n = 2x = 24) of Larix principis-rupprechtii are composed of six pairs of large metacentrics and six pairs of medium-sized submetacentrics. The identification of homologous pairs is hampered by their high degree of similarity at the morphological level in each group. As one of the most extensively used methods in molecular cytogenetics producing chromosome landmarks, fluorescence in situ hybridization (FISH) has significantly facilitated karyotype construction, especially in species with morphologically similar chromosomes. This study developed a simple but effective use of combinatorial labeling probes to distinguish chromosomes of Larix principis-rupprechtii by multicolor FISH. Three highly repetitive sequences in Larix were selected: 25S rDNA hybridized at all of the secondary constrictions of two pairs of metacentrics and the largest pair of submetacentrics; 5S rDNA hybridized at subtelomeric sites of one pair of metacentrics that also harboured 25S rDNA on different arms; LPD family sequences are tandem repeats hybridized at proximal regions of 22 chromosomes. The three different probes were labeled with only two different labels, hybridized to metaphase chromosomes of Larix principis-rupprechtii, simultaneously visualized, and unequivocally distinguished in a single FISH experiment. These multicolor FISH marks largely improved the karyotype analysis of Larix principis-rupprechtii.     

Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries

Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few megabasepairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of overlapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wavelength intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific microdissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region-specific paints, but do not readily allow positioning of breakpoints on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.     

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.     

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei¹. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis². Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene^3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application⁷. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)⁸. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)⁹. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers^8,10.     

An improved method for generating BAC DNA suitable for FISH

Fluorescence in situ hybridization (FISH) is commonly used to identify chromosomal aberrations such as translocations, deletions, duplications, gene fusions, and aneuploidies. It relies on the hybridization of fluorescently labeled DNA probes onto denatured metaphase chromosomes or interphase nuclei. These probes are often generated from DNA sequences cloned within bacterial artificial chromosomes (BACs). Growing these BACs in adequate amounts for FISH can be demanding. We describe FISH performed with bacteriophage Phi29 DNA polymerase amplified BAC DNA. Generating this material required significantly smaller cultures and less time than standard methods. The FISH results obtained were comparable with those obtained from standard BAC DNA. We believe this method of BAC DNA generation is useful for the entire FISH community as it improves considerably on prior methods.     

Localization of BAC clones on mitotic chromosomes of <Emphasis Type="Italic">Musa acuminata</Emphasis> using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization

A bacterial artificial chromosome (BAC) library of banana (Musa acuminata) was used to select BAC clones that carry low amounts of repetitive DNA sequences and could be suitable as probes for fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes. Out of eighty randomly selected BAC clones, only one clone gave a single-locus signal on chromosomes of M. acuminata cv. Calcutta 4. The clone localized on a chromosome pair that carries a cluster of 5S rRNA genes. The remaining BAC clones gave dispersed FISH signals throughout the genome and/or failed to produce any signal. In order to avoid the excessive hybridization of repetitive DNA sequences, we subcloned nineteen BAC clones and selected their ‘low-copy’ subclones. Out of them, one subclone gave specific signal in secondary constriction on one chromosome pair; three subclones were localized into centromeric and peri-centromeric regions of all chromosomes. Other subclones were either localized throughout the banana genome or their use did not result in visible FISH signals. The nucleotide sequence analysis revealed that subclones, which localized on different regions of all chromosomes, contained short fragments of various repetitive DNA sequences. The chromosome-specific BAC clone identified in this work increases the number of useful cytogenetic markers for Musa.     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

Multicolor FISH mapping of the dioecious model plant,<Emphasis Type="Italic"> Silene latifolia</Emphasis>

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.Communicated by J.S. Heslop-Harrison     

Analysis of repetitive DNA in chromosomes by flow cytometry

We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.     

The use of repetitive DNA in cytogenetic studies of plant sex chromosomes

The structure of sex chromosomes in plants was analyzed by fluorescent in situ hybridization (FISH) with repetitive DNAs. FISH probes were successfully obtained from DNA libraries that were amplified from microdissected sex chromosomes. Some probes hybridized to the subtelomeric regions, where many kinds of repetitive DNAs are located with intrachromosomal similarity of their repeat units rather than interchromosomal similarity. For example, FISH with the subtelomeric repetitive sequence can easily show the location of the pseudoautosomal region (PAR) on the X chromosome of Silene latifolia. The other probes were localized on the interstitial region of the sex chromosomes. The interstitial region contains chloroplast DNAs or neighboring sequences of the internal telomeres, suggesting insertion or translocation occurred during differentiation of the sex chromosome. These data are very informative for understanding the structure of the plant sex chromosomes and their evolutionary process.