Optimization of in situ hybridization to human metaphase chromosomes

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.

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Karyotypes and Distribution of Tandem Repeat Sequences in Brassica nigra Determined by Fluorescence in situ Hybridization

Cytogenetic and Genome Research ◽

10.1159/000479179 ◽

2017 ◽

Vol 152 (3) ◽

pp. 158-165 ◽

Cited By ~ 6

Author(s):

Gui-xiang Wang ◽

Qun-yan He ◽

Jiri Macas ◽

Petr Novák ◽

Pavel Neumann ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Fluorescence Intensity ◽

Tandem Repeat ◽

Brassica Nigra ◽

Metaphase Chromosomes ◽

Repeat Sequences ◽

Tandem Repeat Sequences ◽

Black Mustard

Whole-genome shotgun reads were analyzed to determine the repeat sequence composition in the genome of black mustard, Brassica nigra (L.) Koch. The analysis showed that satellite DNA sequences are very abundant in the black mustard genome. The distribution pattern of 7 new tandem repeats (BnSAT13, BnSAT28, BnSAT68, BnSAT76, BnSAT114, BnSAT180, and BnSAT200) on black mustard chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of BnSAT13 and BnSAT76 provided useful cytogenetic markers; their position and fluorescence intensity allowed for unambiguous identification of all 8 somatic metaphase chromosomes. A karyotype showing the location and fluorescence intensity of these tandem repeat sequences together with the position of rDNAs and centromeric retrotransposons of Brassica (CRB) was constructed. The establishment of the FISH-based karyotype in B. nigra provides valuable information that can be used in detailed analyses of B. nigra accessions and derived allopolyploid Brassica species containing the B genome.

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Fluorescence In Situ Hybridization for Glycine max Metaphase Chromosomes

Current Protocols in Plant Biology ◽

10.1002/cppb.20045 ◽

2017 ◽

Vol 2 (1) ◽

pp. 89-107 ◽

Cited By ~ 1

Author(s):

Seth D. Findley ◽

James A. Birchler ◽

Gary Stacey

Keyword(s):

In Situ Hybridization ◽

Glycine Max ◽

Fluorescence In Situ Hybridization ◽

Metaphase Chromosomes

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Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization

Molecular and Cellular Biology ◽

10.1128/mcb.2.3.308-319.1982 ◽

1982 ◽

Vol 2 (3) ◽

pp. 308-319

Author(s):

G M Wahl ◽

L Vitto ◽

R A Padgett ◽

G R Stark

Keyword(s):

In Situ Hybridization ◽

Syrian Hamster ◽

Specific Radioactivity ◽

Large Chromosome ◽

Hybridization Technique ◽

Wild Type ◽

Aspartate Transcarbamylase ◽

Metaphase Chromosomes ◽

Cad Gene

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.

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In Situ Hybridization on Plant Metaphase Chromosomes

Gene Transfer to Plants ◽

10.1007/978-3-642-79247-2_26 ◽

1995 ◽

pp. 264-272

Author(s):

I. Farbos ◽

A. Mouras

Keyword(s):

In Situ Hybridization ◽

Metaphase Chromosomes

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Isolation, characterization, and chromosomal location of the tRNAMet genes in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta)

Genome ◽

10.1139/g99-084 ◽

2000 ◽

Vol 43 (1) ◽

pp. 185-190 ◽

Cited By ~ 3

Author(s):

J Perez ◽

P Moran ◽

E Garcia-Vazquez

Keyword(s):

In Situ Hybridization ◽

Atlantic Salmon ◽

Salmo Salar ◽

Brown Trout ◽

Salmo Trutta ◽

Genomic Library ◽

Metaphase Chromosomes ◽

Atlantic Salmon Salmo Salar ◽

Brown Trout Salmo Trutta

This work describes the isolation, characterization, and physical location of the methionine tRNA in the genome of Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.). An Atlantic salmon genomic library was screened using a tRNAMet probe from Xenopus laevis. Two cosmid clones containing the Atlantic salmon tRNAMet gene were isolated, subcloned and sequenced. The tRNAMet was mapped to metaphase chromosomes by fluorescence in situ hybridization (FISH). Chromosomal data indicated that the tDNA of methionine is tandemly repeated in a single locus in both species. Analysis of genomic DNA by Southern hybridization confirmed the tandem organization of this gene. Key words: cosmids, cloning, in situ hybridization, tRNAMet.

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Horse Radish Peroxidase Labelled Oligonucleotides and Tyramide Signal Amplification for Fluorescence in Situ Hybridization to Low Abundant Cytokine Mrnas

Microscopy and Microanalysis ◽

10.1017/s143192760000790x ◽

1997 ◽

Vol 3 (S2) ◽

pp. 203-204

Author(s):

Mariette van de Corput ◽

Rob van Gijlswijk ◽

Mark Bobrow ◽

Tom Erickson ◽

Roel Dirks ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Signal Amplification ◽

Signal To Noise Ratio ◽

High Specificity ◽

Tyramide Signal Amplification ◽

Horse Radish Peroxidase ◽

Metaphase Chromosomes ◽

Generation Capacity

In recent years, Tyramide Signal Amplification (TSA) has gained acclaim as a very sensitive detection method for immunocytochemsitry and fluorescence in situ hybridization (FISH). To maximally exploit the great signal generation capacity of TSA in mRNA-FISH, minimizing signals emanating from non-specifically bound nucleic acid probe becomes of prime importance, because a specificity check of the signals observed in the cytoplasm is virtually impossible. We reasoned that utilization of synthetic oligonucleotides (ONTs) in stead of commonly used cDNAs or cRNAs would diminish non-specific probe binding and that direct Horse Radish Peroxidase (HRP) labelling of ONTs and TSA would enable their in situ detection.This approach was first tested in metaphase DNA-FISH using chromosome-specific repeats as targets. Using bifunctional crosslinking chemistry and HPLC, 5’-hexylamino oligonucleotides for chromosome specific simple satellite and alphoid sequences were conjugated to HRP and purified. Following 15 - 20 min of situ hybridization of a single HRP-ONT probe to metaphase chromosomes and a direct flurochrome-tyramide detection step, such repeat targets could be visualized with high specificity and excellent signal-to noise ratio.

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Detection of myc translocations in lymphoma cells by fluorescence in situ hybridization with yeast artificial chromosomes

Blood ◽

10.1182/blood.v85.8.2132.bloodjournal8582132 ◽

1995 ◽

Vol 85 (8) ◽

pp. 2132-2138 ◽

Cited By ~ 23

Author(s):

ML Veronese ◽

M Ohta ◽

J Finan ◽

PC Nowell ◽

CM Croce

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Yeast Artificial Chromosome ◽

Human Lymphocytes ◽

Artificial Chromosome ◽

Chromosome 8 ◽

Metaphase Chromosomes ◽

Artificial Chromosomes ◽

Myc Gene

Translocations involving chromosome 8 at band q24 and one of the Ig loci on chromosomes 14q32, 22q11, and 2p11 are the hallmark of Burkitt's lymphoma (BL). It has been previously observed that the exact localization of the breakpoints at chromosome 8q24 can vary significantly from patient to patient, scattering over a distance of more than 300 kb upstream of c-myc and about 300 kb downstream of c-myc. To generate probes for fluorescence in situ hybridization (FISH) that detect most c-myc translocations, we screened a yeast artificial chromosome (YAC) library from normal human lymphocytes by colony hybridization, using three markers surrounding the c-myc gene as probes. We obtained 10 YAC clones ranging in size between 500 and 200 kb. Two nonchimeric clones were used for FISH on several BL cell lines and patient samples with different breakpoints at 8q24. Our results show that the YAC clones detected translocations scattered along approximately 200 kb in both metaphase chromosomes and interphase nuclei. The sensitivity, rapidity, and feasibility in nondividing cells render FISH an important diagnostic tool. Furthermore, the use of large DNA fragments such as YACs greatly simplifies the detection of translocations with widely scattered breakpoints such as these seen in BL.

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Single-copy and amplified CAD genes in Syrian hamster chromosomes localized by a highly sensitive method for in situ hybridization.

Molecular and Cellular Biology ◽

10.1128/mcb.2.3.308 ◽

1982 ◽

Vol 2 (3) ◽

pp. 308-319 ◽

Cited By ~ 47

Author(s):

G M Wahl ◽

L Vitto ◽

R A Padgett ◽

G R Stark

Keyword(s):

In Situ Hybridization ◽

Syrian Hamster ◽

Specific Radioactivity ◽

Large Chromosome ◽

Hybridization Technique ◽

Wild Type ◽

Aspartate Transcarbamylase ◽

Metaphase Chromosomes ◽

Cad Gene

Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a specific inhibitor of the aspartate transcarbamylase activity of the multifunctional protein CAD, overproduce this protein as a result of amplification of the CAD gene. We have used a sensitive in situ hybridization technique to localize CAD genomes in spreads of metaphase chromosomes from several independent PALA-resistant lines and from wild-type PALA-sensitive cells. The amplified genes were always found within chromosomes, usually in an expanded region of the short arm of chromosome B9. In wild-type cells, the CAD gene was also on the short arm of chromosome B9. In one mutant line, 90 to 100 CAD genes were found within an expanded B9 chromosome and 10 to 15 more were near the distal end of one arm of several different chromosomes. Another line contained most the genes in a telomeric chromosome or large chromosome fragment. The amplified genes were in chromosomal regions that were stained in a banded pattern by trypsin-Giemsa. A few double minute chromosomes were observed in a very small fraction of the total spreads examined. The it situ hybridizations were performed in the presence of 10% dextral sulfate 500, which increases the signal by as much as 100-fold. Using recombinant DNA plasmids nick-translated with [125I]dCTP to high specific radioactivity, 10 CAD genes in a single chromosomal region were revealed after 1 week of autoradiographic exposure, and the position of the unique gene could be seen after 1 month.

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

Genome ◽

10.1139/g98-054 ◽

1998 ◽

Vol 41 (4) ◽

pp. 560-565 ◽

Cited By ~ 12

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.

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