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Autoři uvádějí své zkušenosti s neradioaktivní DNA-diagnostikou Duchennovy a Beckerovy muskulární dystrofie, při které využili polymorfismu krátkých tandemových repeticí ze čtyř intronových oblastí (introny 44, 45, 49, 50) a dvou oblastí 5'-konce dystrofínového genu. Oblasti s tandemovými repeticemi byly amplifikovány pomocí polymerázové řetězové reakce a po rozdělení v denaturačním polyakrylamidovém gelu byly identifikovány barvením stříbrem. Předkládané výsledky ukazují spolehlivost a jednoduchost neradioaktivní diagnostiky, kterou je možno zavést v každé DNA-laboratoři.
The authors present their experience with non-radioactive diagnostics of Duchenne and Becker muscular dystrophy using short tandem repeat polymorphism from four intron loci (introns 44, 45, 49, 50) and two 5'terminus regions of the dystrophine gene. Regions with tandem repeats were amplified by the polymerase chain reaction and after senaration in denaturin£f polvacrylamide gel identified by silver staining. The results presented show the reliability and simplicity of non-radioactive diagnostics, which could be introduced in every DNA-laboratory.
- MeSH
- barvení stříbrem MeSH
- dítě MeSH
- DNA analýza genetika MeSH
- dospělí MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- genetické testování metody normy statistika a číselné údaje MeSH
- introny MeSH
- lidé MeSH
- minisatelitní repetice genetika MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
Východisko. Dynamické mutace lidského genomu jsou novou třídou genových mutací, reprezentovaných nestabilním počtem trinukleotidových opakování, způsobující závažná dědičná neuromuskulární a neurodegenerativní onemocnění u lidí. Identifikace patologických expandovaných alel na molekulární úrovni je důležitá pro klinickou diagnózu. Metody a výsledky. Pro molekulární diagnostiku expandovaných tandemových repeticí trinukleotidových sekvencí jsme zavedli efektivní TP-PCR metodu podleWarnera et al. (13). Pro rychlou detekci expandovaných CTG alel genu DMPK (myotonická dystrofie –MD) jsme tutometodu modifikovali do dvoufázového protokolu; nejprve byly pomocí PCR selektovány vzorky DNA heterozygotů s využitím primerů P1 a P2, ohraničujících trakt s repeticemi, druhé vyšetření metodou TP-PCR bylo zaměřeno především na identifikaci patologické alely. Fluorescenčně značený specifický primer v TP-PCR byl použit pro přesné určení počtu CAGopakování v genu IT-15 (Huntingtonova choroba – HD) v diagnosticky důležité oblasti šedé zóny (rozmezí 35–39 CAG). Reprodukovatelnost výsledků PCR byla ověřena na kontrolních vzorcích DNA o známém genotypu a v případě MD také Southernovou hybridizací. Zejména jsme ukázalimožnost levnějšího PCR-P1/P2 a TP-PCR protokolu, který využívá barvení PCRproduktů separovaných na polyakrylamidových gelech stříbrem. Závěr. Naše zkušenosti se zavedenímvýše uvedených PCRmetod do laboratorní praxe dokumentují obecnémožnosti jejich významného uplatnění pro molekulární diagnostiku dědičných chorob charakterizovaných nestabilitou v trinukleotidových traktech.
Background. Human genome dynamic mutations are a new class of gene mutations represented by an unstable number of trinucleotide repeats and causing severe human hereditary neuromuscular and neurodegenerative diseases. The identification of pathological expanded alleles on the molecular level is important for clinical diagnostics. Methods and Results. For the molecular diagnostics of expanded tandem repeat trinucleotide sequences we have introduced a fast and efficient TP-PCR fluorescent method according toWarner et al. (1996).We have modified this TP-PCR method for a rapid detection of expanded CTG alleles of the DMPK gene (myotonic dystrophy, MD) into a two-level protocol; first, the heterozygote sample DNAs were selected using P1/P2 primers flanking repeat tracts and, second, the TP-PCR protocol used was focused above all on the identification of a pathological allele. A fluorescent-labelled specific primer in TP-PCR was used for the exact determination of the number of CAG repeats of the gene IT-15 (Huntington’s disease – HD) in the diagnostically important region of the grey zone (35 to 39 CAG). The reproducibility of the PCR results was demonstrated on control DNA samples with the known genotype and, in the case of MD, also by Southern blot analysis. We have especially shown the possibility of a cheaper PCR-P1/P2 and TP-PCR protocol, which can be used, with silver staining of separated PCR products on polyacrylamide gels. Conclusions. Our experience with introducing the above-mentioned PCR methods into laboratory practice clearly documents the possibilities of their general applicability in the molecular diagnostics of hereditary diseases characterised by instability of the trinucleotide repeat tracts.
DNA is a fundamentally important molecule for all cellular organisms due to its biological role as the store of hereditary, genetic information. On the one hand, genomic DNA is very stable, both in chemical and biological contexts, and this assists its genetic functions. On the other hand, it is also a dynamic molecule, and constant changes in its structure and sequence drive many biological processes, including adaptation and evolution of organisms. DNA genomes contain significant amounts of repetitive sequences, which have divergent functions in the complex processes that involve DNA, including replication, recombination, repair, and transcription. Through their involvement in these processes, repetitive DNA sequences influence the genetic instability and evolution of DNA molecules and they are located non-randomly in all genomes. Mechanisms that influence such genetic instability have been studied in many organisms, including within human genomes where they are linked to various human diseases. Here, we review our understanding of short, simple DNA repeats across a diverse range of bacteria, comparing the prevalence of repetitive DNA sequences in different genomes. We describe the range of DNA structures that have been observed in such repeats, focusing on their propensity to form local, non-B-DNA structures. Finally, we discuss the biological significance of such unusual DNA structures and relate this to studies where the impacts of DNA metabolism on genetic stability are linked to human diseases. Overall, we show that simple DNA repeats in bacteria serve as excellent and tractable experimental models for biochemical studies of their cellular functions and influences.
- MeSH
- Bacteria genetika MeSH
- DNA genetika ultrastruktura MeSH
- genom bakteriální genetika MeSH
- genom lidský genetika MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- mikrosatelitní repetice genetika MeSH
- nestabilita genomu genetika MeSH
- repetitivní sekvence nukleových kyselin genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Reference genomes of important cereals, including barley, emmer wheat and bread wheat, were released recently. Their comparison with genome size estimates obtained by flow cytometry indicated that the assemblies represent not more than 88-98% of the complete genome. This work is aimed at identifying the missing parts in two cereal genomes and proposing techniques to make the assemblies more complete. We focused on tandemly organised repetitive sequences, known to be underrepresented in genome assemblies generated from short-read sequence data. Our study found arrays of three tandem repeats with unit sizes of 1242 to 2726 bp present in the bread wheat reference genome generated from short reads. However, this and another wheat genome assembly employing long PacBio reads failed in integrating correctly the 2726-bp repeat in the pseudomolecule context. This suggests that tandem repeats of this size, frequently incorporated in unassigned scaffolds, may contribute to shrinking of pseudomolecules without reducing size of the entire assembly. We demonstrate how this missing information may be added to the pseudomolecules with the aid of nanopore sequencing of individual BAC clones and optical mapping. Using the latter technique, we identified and localised a 470-kb long array of 45S ribosomal DNA absent from the reference genome of barley.
Ever since the introduction of high-throughput sequencing following the human genome project, assembling short reads into a reference of sufficient quality posed a significant problem as a large portion of the human genome-estimated 50-69%-is repetitive. As a result, a sizable proportion of sequencing reads is multi-mapping, i.e., without a unique placement in the genome. The two key parameters for whether or not a read is multi-mapping are the read length and genome complexity. Long reads are now able to span difficult, heterochromatic regions, including full centromeres, and characterize chromosomes from "telomere to telomere". Moreover, identical reads or repeat arrays can be differentiated based on their epigenetic marks, such as methylation patterns, aiding in the assembly process. This is despite the fact that long reads still contain a modest percentage of sequencing errors, disorienting the aligners and assemblers both in accuracy and speed. Here, I review the proposed and implemented solutions to the repeat resolution and the multi-mapping read problem, as well as the downstream consequences of reference choice, repeat masking, and proper representation of sex chromosomes. I also consider the forthcoming challenges and solutions with regards to long reads, where we expect the shift from the problem of repeat localization within a single individual to the problem of repeat positioning within pangenomes.
- MeSH
- centromera chemie MeSH
- délka genomu MeSH
- genom lidský * MeSH
- lidé MeSH
- mapování chromozomů metody MeSH
- metylace DNA MeSH
- mikrosatelitní repetice * MeSH
- pohlavní chromozomy chemie MeSH
- telomery chemie MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The (GA)(n) microsatellite has been known from previous studies to adopt unusual, ordered, cooperatively melting secondary structures in neutral aqueous solutions containing physiological concentrations of salts, at acid pH values or in aqueous ethanol solutions. To find more about the primary structure specificity of these structures, we performed parallel comparative studies of related tetranucleotide repeats (GAGC)(5), (GAGT)(5), and (GACA)(5). The general conclusion following from these comparative studies is that the primary structure specificity is fairly high, indicating that not only guanines but also adenines play a significant role in the stabilization of these unusual structures. (GAGC)(5) is a hairpin or a duplex depending on DNA concentration. Neither acid pH nor ionic strength or the presence of ethanol changed the secondary structure of (GAGC)(5) in a significant way. (GACA)(5) forms a weakly stable hairpin in neutral aqueous solutions but forms a duplex at acid pH where cytosine is protonated. (GAGT)(5) behaves most similar to (GAGA)(5). Salt induces its hairpin to duplex transition at neutral pH and an isomerization into another, probably parallel stranded, duplex takes place at acid pH. (GAGT)(5) is the only of the three present 20-mers that responds to ethanol like (GAGA)(5).
Ankyrin repeat proteins (ARPs) appear to be abundant in organisms from all phyla, and play critical regulatory roles, mediating specific interactions with target biomolecules and thus ordering the sequence of events in diverse cellular processes. ARPs possess a non-globular scaffold consisting of repeating motifs named ankyrin (ANK) repeats, which stack on each other. The modular architecture of ARPs provides a new paradigm for understanding protein stability and folding mechanisms. In the present study, the stability of various C-terminal fragments of the ARP p18(INK4c) was investigated by all-atomic 450 ns molecular dynamics (MD) simulations in explicit water solvent. Only motifs with at least two ANK repeats made stable systems in the available timescale. All smaller fragments were unstable, readily losing their native fold and alpha-helical content. Since each non-terminal ANK repeat has two hydrophobic sides, we may hypothesize that at least one hydrophobic side must be fully covered and shielded from the water as a necessary, but not sufficient, condition to maintain ANK repeat stability. Consequently, at least two ANK repeats are required to make a stable ARP.
The study of fish cytogenetics has been impeded by the inability to produce G-bands that could assign chromosomes to their homologous pairs. Thus, the majority of karyotypes published have been estimated based on morphological similarities of chromosomes. The reason why chromosome G-banding does not work in fish remains elusive. However, the recent increase in the number of fish genomes assembled to the chromosome level provides a way to analyse this issue. We have developed a Python tool to visualize and quantify GC percentage (GC%) of both repeats and unique DNA along chromosomes using a non-overlapping sliding window approach. Our tool profiles GC% and simultaneously plots the proportion of repeats (rep%) in a color scale (or vice versa). Hence, it is possible to assess the contribution of repeats to the total GC%. The main differences are the GC% of repeats homogenizing the overall GC% along fish chromosomes and a greater range of GC% scattered along fish chromosomes. This may explain the inability to produce G-banding in fish. We also show an occasional banding pattern along the chromosomes in some fish that probably cannot be detected with traditional qualitative cytogenetic methods.
- MeSH
- genom * MeSH
- Gorilla gorilla klasifikace genetika MeSH
- karyotypizace metody MeSH
- kočky MeSH
- mapování chromozomů metody statistika a číselné údaje MeSH
- pruhování chromozomů MeSH
- ryby klasifikace genetika MeSH
- software * MeSH
- tandemové repetitivní sekvence MeSH
- zastoupení bazí * MeSH
- zvířata MeSH
- Check Tag
- kočky MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We have used CD spectroscopy, polyacrylamide gel electrophoresis, and UV absorption spectroscopy to study conformational properties of DNA fragments containing (CCA)n and (TGG)n repeats, which are the most length-polymorphic microsatellite sequences of the human genome. The (CCA)n fragments are random single strands at neutral and alkaline pH but they fold into intramolecular intercalated cytosine tetraplexes at mildly acid pH values. More acid values stabilize intermolecular tetraplex formation. The behavior of (TGG)n repeats is more complex. They form hairpins or antiparallel homoduplexes in low salt solutions which, however, are transformed into parallel-stranded guanine tetraplexes at physiological KCl concentrations. Their molecularity depends on the repeat number: (TGG)4 associates into an octameric complex, (TGG)8 forms tetramolecular complexes. (TGG)n with odd repeat numbers (5, 7, and 9) generate bimolecular and tetramolecular tetraplexes. The only (TGG)7 folds into an intramolecular tetraplex at low KCl concentrations, which is antiparallel-stranded. Moreover, the (TGG)(n) fragments provide various mutually slipped conformers whose population increases with salt concentration and with the increasing repeat number. However, the self-structures of both strands disappear in the presence of the complementary strand because both (TGG)n and (CCA)n prefer to associate into the classical heteroduplex. We suppose that the extreme conformational variability of the DNA strands stands behind the length polymorphism which the (CCA)n/(TGG)n repeats exhibit in the human genome.
- MeSH
- chlorid draselný farmakologie MeSH
- cirkulární dichroismus MeSH
- cytosin chemie MeSH
- denaturace nukleových kyselin MeSH
- DNA chemie MeSH
- EDTA chemie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- financování organizované MeSH
- genom lidský MeSH
- koncentrace vodíkových iontů MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- lidé MeSH
- mikrosatelitní repetice MeSH
- oligonukleotidy MeSH
- polymorfismus genetický MeSH
- soli farmakologie MeSH
- spektrofotometrie MeSH
- teplota MeSH
- trinukleotidové repetice MeSH
- ultrafialové záření MeSH
- Check Tag
- lidé MeSH
