Laser Microdissection and Laser Pressure Catapulting for the Generation of Chromosome-Specific Paint Probes

Schermelleh, Lothar; Thalhammer, Stefan; Heckl, Wolfgang M.; Pösl, H.; Cremer, Thomas; Schütze, Karin

doi:10.2144/99272rr04

Cited by 66 publications

(42 citation statements)

References 6 publications

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“…Alternatively, the isolation of sequences specific to chromosome fragments or whole chromosome painting probes by microdissection might be the method of choice to generate a complete set of chromosome specific probes. The feasibility of such an approach is widely reported in humans [3,29,36]. These probes would constitute promising tools for analysing the chromosomes of embryonic cells which are now widely used in biotechnology.…”

Section: Discussionmentioning

confidence: 99%

A repetitive probe for FISH analysis of bovine interphase nuclei

et al. 2000

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-The purpose of this study was to generate repetitive DNA sequence probes for the analysis of interphase nuclei by fluorescent in situ hybridisation (FISH). Such probes are useful for the diagnosis of chromosomal abnormalities in bovine preimplanted embryos. Of the seven probes (E1A, E4A, Ba, H1A, W18, W22, W5) that were generated and partially sequenced, five corresponded to previously described Bos taurus repetitive DNA (E1A, E4A, Ba, W18, W5), one probe (W22) shared no homology with other DNA sequences and one (H1A) displayed a significant homology with Rattus norvegicus mRNA for secretin receptor transmembrane domain 3. Fluorescent in situ hybridisation was performed on metaphase bovine fibroblast cells and showed that five of the seven probes hybridised most centromeres (E1A, E4A, Ba, W18, W22), one labelled the arms of all chromosomes (W5) and the H1A probe was specific to three chromosomes (ch14, ch20, and ch25). Moreover, FISH with H1A resulted in interpretable signals on interphase nuclei in 88% of the cases, while the other probes yielded only dispersed overlapping signals. la sonde W22 ne présenteà ce jour aucune homologie avec les séquences connues dans "Genbank" et la dernière, H1A (3,5 kb isolée après digestion par l'enzyme HindIII) présente une homologie significative sur 158 paires de base avec l'ARNm codant pour le 3 e domaine transmembranaire du récepteur de la secrétine de rat (Rattus norvegicus). L'hybridation in situ fluorescente sur des fibroblastes bovins en métaphase a montré que cinq sondes (E1A, E4A, Ba, W18, W22) hybrident la plupart des centromères, que la sonde W5 marque les bras de tous les chromosomes, et que la sonde H1A est spécifique de trois chromosomes bovins (ch14, ch20 et ch25). De plus, sur noyaux interphasiques, l'utilisation de H1A a permis d'obtenir des signaux interprétables dans 88 % des cas, contrairement aux autres sondes qui donnent des signaux superposés difficilesà interpréter. ADN satellite / FISH / bovin / centromère

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Section: Discussionmentioning

confidence: 99%

A repetitive probe for FISH analysis of bovine interphase nuclei

et al. 2000

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“…22 Another group of workers has reported the use of the ultraviolet microbeam microdissection method in combination with laser pressure catapulting to isolate single chromosomes for the development of chromosome specific paint probes. 23 …”

Section: Other Laser Based Microdissection Systemsmentioning

confidence: 99%

Laser Capture Microscopy

2002

Methods in Enzymology

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Human tissues are composed of complex admixtures of diVerent cell types and their biologically meaningful analysis necessitates the procurement of pure samples of the cells of interest. Many approaches have been used in attempts to overcome this diYculty, including a variety of microdissection methods. This review concerns a recent advance in microdissection techniques, namely laser capture microdissection (LCM). The principle underlying this technique is outlined, and practical issues pertaining to LCM are considered. In addition, the literature relating to LCM is reviewed, with examples of research applications of this technique being outlined. (J Clin Pathol: Mol Pathol 2000;53:64-68)

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“…The conventional approach uses fine extended glass needles for collection of whole chromosomes or subchromosomal regions (Meltzer et al 1992). In turn, laser microdissection employs a UV-laser microbeam in combination with laser-pressure catapulting (LPC) to isolate and eject the chromosome onto a collection device, speeding up the process (Schermelleh et al 1999).…”

Section: Introductionmentioning

confidence: 99%

Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

Passamani

Carvalho

Soares

2017

An. Acad. Bras. Ciênc.

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Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

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Laser Microdissection and Laser Pressure Catapulting for the Generation of Chromosome-Specific Paint Probes

Cited by 66 publications

References 6 publications

A repetitive probe for FISH analysis of bovine interphase nuclei

A repetitive probe for FISH analysis of bovine interphase nuclei

Laser Capture Microscopy

Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

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