共查询到20条相似文献,搜索用时 31 毫秒
1.
Padilla-Nash HM Barenboim-Stapleton L Difilippantonio MJ Ried T 《Nature protocols》2006,1(6):3129-3142
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. 相似文献
2.
Tönnies H 《Trends in molecular medicine》2002,8(6):246-250
During the past decade, fluorescence in situ hybridization (FISH) has become an important complementing application in genetic diagnostics. The use of variable FISH techniques enhances the thorough interpretation of numerical and complex chromosome aberrations, bridging the gap between conventional chromosome banding analysis and molecular genetic DNA studies. This review gives a brief overview of the different molecular cytogenetic FISH techniques and applications currently used in routine genetic diagnostics and focus on their advantages and limitations. 相似文献
3.
JM Rodríguez-Domínguez LL Ríos-Lara E Tapia-Campos R Barba-Gonzalez 《Biotechnic & histochemistry》2017,92(3):159-166
Preparations that contain well-spread metaphase chromosomes are critical for plant cytogenetic analyses including chromosome counts, banding procedures, in situ hybridization, karyotyping and construction of ideograms. Chromosome spreading is difficult for plants with large and numerous chromosomes. We report here a technique for obtaining cytoplasm-free, well-spread metaphases from two Amaryllidaceae species: Sprekelia formosissima (2n = 120) and Hymenocallis howardii (2n = 96). The technique has three main steps: 1) pretreatment to cause chromosome condensation, 2) dripping onto tilted slides coated with a thin layer of pure acetic acid and 3) application of steam and acetic acid to produce cytoplasmic hydrolysis, which spreads the chromosomes. 相似文献
4.
The identification of unbalanced structural chromosome rearrangements using conventional cytogenetic techniques depends on recognition of the unknown material from its banding pattern. Even with optimally banded chromosomes, when large chromosome segments are involved, cytogeneticists may not always be able to determine the origin of extrachromosomal material and supernumerary chromosomes. We report here on the application of comparative genomic hybridization (CGH), a new molecular-cytogenetic assay capable of detecting chromosomal gains and losses, to six clinical samples suspected of harboring unbalanced structural chromosome abnormalities. CGH provided essential information on the nature of the unbalanced aberration investigated in five of the six samples. This approach has proved its ability to resolve complex karyotypes and to provide information when metaphase chromosomes are not available. In cases where metaphase chromosome spreads were available, confirmation of CGH results was easily obtained by fluorescence in situ hybridization (FISH) using specific probes. Thus the combined use of CGH and FISH provided an efficient method for resolving the origin of aberrant chromosomal material unidentified by conventional cytogenetic analysis. 相似文献
5.
Loja T Kuglik P Oltova A Smuharova P Zitterbart K Bajciova V Veselska R 《Cytogenetic and genome research》2007,116(3):186-193
Spectral karyotyping (SKY) represents an important tool for the investigation of the complex chromosomal rearrangements (CCRs) in many human malignancies which may be difficult to characterize by conventional banding techniques. The main goal of our work was to optimize the most important steps in the preparation of molecular cytogenetic slides for a SKY protocol. This approach consisted of optimization of both the aging procedure and protease pretreatment of the slides, with special regard given to the preservation of chromosome structure and shape, as well as to the intensity of hybridization signals. The best results were obtained with a chemical aging procedure using SSC or ethanol in combination with trypsin pretreatment applied at a higher concentration for a shorter period of pretreatment. A resulting protocol for SKY also applicable to human solid tumour cells was subsequently proposed. The practical potential of the SKY technique was demonstrated on examples of two types of human embryonal tumours--neuroblastoma and Wilms' tumour, in which some kinds of chromosomal aberrations were not detectable by means of classic cytogenetic methods. 相似文献
6.
Budny B Kanik M Latos-Bieleńiska A 《Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society》2002,40(2):107-108
The methods of molecular cytogenetics, in particular fluorescence in situ hybridization (FISH), are widely applied in cytogenetics for identification of numerical and structural chromosomal abnormalities, which are difficult to detect by routine cytogenetic techniques. Due to many advantages, FISH is used in research (gene mapping, gene expression studies, interspecies chromosome homology), and clinical diagnostics (chromosomal aberrations analysis in pre- and postnatal diagnostics, oncology). The techniques of in situ hybridization (ISH) are often employed in addition to classical banding techniques, in case where banding pattern is not reliable. This paper focuses on particular clinical examples, where FISH was successfully used to identify structural and numerical chromosomal aberrations. 相似文献
7.
Prenatal detection of aneuploidies using fluorescencein situ hybridization: A preliminary experience in an Indian set up 总被引:7,自引:0,他引:7
Fluorescencein situ hybridization (FISH) is a powerful molecular cytogenetic technique which allows rapid detection of aneuploidies on interphase
cells and metaphase spreads. The aim of the present study was to evaluate FISH as a tool in prenatal diagnosis of aneuploidies
in high risk pregnancies in an Indian set up. Prenatal diagnosis was carried out in 88 high-risk pregnancies using FISH and
cytogenetic analysis. Multicolour commercially available FISH probes specific for chromosomes 13, 18, 21, X and Y were used.
Interphase FISH was done on uncultured cells from chorionic villus and amniotic fluid samples. FISH on metaphase spreads was
done from cord blood samples. The results of FISH were in conformity with the results of cytogenetic analysis in all the normal
and aneuploid cases except in one case of structural chromosomal abnormality. The hybridization efficiency of the 5 probes
used for the detection of aneuploidies was 100%. Using these probes FISH assay yielded discrete differences in the signal
profiles between cytogenetically normal and abnormal samples. The overall mean interphase disomic signal patterns of chromosomes
13, 18, 21, X and Y were 94.45%; for interphase trisomic signal pattern of chromosome 21 was 97.3%. Interphase FISH is very
useful in urgent high risk cases. The use of FISH overcomes the difficulties of conventional banding on metaphase spreads
and reduces the time of reporting. However, with the limited number of probes used, the conventional cytogenetic analysis
serves as a gold standard at present. It should be employed as an adjunctive tool to conventional cytogenetics 相似文献
8.
9.
Thomas Eggermann Hartmut Engels Barbara Moskalonek Markus M. Nöthen Jutta Müller-Navia Engelhardt Schleiermacher Gesa Schwanitz Sabine Stengel-Rutkowski 《Human genetics》1996,97(5):568-572
We report a de novo supernumerary isochromosome 18p in a child with tetrasomy 18p, analyzed by a straightforward combination
of cytogenetic and molecular cytogenetic methods. The diagnostic procedure consisted of standard banding techniques and fluorescence
in situ hybridization (FISH) with centromere and library DNA probes for chromosome 18, and 18p-specific FISH probes prepared
by chromosome microdissection and in vitro amplification. The maternal origin as well as the most probable cell stages of
formation of the supernumerary isochromosome were determined by typing of short sequence repeats (SSRs). The pattern of allelic
distribution suggests a nondisjunction during meiosis followed by a centromeric misdivision in an early postzygotic mitosis
as the most probable mode of isochromosome 18p formation. The combination of the applied methods represents a powerful tool
to investigate the nature and the origin of de novo marker chromosomes.
Received: 28 August 1995 / Revised: 3 November 1995; 20 December 1995 相似文献
10.
A colored banding pattern for human chromosomes is described that distinguishes each chromosome in a single fluorescence
in situ hybridization with a set of subregional DNA probes. Alu/polymerase chain reaction products of various human/rodent
somatic cell hybrids (fragment hybrids) were pooled into two probe sets that were labeled differentially and detected by red
and green fluorescence. Chromosome regions hybridized by DNA present in both pools appeared yellow. The result was a multi-color
set of 110 distinct signals per haploid chromosome set for the human karyotype. Each individual chromosome showed a unique
sequence of signals, a result termed the “chromosome bar code”. The reproducibility of the hybridization pattern in various
labeling and hybridization experiments was analyzed by computer densitometry. We have applied the chromosome bar code both
in diagnostic cytogenetics and in genome studies. The approach allows the rapid identification of chromosomes and chromosome
rearrangements. Although not yet showing the resolution of classical banding patterns, the present experiments demonstrate
various applications in which the present multi-color bar code can significantly add to the spectrum of cytogenetic techniques.
Received: 18 December 1996 / Accepted: 10 February 1997 相似文献
11.
BACKGROUND: The recently developed tissue microarray (TMA) technology allows the arrangement of up to a thousand tissue specimens on a single microscope slide. This technology enables researchers to perform gene copy number studies on very large series of archival formalin-fixed tissues using fluorescence in situ hybridization (FISH). However, the hybridization properties of individual archival specimens can vary considerably. Therefore a highly optimized protocol is needed to fulfill the task of producing evaluable hybridization signals simultaneously in hundreds of specimens in a TMA. METHODS: The performance of two different FISH protocols, the standard protocol for paraffin embedded tissues and our new optimized protocol, was tested on TMAs using probes for the HER-2 and ZNF217 genes as well as the chromosome 17 centromere. RESULTS: The new protocol resulted in greatly increased signal intensity and an almost 30% increase in the number of tissue samples with evaluable hybridization signals. CONCLUSIONS: Our improved protocol for FISH on TMAs provides standardized hybridization conditions leading to high-quality hybridization signals in the majority of specimens. The increases in the signal intensity and the number of evaluable samples are extremely important for the successful analyses of TMAs by FISH and will allow the utilization of the TMA technology in its full potential. 相似文献
12.
Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale. 相似文献
13.
Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established. 相似文献
14.
Different chromosome Y abnormalities in Turner syndrome. 总被引:2,自引:0,他引:2
A 17-year-old phenotypically female girl was referred for evaluation because of short stature and primary amenorrhea. Cytogenetic analysis showed a mosaic 46,XY/45,X/47,XYY/46,X,idic(Yq)/47,XY,idic(Yq)/48,XXY,idic(Yq)/46,X,t(C;Y) karyotype. Conventional cytogenetic results were supplemented with fluorescence in situ hybridization (FISH) techniques to ensure a better characterization of abnormalities. By using FISH, a supernumerary marker chromosome derived from chromosome Y which could not be detected by conventional cytogenetics was revealed. Furthermore, additional abnormalities and their frequencies were highlighted by the application of DNA probes specific for X and Y chromosomes. Thus, FISH proved useful in determining low frequency cell lines which would need analysis of a large number of good quality metaphase spreads by conventional cytogenetic techniques: it helped in identifying the nature and the origin of unknown markers and rearrangements which have important implication in sexual differentiation and development of gonadal tumours. 相似文献
15.
In situ chromosome preparation technique for simultaneous cytogenetic and immunocytochemical studies on cell cultures of solid tumors 总被引:2,自引:1,他引:1
Wolfram Henn Jutta Lehr Gerhard Unteregger Ayhan Ermis Klaus D. Zang 《Human genetics》1990,85(5):551-554
Summary Immunophenotyping of cultured cancer cells requires intact antigenic structures; these are mostly destroyed by conventional chromosome preparation techniques. Thus, the simultaneous cytogenetic and immunocytochemical characterization of solid tumor cells appears unfeasible. Here, we describe a novel method that allows in situ chromosome preparation from monolayer cultures of solid tumor cells without affecting their immunological features. Using this technique, it is possible to achieve detailed cytogenetic data including chromosome banding together with the demonstration of cytoplasmic and nuclear antigens within the same tumor cell. 相似文献
16.
Distinct cell-cycle-dependent changes in the conformation of centromeric chromatin in a specific human chromosome containing alpha-satellite DNA have been demonstrated by fluorescence in situ hybridization (FISH). This method, based upon specific FISH signal morphology, allows simultaneous analysis of chromosomal aneuploidy and detection of specific cell-cycle stage(s) of human tumor and/or normal cell populations in a single preparation of interphase cells. This interphase cytogenetic procedure might prove useful for both basic and clinical research involving human cells. 相似文献
17.
Highly effective cell synchronization in plant roots by hydroxyurea and amiprophos-methyl or colchicine. 总被引:5,自引:0,他引:5
Effective somatic cell synchronization in root-tip meristems and improved chromosome spreading were achieved in white campion, wheat, rye, and barley by application of hydroxyurea and amiprophos-methyl or colchicine, combined with a pretreatment of ice water and modified fixative, as well as enzymatic digestion of the meristems. The protocol provides metaphase indices of approximately 50%. The chromosomes and chromosomal DNA were with minimum distortion, providing useful material for chromosome banding studies, in situ DNA-DNA hybridization, microdissection, and microcloning. 相似文献
18.
Primary amenorrhoea is defined as the absence of menstruation in phenotypic women aged 16 years or older, if secondary sexual characteristics are present. X chromosome abnormalities probably comprise about one half of all cases, including Turner syndrome and X chromosome rearrangements. Conventional banding cytogenetic methods might miss the accurate detection of structural chromosome abnormalities. The fluorescence in situ hybridization (FISH) and multicolor FISH techniques are required to interpret specific chromosomal rearrangement. As far as we know, we report the first case with chromosome mosaicism for monosomy X and terminal deletion of Xq26 with duplication of Xp11-->pter. In spite of the fact that a 45,X karyotype was detected in 46% of lymphocytes, she was tall and her secondary sexual characteristics were moderately developed, including breast, pubic and axillary hair stages. Cytogenetic and FISH analyses should be considered for patients presenting primary amenorrhoea even if there are no other clinical features suggestive of chromosome abnormality. 相似文献
19.
All hematological malignancies are characterized by considerable clinical heterogeneity. The diverse entities can be subdivided into a variety of prognosis-defining subtypes on the basis of cytogenetic aberrations and molecular mutations. To adapt the intensity of treatment to the patient’s individual risk profile, an exact classification of the subtypes on the basis of genetic markers is essential. Diverse fluorescent in situ hybridization (FISH) techniques thereby play a central role in interaction with classic chromosome banding analyses for clarifying findings of chromosome analyses, such as in the acute leukemias, or for classifying the diverse subtypes, as in the non-Hodgkin’s lymphomas. Depending on the disease, the clinical impact of FISH varies. It is used as the method of choice for genetic characterization (e.g., in multiple myeloma) or is used in combination with chromosome banding analysis. Furthermore, interphase FISH is essential when rapid confirmation of the diagnosis is needed, as in acute promyelocytic leukemia with the t(15;17)/PML-RARA rearrangement, for which therapy with all-trans retinoic acid (ATRA) should be immediately started. 相似文献
20.
Epithelial tumour karyotypes are often difficult to study by standard cytogenetic methods because of poor chromosome preparation quality and the high complexity of their genomic rearrangements. Subtelomeric fluorescence in situ hybridisation (FISH) has proved to be a useful method for detecting cryptic constitutional chromosomal rearrangements but little is known about its usefulness for tumour cytogenetic analysis. Using a combination of chromosome banding, multicolour karyotyping and subtelomeric FISH, five colorectal cancer cell lines were characterised. The resulting data were compared to results from previous studies by comparative genomic hybridisation and spectral karyotyping or multicolour FISH. Subtelomeric FISH made it possible to resolve several highly complex chromosome rearrangements, many of which had not been detected or were incompletely characterised by the other methods. In particular, previously undetected terminal imbalances were found in the two cell lines not showing microsatellite instability. 相似文献