DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1994 ◽  
Vol 52 ◽  
pp. 80-81
Author(s):  
Barbara Trask ◽  
Hiroki Yokota ◽  
Richard Esposito ◽  
Cynthia Friedman ◽  
Hillary Massa ◽  
...  
Keyword(s):  
Gene Mapping ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Large Scale ◽  
Dual Band ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

By using FISH, the positions of DNA sequences can be discretely marked with a fluorescent spot. For this reason, FISH has proven useful in gene mapping, studying large-scale variation in the human genome, detecting chromosome rearrangements associated with disease, and in understanding the organization of the cell nucleus.The applications of FISH in gene mapping and cytogenetics requires a basic understanding of the organization of the interphase nucleus. These applications are constrained by the efficiency of hybridization, the spatial resolution of hybridization sites in nuclei, and the correlation (if any) between the separation of sequences in interphase chromatin and their separation on the linear DNA molecule.The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ∼0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

Organization and distribution of a Sau3A tandem repeated DNA sequence in Picea (Pinaceae) species

Genome ◽  
1998 ◽  
Vol 41 (4) ◽  
pp. 560-565 ◽  
Author(s):  
Garth R Brown ◽  
Craig H Newton ◽  
John E Carlson
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Picea Glauca ◽  
Tandem Repeats ◽  
Picea Sitchensis ◽  
Repeated Dna ◽  
Metaphase Chromosomes ◽  
Genes Encoding ◽  
Repeated Dna Sequence

Repeated DNA families contribute to the large genomes of coniferous trees but are poorly characterized. We report the analysis of a 142 bp tandem repeated DNA sequence identified by the restriction enzyme Sau3A and found in approximately 20 000 copies in Picea glauca. Southern hybridization indicated that the repeated DNA family is specific to the genus, was amplified early in its evolution, and has undergone little structural alteration over evolutionary time. Fluorescence in situ hybridization localized arrays of the Sau3A repeating element to the centromeric regions of different subsets of the metaphase chromosomes of P. glauca and the closely related Picea sitchensis, suggesting that mechanisms leading to the intragenomic movement of arrays may be more active than those leading to mutation of the repeating elements themselves. Unambiguous identification of P. glauca and P. sitchensis chromosomes was made possible by co-localizing the Sau3A tandem repeats and the genes encoding the 5S and 18S-5.8S-26S ribosomal RNAs.Key words: Picea, repeated DNA, in situ hybridization, centromere.

In situ localization of two repetitive DNA sequences to surface-spread pachytene chromosomes of rye

Genome ◽  
1992 ◽  
Vol 35 (4) ◽  
pp. 551-559 ◽  
Author(s):  
S. M. Albini ◽  
T. Schwarzacher
Keyword(s):  
In Situ Hybridization ◽  
Synaptonemal Complex ◽  
Dna Sequences ◽  
Meiotic Prophase ◽  
Hybridization Signal ◽  
Metaphase Chromosomes ◽  
Somatic Metaphase ◽  
Rdna Signal ◽  
Surface Spread

Surface-spread pollen mother cells at meiotic prophase from Secale cereale (rye) were used for fluorescent DNA:DNA in situ localization of two tandemly repeated DNA sequences: pTa71, a wheat rDNA clone, and pSc119.2, a cloned 120-bp repeat from rye heterochromatin. The fluorescent hybridization signal, consisting of many yellow-green dots, was closely associated with the bivalent axes, corresponding to the synaptonemal complex, and located in the surrounding chromatin. The rDNA signal was associated with one bivalent, the smallest of the seven, at a distance about 13% of the bivalent length from the telomere. This corresponded to the position of the nucleolar organizing region of silver-stained synaptonemal complexes analyzed under the electron microscope and published data for somatic metaphase chromosomes. The relative length of the axis covered with the rDNA signal is less than expected from somatic metaphases, but it corresponds more closely to the proportion of the sequences in the genome. The hybridization signal with the 120-bp repeat was located mainly at the telomeric regions of several bivalents that showed thickenings of the axis after DAPI staining, probably corresponding to somatic C-bands. These major and some minor intercalary sites agree with the distribution of the 120-bp repeat in somatic metaphase. Fluorescent in situ hybridization to plant surface-spread pachytene chromosomes, which can be obtained in large numbers, has great potential for studying meiotic prophase, high-resolution mapping of DNA sequences, and investigating the relationship of DNA sequences to the synaptonemal complex.Key words: in situ hybridization, cereals, pachytene, meiosis, synaptonemal complex, physical mapping.

Detection of maize DNA sequences amplified in wheat

Genome ◽  
1995 ◽  
Vol 38 (5) ◽  
pp. 946-950 ◽  
Author(s):  
Juan Zhang ◽  
Bernd Friebe ◽  
Bikram S. Gill
Keyword(s):  
Triticum Aestivum ◽  
Zea Mays ◽  
In Situ Hybridization ◽  
Dna Sequences ◽  
Hexaploid Wheat ◽  
Chinese Spring ◽  
Genomic Dna ◽  
Genomic In Situ Hybridization ◽  
Metaphase Chromosomes

Genomic in situ hybridization to somatic metaphase chromosomes of hexaploid wheat cv. Chinese Spring using biotinylated maize genomic DNA as a probe revealed the existence of amplified maize DNA sequences in five pairs of chromosomes. The in situ hybridization sites were located on chromosomes 1A, 7A, 2B, 3B, and 7B. One pair of in situ hybridization sites was also observed in hexaploid oat. The locations and sizes of in situ hybridization sites varied among progenitor species.Key words: Triticum aestivum, Zea mays, shared DNA sequences, genomic in situ hybridization.

Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research

Genome ◽  
2006 ◽  
Vol 49 (9) ◽  
pp. 1057-1068 ◽  
Author(s):  
Jiming Jiang ◽  
Bikram S. Gill
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Molecular Cytogenetics ◽  
Plant Genome ◽  
Current Status ◽  
Sequence Information ◽  
Resolution Mapping

Fluorescence in situ hybridization (FISH), which allows direct mapping of DNA sequences on chromosomes, has become the most important technique in plant molecular cytogenetics research. Repetitive DNA sequence can generate unique FISH patterns on individual chromosomes for karyotyping and phylogenetic analysis. FISH on meiotic pachytene chromosomes coupled with digital imaging systems has become an efficient method to develop physical maps in plant species. FISH on extended DNA fibers provides a high-resolution mapping approach to analyze large DNA molecules and to characterize large genomic loci. FISH-based physical mapping provides a valuable complementary approach in genome sequencing and map-based cloning research. We expect that FISH will continue to play an important role in relating DNA sequence information to chromosome biology. FISH coupled with immunoassays will be increasingly used to study features of chromatin at the cytological level that control expression and regulation of genes.

Use of fluorescence in situ hybridization to study the arrangement of DNA sequences in normal and disease-related chromosomes

1995 ◽  
Vol 53 ◽  
pp. 748-749
Author(s):  
Barbara J. F. Trask ◽  
Hillary Massa ◽  
Cynthia Friedman ◽  
Richard Esposito ◽  
Ger van den Engh ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequences ◽  
Single Copy ◽  
Two Dimensions ◽  
Genetic Maps ◽  
Epifluorescence Microscopy ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei

The sites of specific DNA sequences can be fluorescently tagged by fluorescence in situ hybridization (FISH). Different sequences can be labeled with different fluorochromes so that their arrangement can be studied using epifluorescence microscopy. The distances between points on the same or different chromosomes can be determined easily in a large number of interphase nuclei or metaphase chromosomes. A variety of probe types, ranging from single-copy sequences to highly repeated sequences can be employed. Our work has focussed on the analysis of hybridization patterns in two dimensions using conventional fluorescence microscopy.We have used FISH to study various aspects of genome organization that are difficult to study using other techniques. Examples of these applications will be presented.FISH is now the method of choice for determining the chromosomal location of DNA sequences. DNA sequences can be positioned in the genome with <1:1000 accuracy (to a 3-Mbp region within a 3000-Mbp genome). Through FISH, the cytogenetic, physical and genetic maps of chromosomes can be linked.

Histochemical aspects of nucleic acid detection

1995 ◽  
Vol 53 ◽  
pp. 746-747
Author(s):  
B. A. Hamkalo ◽  
Elizabeth R. Unger
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
High Resolution ◽  
Dna Sequences ◽  
Single Copy ◽  
Nucleic Acid Detection ◽  
Metaphase Chromosomes ◽  
Potential Applications ◽  
Nucleic Acid Sequences

This symposium brings together several approaches for the detection of specific nucleic acid sequences that have potential applications at the histochemical level.Trask et al. report on the use of fluorescence in situ hybridization (FISH) techniques to study the arrangement of DNA sequences in normal and diseaserelated chromosomes. The sites of specific DNA sequences can be fluorescently tagged. Different sequences can be labeled with different fluorochromes so that their arrangement can be studied using fluorescence microscopy. The distances between points on the same or different chromosomes can be determined in a large number of interphase nuclei or metaphase chromosomes. A variety of probe types, ranging from single-copy sequences to highly repeated sequences can be employed.Hamkalo and co-workers have used non-radioactive methods at the EM level for the detection of nucleic acid sequences by in situ hybridization. Analysis of metaphase chromosomes by electron microscopy allows for high resolution mapping of chromosomes. A variety of labelling procedures have been employed to illustrate the utility of high resolution nucleic acid sequence mapping in these preparations.

Chromosome preparations from protoplasts: in situ hybridization banding pattern of a dispersed DNA sequence in rye (Secale cereale L.)

Genome ◽  
1991 ◽  
Vol 34 (4) ◽  
pp. 524-527 ◽  
Author(s):  
N. Jouve ◽  
C. L. McIntyre ◽  
J. P. Gustafson
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Banding Pattern ◽  
Banding Technique ◽  
Differential Distribution ◽  
Cellular Debris ◽  
Biotin Labeling ◽  
Secale Cereale L

The utilization of genome-specific DNA sequences coupled with in situ hybridization for chromosome karyotyping in wheat, rye, and triticale has been of limited value because of the presence of cellular and cytoplasmic debris. The use of protoplasts, thus eliminating cellular debris, has been shown to improve the level of detection of low-copy and unique DNA sequences in cereals. Therefore, the use of protoplasts could represent an appropriate tool to improve the results of karyotyping cereal chromosomes with genome-specific DNA sequences. This paper describes the results on the comparative application of protoplasts and squash preparations in the analysis of physical mapping of a dispersed DNA sequence (pSc119.1) to rye chromosomes by in situ hybridization. Individual chromosomes of rye were not distinguishable by their hybridization patterns to pSc119.1 when squash preparations were used. These showed an undefined distribution of the DNA probe that covered apparently the entire length of each rye chromosome. However, considerable improvement was observed for the differential distribution of the pSc119.1 DNA sequence in protoplast preparations. The karyotypic banding pattern of pSc119.1 showed a better banding pattern than can be observed using the C-banding technique. Therefore, the use of protoplasts hybridized with dispersed DNA markers could be of more value in monitoring chromosome karyotypes than existing cytological techniques.Key words: biotin labeling, dispersed sequences, rye.

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years

Genome ◽  
1994 ◽  
Vol 37 (5) ◽  
pp. 717-725 ◽  
Author(s):  
Jiming Jiang ◽  
Bikram S. Gill
Keyword(s):  
In Situ Hybridization ◽  
Physical Mapping ◽  
Dna Sequences ◽  
Genome Mapping ◽  
Molecular Cytogenetics ◽  
Gene Families ◽  
Single Copy ◽  
Plant Genome ◽  
Metaphase Chromosomes

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.Key words: in situ hybridization, physical mapping, genome mapping, molecular cytogenetics.

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