Primed in situ labelling facilitates flow sorting of similar sized chromosomes

Flow karyotyping and sorting of individual chromosome types is difficult when chromosomes of a complement do not differ sufficiently in DNA content. A strategy for sorting chromosomes of similar size has been developed. For this purpose oligonucleotide primed in situ (PRINS)-labelling was adapted to field bean chromosomes in suspension. With a primer designed according to a tandemly repetitive sequence ( Fokl element) PRINS-labelling resulted in fluorescence signals specific in position and intensity for each chromosome. A bivariate sorting mode combining fluorescence pulse areas obtained from propidium iodide staining (representing DNA content) and fluorescein isothiocyanate signals (representing chromosome-specific label) allowed chromosomes deviating in length by less than 1% of the haploid metaphase complement to be sorted. The average purity of sorted fractions was 95%. This technique should be applicable also to chromosomes of other species for obtaining chromosome-specific painting probes, for construction of chromosome-specific libraries (both without additional DNA amplification), and for gene mapping.     

Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labeled via linear amplification.

The ability to identify large numbers of yeast artificial chromosomes (YACs) specific to any given genomic region rapidly and efficiently enhances both the construction of clone maps and the isolation of region-specific landmarks (e.g., polymorphic markers). We describe a method of preparing region-specific single-stranded hybridization probes from Alu element-mediated polymerase chain reaction (Alu-PCR) products of somatic cell hybrids for YAC library screening. Pools of up to 50 cloned Alu-PCR products from an irradiation-reduced hybrid containing 22q11.2-q13.1 were labeled to high specific activity by linear amplification using a single vector primer. The resulting single-stranded probes were extensively competed to remove repetitive sequences, while retaining the full complexity of the probe. Extensive coverage of the region by YACs using multiple probe pools was demonstrated as many YACs were detected more than once. In situ analysis using chosen YACs confirmed that the clones were specific for the region. Thus, this pooled probe approach constitutes a rapid method to identify large numbers of YACs relevant to a large chromosomal region.     

Purifying human Y chromosomes by flow cytometry and sorting

A method of producing an enriched sample of human Y chromosomes from peripheral blood lymphocytes is described. Metaphase chromosomes were prepared from peripheral blood lymphocytes donated by 17 normal male individuals. A suspension of chromosomes in a polyamine buffer was produced from each sample, stained with the fluorescent dye Hoechst 33258, and passed through a flow cytometer and sorter. Following analysis of the 17 fluorescence distributions, a single donor was found giving a separate peak corresponding to the Y chromosome. Seventy percent of the chromosomes sorted from this peak were identified as Y chromosomes. Batches of a million Y chromosomes were produced from each of several 40 ml donations of peripheral blood. These were assessed for the amount of Y DNA present and used to construct a DNA library.     

Rapid cloning and characterization of new chromosome 10 DNA markers by Alu element-mediated PCR

Alu element-mediated polymerase chain reaction is a strategy for rapidly cloning and mapping human DNA markers from mixed DNA sources. A novel primer homologous to the 3' end of the human Alu repeat element provides the basis for preferential synthesis of human DNA fragments from human/rodent somatic cell hybrid DNA template. This approach has been used to isolate a series of new markers from chromosome 10. The Alu element-mediated PCR probes were regionally assigned on chromosome 10 by hybridization to Southern blots of Alu PCR-synthesized DNA derived from somatic cell hybrid template DNA. Alu element-mediated PCR is generally applicable and makes possible the analysis of complex genomes with a speed and sensitivity that has not been previously possible.     

Fluorescent in situ hybridisation to amphioxus chromosomes

We describe an efficient protocol for mapping genes and other DNA sequences to amphioxus chromosomes using fluorescent in situ hybridisation. We apply this method to identify the number and location of ribosomal DNA gene clusters and telomere sequences in metaphase spreads of Branchiostoma floridae. We also describe how the locations of two single copy genes can be mapped relative to each other, and demonstrate this by mapping an amphioxus Pax gene relative to a homologue of the Notch gene. These methods have great potential for performing comparative genomics between amphioxus and vertebrates.     

Fluorescent in situ hybridization to ascidian chromosomes

The draft genome of the ascidian Ciona intestinalis has been sequenced. Mapping of the genome sequence to the Ciona 14 haploid chromosomes is essential for future studies of the genome-wide control of gene expression in this basal chordate. Here we describe an efficient protocol for fluorescent in situ hybridization for mapping genes to the Ciona chromosomes. We demonstrate how the locations of two BAC clones can be mapped relative to each other. We also show that this method is efficient for coupling two so-far independent scaffolds into one longer scaffold when two BAC clones represent sequences located at either end of the two scaffolds.     

On-line sorting of human chromosomes by centromeric index,and identification of sorted populations by GTG-banding and fluorescent in situ hybridization

Summary Using slit-scan flow cytometry, the shape of human metaphase chromosomes, as expressed in their centromeric index (CI), and the DNA content of the chromosomes have been used as parameters in bivariate flow karyotyping. The resolution of the DNA vs CI flow karyogram of the larger chromosomes up to chromosome 13 is much higher than the resolution obtained in the DNA-based monovariate flow karyogram. Chromosome length appears to be an important factor in the resolution of the DNA vs CI-based flow karyogram. A method has been developed to obtain chromosomes in suspension that are long enough for adequate analysis. Several chromosomes that cannot be distinguished or are difficult to discriminate in the DNA-based karyogram can now be distinguished as individual peaks, e.g., chromosomes 1 and 2. The peak of chromosomes 9–12 can be separated into two peaks formed by chromosomes 9 and 11, and 10 and 12, respectively. The advantage of the system applied in this study is that the DNA vs CI analysis is performed on-line, allowing chromosomes to be sorted on the bases of their CI. Pulse shapes of the selected chromosomes can be recorded simultaneously with the transmission of the sorting command. The purity of the sorted fraction can be estimated from the offline inspection of these pulse shapes. Fractions of chromosome 1 have been sorted out on the basis of the CI information, centrifuged on slides, fixed and subsequently banded with trypsin and Giemsa or hybridized with the chromosome 1 specific probe, pUC 1.77. The observed purity under the selected conditions ranges from 80%–99% and is in accordance with the estimates of the purities made on the basis of the simultaneously recorded pulse shapes. Fixation of the chromosome suspension prior to flow cytometric analysis and sorting appears to be essential for the preservation of their morphology and has no adverse influence on the resolution of Giemsa banding or on the quality of in situ hybridization.     

In situ nick translation of human chromosomes using Alu I: unmasking of recognition sites by proteinase K pretreatment

Human chromosomes prepared according to routine methods were treated with the restriction endonuclease Alu I followed by staining with Giemsa solution or fluorescent dyes. This procedure results in a C-band-like appearance of the chromosomes due to removal of DNA from euchromatic chromosomal regions. The resistance of heterochromatic regions against cleavage by the enzyme has mainly been interpreted by the absence or rareness of recognition sites for this particular enzyme in these regions. Proteinase K pretreatment followed by a nick translation procedure with Alu I was combined to check this hypothesis. The results show that heterochromatic chromosomal regions can also be labelled. Thus, they are not characterized by a lack of recognition sites. Gradual deproteinisation of chromosomes changes the labelling pattern from a reverse C-banding pattern to a C-band-like appearance. The resistance of heterochromatic chromosomal parts revealed by the technique is mainly due to local chromatin configuration rather than to the underlying DNA sequence itself.     

Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry.

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.     

The use of peptide nucleic acids for in situ identification of human chromosomes.

The peptide nucleic acids (PNAs) constitute a remarkable new class of synthetic nucleic acid analogues, based on their peptide-like backbone. This structure gives to PNAs the capacity to hybridize with high affinity and specificity to complementary RNA and DNA sequences and a great resistance to nucleases and proteinases. Originally conceived as ligands for the study of double-stranded DNA, the unique physicochemical properties of PNAs have led to the development of a large variety of research and diagnostic assays, including antigene and antisense therapy, genome mapping, and mutation detection. Over the past few years, PNAs have been shown to be powerful tools in cytogenetics for the rapid in situ identification of human chromosomes and the detection of aneuploidies. Recent studies have reported the successful use of chromosome-specific PNA probes on human lymphocytes, amniocytes, and spermatozoa, as well as on isolated oocytes and blastomeres. Multicolor PNA protocols have been described for the identification of several human chromosomes, indicating that PNAs could become a powerful complement to FISH for in situ chromosomal investigation.     

Optimization of conditions for isolating chromosomes in flow sorting

Factors for purity and efficiency of flow chromosome sorting were analysed on the base of quantitative analysis. The sorting rate and relative purity of individual chromosome fractions are determined both by the quality of initial chromosome suspension, instrument parameters and gate position on experimental histograms. The described procedure of analysing sorting efficiency and fraction purity allowed to formulate general tips for optimization of sorting conditions depending on a given strategy: maximization of quantity of obtained material, or achieving maximum purity for sorted fraction. The analysis is carried out on the bases of chromosome distribution parameters: their relative halfwidths and distances. These parameters can be obtained by the quantitative analysis programs for flow cytometry data. It is shown that the critical parameter for sorting purity is the level of contaminated objects in a zone of sorted chromosome signal registration. In addition, the fraction purity depends on the cover extent between different chromosome distributions. Created procedure allows to build up nomograms linking the sorting efficiency with fraction purity, depending on the position of sorting gates. These monograms permit to determine the position of sorting gates in relation to one or another strategy: 1) maximum rate for chromosome material obtaining, 2) maximum fraction purity, or 3) compromise between the two. The presented analysis allows to optimize the chromosome sorting process for subsequent genome investigations providing chromosome material with controlled characteristics.     

The identification of human chromosomes by quinacrine fluorescence after hybridisation in situ

Summary A method is described for producing fluorescent bands on human chromosomes by staining with quinacrine after hybridisation in situ. The advantages of the method include the elimination of artefacts arising from staining before hybridisation, the fact that there is no reduction in sample number between staining and autoradiography, the ease with which autoradiographic grains can be identified and counted, and the reduction in exposure time.Offprint requests to: S.S. Lawrie     

AcroM fluorescent in situ hybridization analyses of marker chromosomes

The presence of a de novo supernumerary marker chromosome (SMC) poses problems in genetic counseling. The consequences of the additional chromosomal material may range from harmless to detrimental. As the composition of a SMC cannot be deciphered by traditional banding analysis, sophisticated methods are needed for their rapid and detailed analyses. A new strategy is presented, which allows the elucidation of the composition of SMCs in one or two hybridizations. One hybridization, termed AcroM-FISH, involves a newly generated probe mix, which consists of painting probes for all acrocentric chromosomes, centromere probes for chromosomes 13/21, 14/22, 15, and a probe specific for rDNA, each labeled with a specific combination of fluorochromes. This probe mix is sufficient to characterize approximately 80% of all SMCs. For the other 20% of SMCs, chromosomes can be analyzed in a second hybridization by multicolor karyotyping, for example, multiplex FISH (M-FISH), to check for the presence of euchromatin of other chromosomes. The potential of AcroM-FISH was tested in various applications.     

Swine chromosomes: flow sorting and spot blot hybridization

Flow cytometry analysis was applied to swine chromosomes prepared from phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes. Flow karyotypes from both sexes and from t(3;7) translocation carrier females were obtained. A certain number of chromosome pairs could be assigned to various peaks. In fact, 13 peaks were observed for 18 autosomal pairs plus X and Y. Moreover, abnormalities owing to the t(3;7) translocation were readily observable. The number of base pairs for chromosomes associated with the various peaks was estimated by comparison with human flow karyotypes. The following four peaks were thus sorted: the peak assumed to represent the translocated chromosome 7 plus the normals associated with it; the corresponding peak from a normal swine; the peak assumed to contain among others the normal chromosome 7; and finally the peak corresponding to swine chromosome 1. Chromosomes of each peak were collected on Pall Biodyne membrane. Following appropriate denaturation and prehybridization, the four samples were hybridized with a human leucocyte antigen (HLA) class I 32P-labelled cDNA probe, representing most of the coding sequence of the HLA B7 gene. The results confirmed previous data from other techniques that assigned the swine MHC(SLA) to chromosome 7. Subsequently, sorted samples were hybridized with a porcine genomic Interferon alpha probe in order to confirm the mapping of this gene family on porcine chromosome 1.     

Purification of the chromosomes of the Indian muntjac by flow sorting.

Metaphase chromosomes were isolated from a male Indian muntjac cell line, were stained with ethidium bromide and were analyzed by flow microfluorometry to establish a deoxyribonucleic acid (DNA)-based karyotype. Five major peaks were evident on the chromosomal DNA distribution corresponding to the five chromosome types in this species. The amount of DNA in each chromosome was confirmed by cytophotometric measurements of intact metaphase spreads. The five chromosome types were separated by flow sorting at rates up to several hundred chromosomes per second. The sorted chromosomes were identified by morphology and by Giemsa banding patterns. The automsomes, Numbers 1, 2 and 3, and the X + 3 composite chromosome were separated with a high degree of purity (90%). The centromere region of the X + 3 chromosome was fragile to mechanical shearing, and during isolation a small proportion of these chromosomes broke into four segiments: the long arm, the short arm, the short arm plus centromere and the centromere region. A large fraction of the constitutive heterochromatin of this species is present in the centromere region of the X + 3 chromosome and in the Y chromosome; these two regions possess similar amounts of DNA and therefore sort together. Chromosome flow sorting is rapid, reproducible and precise; it allows the collection of microgram quantities of purified chromosomes.     

Fluorescent in situ hybridization en suspension (FISHES) using digoxigenin-labeled probes and flow cytometry.

Fluorescent in situ hybridization has become a major technique for visualizing genetic material in fixed cells. Currently, many systems utilize the hybridization of labeled molecular probes to cells that are attached to slides. We have developed a technique that allows for in situ hybridization to be performed using cells in suspension. By using digoxigenin-labeled DNA probes and a fluoresceinated antibody directed against the digoxigenin, we can measure the resulting signal on a flow cytometer and the cells can be attached to microscope slides for visual analysis.     

Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and L1 primers

Summary Specific amplification of human sequences of up to several kb length has recently been accomplished in man-hamster and man-mouse somatic hybrid cell DNA by IRS-PCR (interspersed repetitive sequence — polymerase chain reaction). This approach is based on oligonucleotide primers that anneal specifically to human Alu- or L1-sequences and allows the amplification of any human sequences located between adequately spaced, inverted Alu- or L1-blocks. Here, we demonstrate that probe pools generated from two somatic hybrid cell lines by Alu- and L1-PCR can be used for chromosome painting in normal human lymphocyte metaphase spreads by chromosomal in situ suppression (CISS-) hybridization. The painted chromosomes and chromosome subregions directly represent the content of normal and deleted human chromosomes in the two somatic hybrid cell lines. The combination of IRS-PCR and CISS-hybridization will facilitate and improve the cytogenetic analysis of somatic hybrid cell panels, in particular, in cases where structurally aberrant human chromosomes or human chromosome segments involved in interspecies translocations cannot be unequivocally identified by classical banding techniques. Moreover, this new approach will help to generate probe pools for the specific delineation of human chromosome subregions for use in cytogenetic diagnostics and research without the necessity of cloning.     

Isolation of a human chromosome 14-only somatic cell hybrid: analysis using Alu and LINE-based PCR.

Interspecific somatic cell hybrids containing single human chromosomes are valuable reagents for localization of cloned genes and DNA fragments to specific chromosomes, for the development of chromosome-specific libraries, and for generation of hybrid cell lines containing subchromosomal regions. A CHO somatic cell hybrid containing a single, intact human chromosome 14 (MHR14) was developed and confirmed by LINE PCR amplification gel pattern, by Alu-517 PCR product dot blot hybridization, and by cytogenetic analysis. MHR14 will serve as the chromosome source for the development of a radiation map of human chromosome 14.