Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.

Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps.     

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.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

Detection of DNA sequences in Plasmodium berghei by means of in situ hybridization

Summary A non-radioactive in situ hybridization technique, used to map unique DNA sequences to plant chromosomes, has been adapted for the localization of specific DNA sequences in nuclei of Plasmodium berghei. After hybridization using probes labeled with biotin-11-dUTP, the formed DNA/DNA hybrids were detected by fluorescence microscopy using a specific double-layer antibody technique. Besides its high resolution, this procedure is characterized by a high sensitivity, allowing the detection of a unique sequence as small as 2.5 kb.     

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.     

Karyotyping of Brassica napus L. Based on C0t-1 DNA Banding by Fluorescence In Situ Hybridization

In order to precisely recognize and karyotype Brassica napus L. chromosomes, C0t-1 DNA was extracted from its genomic DNA, labeled with biotin-1 1-dUTP and in situ hybridized. The hybridized locations were detected by Cy3-conjugated streptavidin. Specific fluorescence in situ hybridization （FISH） signal bands were detected on all individual chromosome pairs. Each chromosome pair showed specific banding patterns. The B. napus karyotype has been constructed, for the first time, on the basis of both Cot-1 DNA FISH banding patterns and chromosome morphology.     

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.     

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.     

Fluorescence in situ hybridization (FISH) on maize metaphase chromosomes with quantum dot-labeled DNA conjugates

Semiconductor nanocrystals, also called quantum dots (QDs), are novel inorganic fluorophores which are brighter and more photostable than organic fluorophores. In the present study, highly dispersive QD-labeled oligonucleotide (TAG)₈ (QD-deoxyribonucleic acid [DNA]) conjugates were constructed via the metal-thiol bond, which can be used as fluorescence in situ hybridization (FISH) probes. FISH analysis of maize metaphase chromosomes using the QD-DNA probes showed that the probes could penetrate maize chromosomes and nuclei and solely hybridized to complementary target DNAs. Compared with the conventional organic dyes such as Cy3 and fluorescein isothiocyanate, this class of luminescent labels bound with oligonucleotides is brighter and more stable against photobleaching on the chromosomes after FISH. These results suggest that QD fluorophores may be a more stable and useful fluorescent label for FISH applications in plant chromosome mapping considering their size-tunable luminescence spectra. Lu Ma and Sheng-Mei Wu contributed equally to this work.     

Multicolor chromosome bar codes

Chromosome bar codes are multicolor banding patterns produced by fluorescence in situ hybridization (FISH) with differentially labeled and pooled sub-regional DNA probes. These molecular cytogenetic tools facilitate chromosome identification and the delineation of both inter- and intra-chromosomal rearrangements. We present an overview of the various conceptual approaches which can be largely divided into two classes: Simple bar codes designed for chromosome identification and complex bar codes for high resolution aberration screening of entire karyotypes. We address the issue of color redundancy and how to overcome this limitation by complementation of bar codes with whole chromosome painting probes.     

Detection of sex chromosome aneuploidy in dog spermatozoa by triple color fluorescence in situ hybridization

With the development of a direct visualization of sex chromosome in a single sperm by fluorescence in situ hybridization (FISH) technique, the frequency of aberration (aneuploidy) in spermatozoa in several mammals has been investigated. However, there is no report in the incidence of X-Y aneuploidy in the sperm population of dogs. Therefore, in this study, the aneuploidy in dog spermatozoa was examined by multicolor FISH using specific molecular probes for canine sex chromosomes and autosome. Semen from eight male Labrador retrievers was used as specimen. For decondensation of sperm nuclei, the specimen was treated with 1 M NaOH for 4 minutes at room temperature. Probes for chromosomes X, Y, and 1, labeled with SpectrumGreen, Cy3 and Cy5, respectively, were hybridized with decondensed spermatozoa. Fluorescence in situ hybridization signals in sperm heads were clearly detected in each specimen, regardless of the sperm donor. The FISH signal of at least one of the three probes was detected in all sperm heads examined. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X and Y chromosomes in spermatozoa of all the eight dogs. Mean percentage of sex chromosome aneuploidy was 0.127% (ranged between 0% and 0.316%). This percentage of canine sex chromosome aneuploidy was lower than the one reported in cattle, horses, river buffalo, and goats sperm, but higher than that observed in mice and sheep.     

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.     

Painting the rye genome with genome-specific sequences

We used rye-specific repetitive DNA sequences in fluorescence in situ hybridization (FISH) to paint the rye genome and to identify rye DNA in a wheat background. A 592 bp fragment from the rye-specific dispersed repetitive family R173 (named UCM600) was cloned and used as a FISH probe. UCM600 is dispersed over the seven rye chromosomes, being absent from the pericentromeric and subtelomeric regions. A similar pattern of distribution was also observed on the rye B chromosomes, but with weaker signals. The FISH hybridization patterns using UCM600 as probe were comparable with those obtained with the genomic in situ hybridization (GISH) procedure. There were, however, sharper signals and less background with FISH. UCM600 was combined with the rye-specific sequences Bilby and pSc200 to obtain a more complete painting. With these probes, the rye chromosomes were labeled with distinctive patterns; thus, allowing the rye cultivar 'Imperial' to be karyotyped. It was also possible to distinguish rye chromosomes in triticale and alien rye chromatin in wheat-rye addition and translocation lines. The distribution of UCM600 was similar in cultivated rye and in the wild Secale species Secale vavilovii Grossh., Secale sylvestre Host, and Secale africanum Stapf. Thus, UCM600 can be used to detect Secale DNA introgressed from wild species in a wheat background.     

Reconstruction of the female Gorilla gorilla karyotype using 25-color FISH and multicolor banding (MCB)

The origin of the human and great ape chromosomes has been studied by comparative chromosome banding analysis and, more recently, by fluorescence in situ hybridization (FISH), using human whole-chromosome painting probes. It is not always possible, however, to determine the exact breakpoints and distribution or orientation of specific DNA regions using these techniques. To overcome this problem, the recently developed multicolor banding (MCB) probe set for all human chromosomes was applied in the present study to reanalyze the chromosomes of Gorilla gorilla (GGO). While the results agree with those of most previous banding and FISH studies, the breakpoints for the pericentric inversion on GGO 3 were defined more precisely. Moreover, no paracentric inversion was found on GGO 14, and no pericentric inversions could be demonstrated on GGO 16 or 17.     

The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes

Oligonucleotide probes labeled with fluorescent dyes are used in a variety of in situ applications to detect specific DNA or RNA molecules. It has been described that probe fluorescence might be quenched upon hybridization in a sequence specific way. Here, a set of 17 oligonuleotides labeled with 6-carboxyfluorescein was used to examine the relevance of nucleotide specific quenching for fluorescence in situ hybridization (FISH) to whole fixed bacterial cells. Probes quenched upon hybridization to a guanine-rich region of purified RNA in solution were not quenched upon FISH. Among other factors the high protein concentration within cells may prevent quenching of probe fluorescence in situ.     

Atomic force microscopy and scanning near-field optical microscopy studies on the characterization of human metaphase chromosomes

A better knowledge of biochemical and structural properties of human chromosomes is important for cytogenetic investigations and diagnostics. Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of chromosomal details. Localizing specific gene probes by FISH combined with conventional fluorescence microscopy has reached its limit. Also, microdissecting DNA from G-banded human metaphase chromosomes by either a glass tip or by laser capture needs further improvement. By both atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM), local information from G-bands and chromosomal probes can be obtained. The final resolution allows a more precise localization compared to standard techniques, and the extraction of very small amounts of chromosomal DNA by the scanning probe is possible. Besides new strategies towards a better G-band and fluorescent probe detection, this study is focused on the combination of biochemical and nanomanipulation techniques which enable both nanodissection and nanoextraction of chromosomal DNA.     

Mapping nonisotopically labeled DNA probes to human chromosome bands by confocal microscopy.

A method for mapping nonisotopically labeled probes to human metaphase chromosomes that can be used with laser scanning confocal microscopy has been developed. Only a limited number of wavelengths are available from the argon ion lasers used in most commercial instruments and therefore a method that allowed the visualization of bands on human chromosomes stained with propidium iodide and, simultaneously, the detection of hybridization signals using FITC-labeled antibodies was developed. The confocal microscope was used to map single-copy probes to chromosome bands and the positions of the probes on the R-banded chromosomes corresponded to map positions previously determined on Hoechst 33258-stained chromosomes (G-banded). A comparison of confocal imaging of single-copy hybridization signals with conventional fluorescence microscopy and high-sensitivity video cameras revealed little difference in sensitivity but greater resolution of chromosome bands with the confocal microscope. The polymerase chain reaction was used to prepare nonisotopically labeled probes for in situ hybridization and to amplify Alu and KpnI family repeats from cloned DNA to be used to suppress hybridization of these repeat sequences so that a cosmid probe could be mapped to a chromosome band.     

A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes

Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO-FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO-FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO-FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO-FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution.     

Multiplex FISH analysis of a six-species bacterial biofilm

Established procedures use different and seemingly incompatible experimental protocols for fluorescent in situ hybridization (FISH) with Gram-negative and Gram-positive bacteria. The aim of this study was to develop a procedure, based on FISH and confocal laser scanning microscopy (CLSM), for the analysis of the spatial organization of in vitro biofilms containing both Gram-negative and Gram-positive oral bacteria. Biofilms composed of the six oral species Actinomyces naeslundii, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, and Veillonella dispar were grown anaerobically for 64.5 h at 37 degrees C on hydroxyapatite disks preconditioned with saliva. Conditions for the simultaneous in situ hybridization of both Gram-negative and Gram-positive bacteria were sought by systematic variation of fixation and exposure to lysozyme. After fixation and permeabilization biofilms were labeled by FISH with 16S rRNA-targeted oligonucleotide probes ANA103 (for the detection of A. naeslundii), EUK116 (C. albicans), FUS664 (F. nucleatum), MIT447 and MIT588 (S. oralis), SOB174 (S. sobrinus), and VEI217 (V. dispar). Probes were used as 6-FAM, Cy3 or Cy5 conjugates, resulting in green, orange-red or deep-red fluorescence of target cells, respectively. Thus, with two independent triple-hybridizations with three probes carrying different fluorescence-tags, all six species could be visualized. Results show that the simultaneous investigation by FISH of complex biofilms composed of multiple bacterial species with differential Gram-staining properties is possible. In combination with the optical sectioning properties of CLSM the technique holds great promise for the analysis of spatial alterations in biofilm composition in response to environmental challenges.     

Classifying by colors: FISH-based genome analysis

In recent years a fascinating evolution of different multicolor fluorescence in situ hybridization (FISH) technologies could be witnessed. The various approaches to cohybridize multiple DNA probes in different colors opened new avenues for FISH-based automated karyotyping or the simultaneous analysis of multiple defined regions within the genome. These developments had a remarkable impact on microscopy design and the usage of highly sensitive area imagers. In addition, they led to the introduction of new fluorochromes with appropriate filter combinations, refinements of hybridization protocols, novel probe sets, and innovative software for automated chromosome analysis. This paper attempts to summarize the various multicolor approaches and discusses the application of the individual technologies.