Multicolor fluorescence in situ hybridization on metaphase chromosomes and interphase Halo-preparations using cosmid and YAC clones for the simultaneous high resolution mapping of deletions in the dystrophin gene

Tocharoentanaphol, Chintana; Cremer, Marion; Blonden, Lau; Kilian, Karin; Ried, Thomas

doi:10.1007/bf00212014

Cited by 25 publications

(15 citation statements)

References 29 publications

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“…[6][7][8][9]11 In each case, the carrier status (or disease status) could be correctly identified using FISH. However, there are some restrictions to the use of a FISH-based approach to carrier testing.…”

Section: Discussionmentioning

confidence: 99%

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Identification of female carriers for Duchenne and Becker muscular dystrophies using a FISH-based approach

et al. 2000

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Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular diseases caused by dystrophin gene mutations. Deletions, or more rarely duplications, of single or multiple exons within the dystrophin gene can be detected by current molecular methods in approximately 65% of DMD patients. Mothers of affected males have a two-thirds chance of carrying a dystrophin mutation, whilst approximately one-third of affected males have de novo mutations. Currently, Southern blot analysis and multiplex PCR directed against exons in deletion hot spots are used to determine female carrier status. However, both of these assays depend on dosage assessment to accurately identify carriers since, in females, the normal X chromosome is also present. To obviate quantitation of gene dosage, we have developed exon-specific probes from the dystrophin gene and applied them to a screen for potential carrier females using fluorescence in situ hybridization (FISH). Cosmid clones, representing 16 exons, were identified and used in FISH analysis of DMD/BMD families. Our preliminary work has identified multiple, informative probes for several families with dystrophin deletions and has shown that a FISH-based assay can be an effective and direct method for establishing the DMD/BMD carrier status of females.

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Section: Discussionmentioning

confidence: 99%

“…5 In limited studies, probes ranging in size from cDNAs to genomic inserts cloned into yeast artificial chromosomes (YACs) have been successfully applied towards detection of DMD deletions. [6][7][8][9] FISH offers a number of advantages, including:…”

Section: Introductionmentioning

confidence: 99%

Identification of female carriers for Duchenne and Becker muscular dystrophies using a FISH-based approach

et al. 2000

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“…It contains about 35 kb of the human dystrophin gene, including exon 48 (Tocharoentanaphol et al 1994). The insert is cloned in the BamHI site of the c2RB-derived vector p2CpG (approx.…”

Section: Methodsmentioning

confidence: 99%

High-resolution comparative hybridization to combed DNA fibers

et al. 1997

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Comparative genomic hybridization (CGH) has proven to be a comprehensive new tool to detect genetic imbalances in genomic DNA. However, the resolution of this method carried out on normal human metaphase spreads is limited to low copy number gains and losses of > or = 10 Mb. An improved resolution allowing the detection of copy number representations of single genes would strongly enhance the applicability of CGH as a diagnostic and research tool. This goal may be achieved when metaphase chromosomes are replaced by an array of target DNAs representing the genes of interest. To explore the feasibility of such a development in a model system we used cosmid MA2B3, which encompasses about 35 kb in the vicinity of exon 48 of the human dystrophin gene. Linearized cosmid fibers were attached to a glass surface and aligned in parallel by "molecular combing". Two-color fluorescence in situ suppression hybridization was performed on these cosmid fibers with probe mixtures containing different ratios (ranging from 1:2 to 4:1) of biotin- and digoxigenin-labeled MA2B3 cosmid DNAs. For each mixture fluorescence ratios were determined for 40-50 individual combed DNA molecules. In two series comprising a total of 651 molecules the median fluorescence ratio measurements revealed a linear relationship with the chosen probe ratios. Our study demonstrates that fluorescence ratio measurements on single DNA molecules can be performed successfully.

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“…FISH can detect cryptic chromosomal deletions and rearrangements, not detectable by conventional means: at the chromosomal level 25 26 and at the DNA fibre level 27 28 in both haploinsuYciency syndromes (for example, William's syndrome 26 ) or single gene disorders (for example, Duchenne's muscular dystrophy 28 ) + detection of gene fusion events in malignancy 29 + detection of hidden aneuploidy. 30 Specificity By using a particular probe or probes, chromosomal material of unknown or uncertain origin can be identified: + identification of marker chromosomes 31 + identification of chromosomal variants or polymorphisms.…”

Section: Sensitivitymentioning

confidence: 99%