High-resolution <i>in situ</i> hybridization using DNA halo preparations

Wiegant, J.; Kalle, Wouter; Mullenders, L.H.F.; Brookes, Sharon; Hoovers, J. M. N.; Dauwerse, Johannes G.; Ommen, G.J.B. van; Raap, Anton K.

doi:10.1093/hmg/1.8.587

Cited by 169 publications

(78 citation statements)

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“…The ultimate resolution in cytogenetics is reached by wiping out nuclear organization altogether and conducting FISH on DNA fibers that have been affixed to glass (15)(16)(17)(18). Although morphological information is lost, what was condensed in an interphase or metaphase FISH spot becomes a long fluorescent line in fiber-FISH.…”

Section: Discussionmentioning

confidence: 99%

Single-Step Multicolor Fluorescence In Situ Hybridization Using Semiconductor Quantum Dot-DNA Conjugates

Bentolila

Weiss

2006

CBB

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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.

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Section: Discussionmentioning

confidence: 99%

Single-Step Multicolor Fluorescence In Situ Hybridization Using Semiconductor Quantum Dot-DNA Conjugates

Bentolila

Weiss

2006

CBB

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“…To fine map the breakpoint region at the genomic level, cells from short-term primary tumor cultures of L-1231 were collected in suspension. Linearly extracted, stretched DNA fibers were made from cells using the socalled ''halo technique,'' which has been described for high-resolution FISH mapping (18).…”

Section: Translational Relevancementioning

confidence: 99%

The NFATc2 Gene Is Involved in a Novel Cloned Translocation in a Ewing Sarcoma Variant That Couples Its Function in Immunology to Oncology

Szuhai¹,

IJszenga²,

Jong³

et al. 2009

Clinical Cancer Research

180

130

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Purpose: Ewing sarcoma is an aggressive sarcoma and is the second most common bone sarcoma in childhood. Disease-specific t(11;22) (f85-90%), t(21;22) (f5-10%), or rarer variant translocations with the involvement of chromosome 22 (f5%) are present. At the gene level, the EWSR1 gene fuses with FLI1, ERG, or other ETS transcription factor family members. Thus far, no Ewing sarcoma has been identified with a fusion to transcription factors other than ETS. Experimental Design: Using molecular tools such as multicolor fluorescence in situ hybridization and array comparative genomic hybridization, a ring chromosome containing chromosomes 20 and 22 was identified in four Ewing sarcoma cases. The breakpoint was mapped with (fiber-) fluorescence in situ hybridization and reverse transcription-PCR followed by sequencing of the fusion partners. Results: Molecular karyotyping showed the translocation and amplification of regions of chromosomes 20q13 and 22q12. Cloning of the breakpoint showed an in-frame fusion between the EWSR1 and NFATc2 genes, resulting in loss of the NH 2 -terminal, calcineurin-dependent control region and an intact active domain of NFATc2 controlled by the transactivation domains of EWSR1. Conclusion: A new translocation involving EWSRI and NFATc2 was cloned. NFATc2 is a transcription factor that is not a member of the ETS family and functions inT-cell differentiation and immune response. Direct involvement of NFATc2 has not yet been observed in oncogenesis. We show that due to the shared sequence recognition of NFATc2 and the ETS family, shared transcriptional control is possible using activating protein complex 1.

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“…Several groups applied DNA fiber mapping techniques to investigate the interface between euchromatic and heterochromatic regions as well as the organization of tandem DNA repeats in humans (Wiegant et al 1992;Haaf and Ward 1994b;Shiels et al 1997), animals (Sjöberg et al 1997;Li et al 2000), and plants (Fransz et al 1996;Jackson et al 1998Jackson et al ,1999Zhong et al 1998). The methods applied by these researchers ranged from DNA halo preparations and extended chromatin to highly purified DNA molecules.…”

Section: Overcoming Challenges In Mapping Tandemly Repeated Dna Sequementioning

confidence: 99%

DNA Fiber Mapping Techniques for the Assembly of High-resolution Physical Maps

Weier

2001

J Histochem Cytochem.

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S U M M A R Y High-resolution physical maps are indispensable for directed sequencing projects or the finishing stages of shotgun sequencing projects. These maps are also critical for the positional cloning of disease genes and genetic elements that regulate gene expression. Typically, physical maps are based on ordered sets of large insert DNA clones from cosmid, P1/PAC/BAC, or yeast artificial chromosome (YAC) libraries. Recent technical developments provide detailed information about overlaps or gaps between clones and precisely locate the position of sequence tagged sites or expressed sequences, and thus support efforts to determine the complete sequence of the human genome and model organisms. Assembly of physical maps is greatly facilitated by hybridization of non-isotopically labeled DNA probes onto DNA molecules that were released from interphase cell nuclei or recombinant DNA clones, stretched to some extent and then immobilized on a solid support. The bound DNA, collectively called "DNA fibers," may consist of single DNA molecules in some experiments or bundles of chromatin fibers in others. Once released from the interphase nuclei, the DNA fibers become more accessible to probes and detection reagents. Hybridization efficiency is therefore increased, allowing the detection of DNA targets as small as a few hundred base pairs. This review summarizes different approaches to DNA fiber mapping and discusses the detection sensitivity and mapping accuracy as well as recent achievements in mapping expressed sequence tags and DNA replication sites.

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High-resolution in situ hybridization using DNA halo preparations

Cited by 169 publications

References 0 publications

Single-Step Multicolor Fluorescence In Situ Hybridization Using Semiconductor Quantum Dot-DNA Conjugates

Single-Step Multicolor Fluorescence In Situ Hybridization Using Semiconductor Quantum Dot-DNA Conjugates

The NFATc2 Gene Is Involved in a Novel Cloned Translocation in a Ewing Sarcoma Variant That Couples Its Function in Immunology to Oncology

DNA Fiber Mapping Techniques for the Assembly of High-resolution Physical Maps

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