DNA base sequence
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Druhá a třetí generace sekvenování DNA přinášejí nebo mají potenciál zlepšení důležitých parametrů jako je přesnost, rychlost nebo cena. Každá z konkrétních metod je zaměřena na různě dlouhé fragmenty DNA. Metody první generace vycházejí ze Sangerovy metody. Metody druhé generace k sekvenaci využívají stejný přístup jako generace první a to syntézu komplementárního vlákna, ale liší se principem získání signálu (pyrosekvenování), terminací (pyrosekvenování, sekvenace s využitím reversibilních terminátorů, sekvenace ligací), či způsobem syntézy komplementárního vlákna (sekvenace ligací). Třetí generace přináší i metody s jiným přístupem než je syntéza komplementárního vlákna a to sekvenování přímým zobrazením v elektronovém, či tunelovém mikroskopu a sekvenování pomocí přechodu DNA, nebo jednotlivých nukleotidů přes nanopór. Zatímco některé metody třetí generace jsou stále ve vývoji, jiné metody jsou již komerčně dostupné.
Second and third generation of DNA sequencing bring improvement in important parameters such as accuracy, speed and price. Every specific method is focused to the specific length of DNA fragments. The methods of first generation are based on Sanger‘s principle. The methods of second generation are based on the same approach towards sequencing as the first generation i.e. synthesis of complementary strands. Difference between the first and second generations is in the gaining signal (pyrosequencing), termination (pyrosequencing, The Solexa, SOLiD) or in manner of the sequencing complementary strand (SOLiD). The third generation brings methods with different approach, i.e. sequencing through direct display using electron or tunneling microscope and sequencing by DNA strand, or separate nucleotides passing nanopore. Some of the methods of the third generations are still in the development stage, some are already available.
- Klíčová slova
- první generace, druhá generace, třetí generace,
- MeSH
- lidé MeSH
- sekvenční analýza DNA * metody přístrojové vybavení trendy MeSH
- Check Tag
- lidé MeSH
We present the results of microsecond molecular dynamics simulations carried out by the ABC group of laboratories on a set of B-DNA oligomers containing the 136 distinct tetranucleotide base sequences. We demonstrate that the resulting trajectories have extensively sampled the conformational space accessible to B-DNA at room temperature. We confirm that base sequence effects depend strongly not only on the specific base pair step, but also on the specific base pairs that flank each step. Beyond sequence effects on average helical parameters and conformational fluctuations, we also identify tetranucleotide sequences that oscillate between several distinct conformational substates. By analyzing the conformation of the phosphodiester backbones, it is possible to understand for which sequences these substates will arise, and what impact they will have on specific helical parameters.
sv.
- MeSH
- DNA MeSH
- patenty jako téma * MeSH
- sekvence nukleotidů * MeSH
- Publikační typ
- periodika MeSH
- Konspekt
- Lékařské vědy. Lékařství
- NLK Obory
- genetika, lékařská genetika
The geometry of the phosphodiester backbone was analyzed for 7739 dinucleotides from 447 selected crystal structures of naked and complexed DNA. Ten torsion angles of a near-dinucleotide unit have been studied by combining Fourier averaging and clustering. Besides the known variants of the A-, B- and Z-DNA forms, we have also identified combined A + B backbone-deformed conformers, e.g. with alpha/gamma switches, and a few conformers with a syn orientation of bases occurring e.g. in G-quadruplex structures. A plethora of A- and B-like conformers show a close relationship between the A- and B-form double helices. A comparison of the populations of the conformers occurring in naked and complexed DNA has revealed a significant broadening of the DNA conformational space in the complexes, but the conformers still remain within the limits defined by the A- and B- forms. Possible sequence preferences, important for sequence-dependent recognition, have been assessed for the main A and B conformers by means of statistical goodness-of-fit tests. The structural properties of the backbone in quadruplexes, junctions and histone-core particles are discussed in further detail.
- MeSH
- A-DNA chemie MeSH
- cytosin chemie MeSH
- deoxyribonukleotidy chemie MeSH
- DNA vazebné proteiny chemie MeSH
- DNA chemie MeSH
- financování organizované MeSH
- G-kvadruplexy MeSH
- konformace nukleové kyseliny MeSH
- křížová struktura DNA chemie MeSH
- ligandy MeSH
- nukleozomy chemie MeSH
- RNA chemie MeSH
- sekvence nukleotidů MeSH
The formation of intercalated motifs (iMs) - secondary DNA structures based on hemiprotonated C.C+ pairs in suitable cytosine-rich DNA sequences, is reflected by typical changes in CD and UV absorption spectra. By means of spectroscopic methods, electrophoresis, chemical modifications and other procedures, we characterized iM formation and stability in sequences with different cytosine block lengths interrupted by various numbers and types of nucleotides. Particular attention was paid to the formation of iMs at pH conditions close to neutral. We identified the optimal conditions and minimal requirements for iM formation in DNA sequences, and addressed gaps and inaccurate data interpretations in existing studies to specify principles of iM formation and modes of their folding.
- MeSH
- cytosin chemie metabolismus MeSH
- DNA chemie metabolismus MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- konformace nukleové kyseliny * MeSH
- nukleotidové motivy * MeSH
- párování bází MeSH
- sekvence nukleotidů MeSH
- termodynamika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A nanopore-based devices are extremely sensitive analytical techniques, which uses the electrophoretic translocation of molecules in solution through a nano-scale pores. The nanopores, which mimic the functions of natural ion channels, seems to be the promissing tool for future fast and low-cost DNA sequencing. However, some difficulties in generating usable sequence data have to be solved. In this article the nanopores were reviewed. In the first part the development of nanopore technique was described and the ubiquitous presence of nanopores in living cells was highlighted. Next, the most important part of the principles of nanopore analysis was described, and the knowledges about biological and solid-state nanopores were summarized. Also the pros and cons of both kinds of nanopores and different approaches designed to circumvent the issues were mentioned.
- Klíčová slova
- biopóry, solid-state nanopóry,
- MeSH
- hemolyziny MeSH
- nanopóry * MeSH
- sekvenční analýza DNA * metody přístrojové vybavení MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
[Fe(2)L(3)](4+) (L = C(25)H(20)N(4)) is a synthetic tetracationic supramolecular cylinder (with a triple helical architecture) that targets the major groove of DNA and can bind to DNA Y-shaped junctions. To explore the DNA-binding mode of [Fe(2)L(3)](4+), we examine herein the interactions of pure enantiomers of this cylinder with DNA by biochemical and molecular biology methods. The results have revealed that, in addition to the previously reported bending of DNA, the enantiomers extensively unwind DNA, with the M enantiomer being the more efficient at unwinding, and exhibit preferential binding to regular alternating purine-pyrimidine sequences, with the M enantiomer showing a greater preference. Also, interestingly, the DNA binding of bulky cylinders [Fe(2)(L-CF(3))(3)](4+) and [Fe(2)(L-Ph)(3)](4+) results in no DNA unwinding and also no sequence preference of their DNA binding was observed. The observation of sequence-preference in the binding of these supramolecular cylinders suggests that a concept based on the use of metallosupramolecular cylinders might result in molecular designs that recognize the genetic code in a sequence-dependent manner with a potential ability to affect the processing of the genetic code.
- MeSH
- deoxyribonukleasa I MeSH
- DNA footprinting MeSH
- DNA chemie metabolismus MeSH
- ethidium chemie MeSH
- financování organizované MeSH
- kompetitivní vazba MeSH
- konformace nukleové kyseliny MeSH
- pyridiny chemie MeSH
- restrikční enzymy metabolismus MeSH
- sekvence nukleotidů MeSH
- stereoizomerie MeSH
- superhelikální DNA chemie MeSH
- železnaté sloučeniny chemie MeSH
The conductivity of DNA covalently bonded to a gold surface was studied by means of the STM technique. Various single- and double-stranded 32-nucleotide-long DNA sequences were measured under ambient conditions so as to provide a better understanding of the complex process of charge-carrier transport in natural as well as chemically modified DNA molecules. The investigations focused on the role of several features of DNA structure, namely the role of the negative charge at the backbone phosphate group and the related complex effects of counterions, and of the stacking interactions between the bases in Watson-Crick and other types of base pairs. The measurements have indicated that the best conductor is DNA in its biologically most relevant double-stranded form with Watson-Crick base pairs and charged phosphates equilibrated with counterions and water. All the studied modifications, including DNA with non-Watson-Crick base pairs, the abasic form, and especially the form with phosphate charges eliminated by chemical modifications, lower the conductivity of natural DNA.
- MeSH
- DNA chemie metabolismus MeSH
- elektrická vodivost MeSH
- financování organizované MeSH
- fosfáty chemie metabolismus MeSH
- jednovláknová DNA chemie MeSH
- oligodeoxyribonukleotidy chemie metabolismus MeSH
- párování bází MeSH
- rastrovací tunelová mikroskopie MeSH
- sekvence nukleotidů MeSH
- vodíková vazba MeSH
- zlato chemie MeSH
