hybrid capture
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Laserová záchytná mikrodisekce (Laser capture microdissection, LCM) je rychlá a spolehlivá metoda, která umožňuje izolaci cílových buněk ze specifického komplexu tkáně pro jejich následnou molekulární nebo proteinovou analýzu. Základem LCM je inverzní mikroskop se zabudovaným nízkovýkonnostním infračerveným laserem. Nařezané tkáně jsou upevněny na standardní podložní sklo a termoplastická membrána (TM) je umístuna nad dehydratovaný preparát. V ohnisku laserového mikroskopu je umístněna TM, kterou laser roztaví v požadovaném místě a naváže tak cílovou buňku či strukturu k membráně. V současné době máme k dispozici několik laserových mikrodisekčních systémů, které se liší způsobem zachycení disekovaných buněk, v konfiguraci systému i v jednotlivých aplikacích. Laserovou mikrodisekci lze použít pro izolaci buněk u řady typů buněčných i tkáňových preparátů, včetně zamražených vzorků, formalínem fixovaných parafinizovaných tkání či cytologických preparátů. V závislosti na použitém materiálu je možno z mikrodisekovaných buněk extrahovat DNA, RNA či proteiny v dobré kvalitě. Kombinací s dalšími technikami, jako je například cDNA microarray, LCM pomáhá identifikovat nové diagnostické a prognostické znaky vedoucí ke zlepšení diagnosticko terapeutických metod v léčbě onkologických onemocnění. Na našem pracovišti jsme laserovou mikrodisekcí izolovali buňky z cytologických preparátů adenokarcinomu plic získaných punkční cytologií zmražených či parafinizovaných nádorů a také buněčné linie, např. myeloidní leukemii K562. V této práci popisujeme naše zkušenosti se zavedením LCM s následnou mikroizolací DNA/RNA a lineární amplifikací DNA/RNA pro účely dalších genetických analýz jako je např. komparativní genomická hybridizace, expresní studie, či přímé sekvenování vyšetřovaných genů z biologického materiálu s minimálním obsahem nádorových buněk, či pro studium nádorové heterogenity na jednobuněčné úrovni.
Laser capture microdissection (LCM) is a rapid, reliable method to obtain pure populations of targeted cells from specific microscopic regions of tissue sections for subsequent analysis. LCM is based on the adherence of visually selected cells to a thermoplastic membrane, which overlies the dehydrated tissue section and is focally melted by triggering of a low energy infrared laser pulse. Tissue sections are mounted on standard glass slides, and transparent thermoplastic membrane is then placed over the dry section. The laser provides enough energy to transiently melt this thermoplastic film in to the target cells. Several systems are available for LCM, and vary in cell-capture method, system configuration and applications. LCM was applied to a wide range of cell and tissue preparations including frozen samples, formalin-fixed paraffin-embedded tissues or cytology smears. Depending on the starting material, DNA, good quality mRNA, and proteins can by extracted successfully from captured tissue fragments, down to the single cell level. In combination with another techniques like expression library construction and cDNA array hybridisation, LCM will allow the establishment of new diagnostic and prognostic markers, in order to indicate therapy individually tailored to the molecular profile of a given tumour. In this paper we refer our experiences with the LCM isolation of single cells from cytology smears of lung carcinomas, frozen and paraffin embedded tumour tissues as well as cell line cytospin preparation. Our ultimate goal was to introduce LCM technology in combination with DNA/RNA isolation and linear amplification for subsequent genomic analyses such as comparative genomic hybridisation, RNA expression studies and specific amplifications of investigated genes from tissue specimens with minority of tumour cells and/or for tumour heterogeneity studies based on one the single cell level.
- MeSH
- erbB receptory genetika izolace a purifikace MeSH
- financování organizované MeSH
- geny erbB-1 genetika MeSH
- geny ras genetika MeSH
- hybridizace genetická MeSH
- lasery využití MeSH
- lékařská onkologie metody trendy MeSH
- lidé MeSH
- mikrodisekce metody přístrojové vybavení využití MeSH
- odběr biologického vzorku metody využití MeSH
- srovnávací genomová hybridizace MeSH
- techniky amplifikace nukleových kyselin metody přístrojové vybavení využití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 106-10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 102 CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.
- MeSH
- Escherichia coli O157 klasifikace genetika MeSH
- hybridizace nukleových kyselin MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- Listeria monocytogenes klasifikace genetika MeSH
- multiplexová polymerázová řetězová reakce MeSH
- Salmonella klasifikace genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Chromosomal inversions occur in natural populations of many species, and may underlie reproductive isolation and local adaptation. Traditional methods of inversion discovery are labor-intensive and lack sensitivity. Here, we report the use of three-dimensional contact probabilities between genomic loci as assayed by chromosome-conformation capture sequencing (Hi-C) to detect multi-megabase polymorphic inversions in four barley genotypes. Inversions are validated by fluorescence in situ hybridization and Bionano optical mapping. We propose Hi-C as a generally applicable method for inversion discovery in natural populations.
- MeSH
- financování organizované MeSH
- genetické techniky využití MeSH
- hybridizace genetická genetika MeSH
- lasery využití MeSH
- mikrodisekce metody využití MeSH
- mikroskopie metody trendy využití MeSH
- molekulární biologie metody MeSH
- nádory ultrastruktura MeSH
- techniky amplifikace nukleových kyselin metody využití MeSH
Four accessions of hexaploid Elymus repens from its native Central European distribution area were analyzed using sequencing of multicopy (internal transcribed spacer, ITS) and single-copy (granule-bound starch synthase I, GBSSI) DNA in concert with genomic and fluorescent in situ hybridization (GISH and FISH) to disentangle its allopolyploid origin. Despite extensive ITS homogenization, nrDNA in E. repens allowed us to identify at least four distinct lineages. Apart from Pseudoroegneria and Hordeum, representing the major genome constituents, the presence of further unexpected alien genetic material, originating from species outside the Triticeae and close to Panicum (Paniceae) and Bromus (Bromeae), was revealed. GBSSI sequences provided information complementary to the ITS. Apart from Pseudoroegneria and Hordeum, two additional gene variants from within the Triticeae were discovered: One was Taeniatherum-like, but the other did not have a close relationship with any of the diploids sampled. GISH results were largely congruent with the sequence-based markers. GISH clearly confirmed Pseudoroegneria and Hordeum as major genome constituents and further showed the presence of a small chromosome segment corresponding to Panicum. It resided in the Hordeum subgenome and probably represents an old acquisition of a Hordeum progenitor. Spotty hybridization signals across all chromosomes after GISH with Taeniatherum and Bromus probes suggested that gene acquisition from these species is more likely due to common ancestry of the grasses or early introgression than to recent hybridization or allopolyploid origin of E. repens. Physical mapping of rDNA loci using FISH revealed that all rDNA loci except one minor were located on Pseudoroegneria-derived chromosomes, which suggests the loss of all Hordeum-derived loci but one. Because homogenization mechanisms seem to operate effectively among Pseudoroegneria-like copies in this species, incomplete ITS homogenization in our samples is probably due to an interstitial position of an individual minor rDNA locus located within the Hordeum-derived subgenome.
- MeSH
- Bayesova věta MeSH
- cytogenetické vyšetření metody MeSH
- databáze genetické MeSH
- fylogeneze MeSH
- genetická transkripce MeSH
- hybridizace in situ fluorescenční MeSH
- intergenová DNA MeSH
- lipnicovité genetika MeSH
- modely genetické MeSH
- přenos genů horizontální MeSH
- pseudogeny MeSH
- ribozomální DNA MeSH
- rostlinné geny MeSH
- synthasa škrobu genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hybrid QM/MM methods combine the rigor of quantum mechanical (QM) calculations with the low computational cost of empirical molecular mechanical (MM) treatment allowing to capture dynamic properties to probe critical atomistic details of enzyme reactions. Catalysis by RNA enzymes (ribozymes) has only recently begun to be addressed with QM/MM approaches and is thus still a field under development. This review surveys methodology as well as recent advances in QM/MM applications to RNA mechanisms, including those of the HDV, hairpin, and hammerhead ribozymes, as well as the ribosome. We compare and correlate QM/MM results with those from QM and/or molecular dynamics (MD) simulations, and discuss scope and limitations with a critical eye on current shortcomings in available methodologies and computer resources. We thus hope to foster mutual appreciation and facilitate collaboration between experimentalists and theorists to jointly advance our understanding of RNA catalysis at an atomistic level.
- MeSH
- biofyzika metody MeSH
- fosfáty chemie MeSH
- fosforylace MeSH
- hořčík chemie MeSH
- katalýza MeSH
- konformace nukleové kyseliny MeSH
- kvantová teorie MeSH
- lidé MeSH
- molekulární modely MeSH
- počítačová simulace MeSH
- ribozomy chemie MeSH
- RNA katalytická chemie MeSH
- RNA virová chemie MeSH
- RNA chemie MeSH
- software MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
- Research Support, N.I.H., Extramural MeSH
Hybridization and incomplete lineage sorting are common confounding factors in phylogeny and speciation resulting in mitonuclear disparity. Mitochondrial introgression, a particular case of hybridization, may, in extreme cases, lead to replacement of the mitochondrial genome of one species with that of another (mitochondrial capture). We investigated mitochondrial introgression involving two species of the cyprinid genus Squalius in the western Peloponnese region of Greece using molecular and morphological data. We found evidence of complete mitochondrial introgression of Squalius keadicus into two populations recognized as Squalius peloponensis from the Miras and Pamissos River basins and a divergence of mitochondrial genomes of S. keadicus from the Evrotas basin from that of the introgressed populations dating from the Pleistocene. Secondary contact among basins is a possible factor in connection of the species and the introgression event. Morphological analyses support the hypothesis of mitochondrial introgression, as S. keadicus was different from the other three populations recognized as S. peloponensis, although significant differences were found among the four populations. Isolation by geographical barriers arose during Pleistocene in the western Peloponnese were the source of the evolution of the two reciprocally monophyletic subclades found in the S. keadicus mitochondrial clade, and the morphological differences found among the four populations. Along with the lack of structure in the nuclear genome in the three populations ascribed to S. peloponensis, this suggests an incipient speciation process occurring in these Squalius species in the western Peloponnese.
- MeSH
- Cyprinidae genetika MeSH
- fylogeneze MeSH
- fylogeografie MeSH
- genom mitochondriální MeSH
- hybridizace genetická MeSH
- mitochondriální DNA genetika MeSH
- mitochondrie genetika MeSH
- molekulární evoluce * MeSH
- sekvenční analýza DNA MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Hybridogenesis is a reproductive tool for sexual parasitism. Hybridogenetic hybrids use gametes from their sexual host for their own reproduction, but sexual species gain no benefit from such matings as their genome is later eliminated. Here, we examine the presence of sexual parasitism in water frogs through crossing experiments and genome-wide data. We specifically focus on the famous Central-European populations where Pelophylax esculentus males (hybrids of P. ridibundus and P. lessonae) live with P. ridibundus. We identified a system where the hybrids commonly produce two types of clonal gametes (hybrid amphispermy). The haploid lessonae genome is clonally inherited from generation to generation and assures the maintenance of hybrids through a process, in which lessonae sperm fertilize P. ridibundus eggs. The haploid ridibundus genome in hybrids received from P. ridibundus a generation ago, is perpetuated as clonal ridibundus sperm and used to fertilize P. ridibundus eggs, yielding female P. ridibundus progeny. These results imply animal reproduction in which hybridogenetic taxa are not only sexual parasites, but also participate in the formation of a sexual taxon in a remarkable way. This occurs through a process by which sexual gametes are being captured, converted to clones, and returned to sexual populations in one generation.
- MeSH
- analýza hlavních komponent MeSH
- genetické lokusy MeSH
- genom * MeSH
- haploidie MeSH
- mikrosatelitní repetice genetika MeSH
- Rana esculenta genetika MeSH
- Rana ridibunda genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Distinguishing between hybrid introgression and incomplete lineage sorting causing incongruence among gene trees in that they exhibit topological differences requires application of statistical approaches that are based on biologically relevant models. Such study is especially challenging in hybrid systems, where usual vectors mediating interspecific gene transfers--hybrids with Mendelian heredity--are absent or unknown. Here we study a complex of hybridizing species, which are known to produce clonal hybrids, to discover how one of the species, Cobitis tanaitica, has achieved a pattern of mito-nuclear mosaic genome over the whole geographic range. We appplied three distinct methods, including the method using solely the information on gene tree topologies, and found that the contrasting mito-nuclear signal might not have resulted from the retention of ancestral polymorphism. Instead, we found two signs of hybridization events related to C. tanaitica; one concerning nuclear gene flow and the other suggested mitochondrial capture. Interestingly, clonal inheritance (gynogenesis) of contemporary hybrids prevents genomic introgressions and non-clonal hybrids are either absent or too rare to be detected among European Cobitis. Our analyses therefore suggest that introgressive hybridizations are rather old episodes, mediated by previously existing hybrids whose inheritance was not entirely clonal. Cobitis complex thus supports the view that the type of resulting hybrids depends on a level of genomic divergence between sexual species.
