共查询到20条相似文献,搜索用时 31 毫秒
1.
Cloning DNA typically involves the joining of target DNAs with vector constructs by enzymatic ligation. A commonly used enzyme for this reaction is bacteriophage T4 DNA ligase, which requires ATP as the energy source to catalyze the otherwise unfavorable formation of a phosphodiester bond. Using in vitro selection, we have isolated a DNA sequence that catalyzes the ligation of DNA in the absence of protein enzymes. We have used the action of two catalytic DNAs, an ATP-dependent self-adenylating deoxyribozyme (AppDNA) and a self-ligating deoxyribozyme, to create a ligation system that covalently joins oligonucleotides via the formation of a 3',5'-phosphodiester linkage. The two-step process is conducted in separate reaction vessels wherein the products of deoxyribozyme adenylation are purified before their use as substrates for deoxyribozyme ligation. The final ligation step of the deoxyribozyme-catalyzed sequence of reactions mimics the final step of the T4 DNA ligase reaction. The initial rate constant (k(obs)) of the optimized deoxyribozyme ligase was found to be 1 x 10(-)(4) min(-)(1). Under these conditions, the ligase deoxyribozyme promotes DNA ligation at least 10(5)-fold faster than that generated by a simple DNA template. The self-ligating deoxyribozyme has also been reconfigured to generate a trans-acting construct that joins separate DNA oligonucleotides of defined sequence. However, the sequence requirements of the AppDNA and that of the 3' terminus of the deoxyribozyme ligase limit the range of sequences that can be ligated. 相似文献
2.
Keller S Wang J Chandra M Berger R Marx A 《Journal of the American Chemical Society》2008,130(40):13188-13189
The distinct base pairing property of DNA is an advantageous phenomenon that has been exploited in the usage of DNA as scaffold for directed self-organization to form nanometer-sized objects in a desirable fashion. Herein we report the construction of three-dimensional DNA-based networks that can be generated and amplified by the DNA polymerase chain reaction (PCR). The approach is flexible allowing tuning of the meshes of the network by variation of the size of the template. Additionally, further diversification can be introduced by employment of chemically modified nucleotides in PCR allowing the introduction of functionalities and reporter moieties. 相似文献
3.
DNA is currently explored as a new material for functional, molecular nano-architectures. In this respect, one major question is to transform DNA into a conducting material which has the potential for self-assembly into electronically active networks. The article covers recent insight into how DNA transports positive (holes) and negative (excess electrons) charges. It was found that holes move through DNA over significant distances using a G- and to a lesser extent also A-based hopping mechanism. EPR studies and recent investigations with model systems show that excess electrons can also hop through the duplex. The second part of the article describes how DNA is currently modified, particularly coated with metals, in order to increase the conductivity. 相似文献
4.
Background
Alternative DNA conformations are of particular interest as potential signals to mark important sites on the genome. The structural variability of CA microsatellites is particularly pronounced; these are repetitive poly(CA) · poly(TG) DNA sequences spread in all eukaryotic genomes as tracts of up to 60 base pairs long. Many in vitro studies have shown that the structure of poly(CA) · poly(TG) can vary markedly from the classical right handed DNA double helix and adopt diverse alternative conformations. Here we have studied the mechanism of formation and the structure of an alternative DNA structure, named Form X, which was observed previously by polyacrylamide gel electrophoresis of DNA fragments containing a tract of the CA microsatellite poly(CA) · poly(TG) but had not yet been characterized. 相似文献5.
《Analytical letters》2012,45(12):1897-1927
Abstract Recently there has been an increasing demand to produce systems for the detection of specific DNA sequences that are amenable to non-specialised laboratories. This demand has led to many innovative and novel approaches to DNA analysis which may collectively be termed ′new DNA technology′. Here, we review these advances in relation to their applicability for the production of a DNA biosensor. The present state of the art is described and future possibilities are considered. 相似文献
6.
Betsy M. Sutherland Giovanni Ciarrocchi Marina Ciomei John C. Sutherland 《Photochemistry and photobiology》1986,44(3):391-396
Three principal methods have been developed for measuring femtomoles of damage in nanogram quantities of non-radioactive DNA. Lesions which can be quantified include single and double strand breaks, alkali labile sites including apurinic and apyrimidinic sites, and pyrimidine dimers. The first in vitro method measures the conversion of supercoiled DNA to relaxed or linear molecules, and can detect up to four lesions per molecule. The second in vitro method (supercoil depletion) assesses the fraction of intact linear molecules of homogeneous length, and allows detection of 8 lesions/molecule. The third method, measurement of molecular length distributions of DNAs of heterogeneous length, reveals the extent of DNA damage and repair in vivo or in vitro. 相似文献
7.
We report the new method for detection of DNA hybridization using enzymatic cleavage. The strategy is based on that S1 nuclease is able to specifically cleave only single strand DNA, but not double strand DNA. The capture probe DNA, thiolated single strand DNA labeled with electroactive ferrocene group, was immobilized on a gold electrode. After hybridization of target DNA of complementary and noncomplementary sequences, nonhybridized single strand DNA was cleaved using S1 nuclease. The difference of enzymatic cleavage on the modified gold electrode was characterized by cyclic voltammetry and differential pulse voltammetry. We successfully applied this method to the sequence‐selective discrimination between perfectly matched and mismatched target DNA including a single‐base mismatched target DNA. Our method does not require either hybridization indicators or other exogenous signaling molecules which most of the electrochemical hybridization detection systems require. 相似文献
8.
Determination of the sequence of DNA is one of the most important aspects of modern molecular biology. New sequencing methods currently being developed enable DNA sequence to be determined increasingly faster and more efficiently. One of the major advances in sequencing technology is the development of automated DNA sequencers. These utilize fluorescent rather than radioactive labels. A laser beam excites the fluorescent dyes, the emitted fluorescence is collected by detectors, and the information analyzed by computer. Robotic work stations are being developed to perform template preparation and purification, and the sequencing reactions themselves. Research is currently in progress to develop the technology of mass spectrometry for DNA sequencing. Success in this endeavor would mean that the gel electrophoresis step in DNA sequencing could be eliminated. A major innovation has been the application of polymerase chain reaction (PCR) technology to DNA sequence determination, which has led to the development of linear amplification sequencing (cycle sequencing). This very powerful yet technically simple method of sequencing has many advantages over conventional techniques, and may be used in manual or automated methods. Other recent innovations proposed recently to increase speed and efficiency include multiplex sequencing. This consists of pooling a number of samples and processing them as pools. After electrophoresis, the DNA is transferred to a membrane, and sequence images of the individual samples are obtained by sequential hybridizations with specific labeled oligonucleotides. Multiplex DNA sequencing has been used in conjunction with direct blotting electrophoresis to facilitate transfer of the DNA to a membrane. Chemiluminescent detection can also be used in conjunction with multiplex DNA sequencing to visualize the image on the membrane. 相似文献
9.
Christof M. Niemeyer Dietmar Blohm 《Angewandte Chemie (International ed. in English)》1999,38(19):2865-2869
The complete human genes (ca. 100 000) as well as the whole spectrum of biological diversity should soon be able to be analyzed simultaneously by means of DNA microarrays using the fast technical advances that are occurring in this area. The particular strength of array analysis, typically based on the hybridization of nucleic acid probes attached to microchips with labeled RNA or DNA samples, results from the highly redundant measurement of many parallel hybridization events (see picture), which leads to an extraordinary level of assay validation. 相似文献
10.
TNA, or threose nucleic acid, is capable of Watson-Crick base pairing with DNA, RNA, and TNA; coupled with its chemical simplicity, this suggests that TNA is a possible progenitor of RNA. As an initial step toward developing the molecular tools necessary to investigate the functional capabilities of TNA by in vitro selection, we have screened a variety of DNA polymerases for activity on a TNA template. We report that despite having a repeating unit that is one atom shorter than that of DNA, several polymerases showed surprisingly good ability to copy limited stretches of TNA. 相似文献
11.
The high fidelity detection of multiple DNA sequences in multiplex assays calls for duplexes whose stability is independent of sequence (isostable DNA), forming under universally stringent conditions. Nature did not evolve DNA to form isostable duplexes. Here we report how probe strands can be modified so that an all-A/T target strand is bound with the same or slightly higher affinity than the corresponding all-G/C strand with the same sequence of purines and pyrimidines. We refer to these probes that feature covalently attached ligands as "decorated nucleic acids". Caps, intercalators, and locks were used to stabilize A/T duplexes, and N4-ethylcytosine residues were employed to tune down the stability of G/C duplexes without significantly affecting base pairing selectivity. Near-isostability was demonstrated in solution and on microarrays of high and low density. Further, it is shown that hybridization results involving decorated probes on microarrays can be predicted on the basis of thermodynamic data for duplex formation in solution. Predictable formation of isostable DNA not only benefits microarrays for gene expression analysis and genotyping, but may also improve the sequence-specificity of other applications that rely on the massively parallel formation of Watson-Crick duplexes. 相似文献
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13.
We prepared novel C5-modified triphosphates and phosphoramidites with a diamondoid functionally linked to the nucleobase. Using primer extension experiments with different length templates we investigated whether the modified triphosphates were enzymatically incorporated into DNA and whether they were further extended. We found that all three modified nucleotides can be incorporated into DNA using a single-nucleotide incorporation experiment, but only partially using two templates that demand for multiple incorporation of the modified nucleotides. The modified phosphoramidites were introduced into oligonucleotides utilizing DNA synthesizer technology. The occurring oligonucleotide structures were examined by circular dichroism (CD) and melting temperature (T(m)) measurements and were found to adapt similar helix conformations as their unmodified counterparts. 相似文献
14.
The advent of DNA origami technology greatly simplified the design and construction of nanometer-sized DNA objects. The self-assembly of a DNA-origami structure is a straightforward process in which a long single-stranded scaffold (often from the phage M13mp18) is folded into basically any desired shape with the help of a multitude of short helper strands. This approach enables the ready generation of objects with an addressable surface area of a few thousand nm(2) and with a single "pixel" resolution of about 6 nm. The process is rapid, puts low demands on experimental conditions, and delivers target products in high yields. These features make DNA origami the method of choice in structural DNA nanotechnology when two- and three-dimensional objects are desired. This Minireview summarizes recent advances in the design of DNA origami nanostructures, which open the door to numerous exciting applications. 相似文献
15.
《Angewandte Chemie (International ed. in English)》2017,56(39):12004-12008
DNA hydrogels are of great interest for a variety of biomedical applications owing to their biocompatibility and biodegradability but the advantages of DNA hydrogels have not been exploited yet because of their limited availability. Thus far, DNA hydrogels have been prepared from synthetically derived building blocks, and their production on large scale would be far too expensive. As an alternative, here the generation of DNA hydrogels from plasmid DNA is reported. Plasmid DNA can be prepared on large scale at reasonable costs by a fermentation process. The desired linear DNA building blocks are then obtained from the plasmid DNA by enzymatic digestion. Gel formation is carried out by covalent bond formation between individual building blocks via enzymatic ligation. The generation of pristine DNA hydrogels from plasmid DNA is thus presented for the first time. The viscoelastic properties of the hydrogels were studied by rheology, which confirmed that the gels have storage moduli G′ of >100 Pa. 相似文献
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17.
Li D Yang Z Zhao G Long Y Lv B Li C Hiew S Ng MT Guo J Tan H Zhang H Li T 《Chemical communications (Cambridge, England)》2011,47(26):7479-7481
It is demonstrated that the shapes and magnitudes of DNA writhe can be precisely manipulated solely through maneuvering the nucleotide sequence of DNA and without the assistance of topoisomerases. 相似文献
18.
调控DNA的变性解链过程是DNA扩增与检测的关键步骤。对于传统的热循环DNA扩增策略,由于变温过程中热量分布不均一以及变温速度慢等不利因素,会直接影响DNA变性解链过程,从而降低DNA检测放大的效果、延长检测的时长。因此,探索快速、高效的调控DNA变性解链的方法具有重要的研究意义。本文发展了以胞嘧啶在酸性条件下的质子化反应为基础,通过改变溶液的pH值,来诱导DNA构象在Watson-Crick(WC)碱基对与Hoogsteen(HG)碱基对之间的分子构象转换,从而实现精准、快速、高效的DNA变性解链调控目标。结果表明,相较于传统的温控方法,pH调控方法能显著提高DNA变性的速率约6倍以上。本文发现pH调控方法通过降低双链DNA的反应焓约160 kJ/mol,从而提高双链DNA变性速率和效率。该方法具有用于DNA信号放大与检测等相关应用的潜力。 相似文献
19.
Zauner G Wang Y Lavesa-Curto M MacDonald A Mayes AG Bowater RP Butt JN 《The Analyst》2005,130(3):345-349
A novel electrochemical assay for DNA ligase activity is described. The assay exploits the properties of DNA hairpins tethered at one terminus to a gold electrode and labelled at the other with a ferrocene group for rapid characterisation of DNA status by cyclic voltammetry. Successful ligation of 'nicked' DNA hairpins is indicated by retention of the ferrocene couple when exposure to DNA ligase is followed by conditions that denature the hairpin. The results demonstrate the simplicity of integrating electrochemical detection with hairpin based biosensors and illustrate a new approach to the assay of DNA ligases, of which the NAD(+)-dependent enzymes represent a potential broad spectrum antibacterial drug target. 相似文献