930 resultados para Dna-sequences


Relevância:

70.00% 70.00%

Publicador:

Resumo:

Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the π-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.

First, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.

Using low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.

For effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 μL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.

With the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.

As differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

DNA techniques are increasingly used as diagnostic tools in many fields and venues. In particular, a relatively new application is its use as a check for proper advertisement in markets and on restaurant menus. The identification of fish from markets and restaurants is a growing problem because economic practices often render it cost-effective to substitute one species for another. DNA sequences that are diagnostic for many commercially important fishes are now documented on public databases, such as the National Center for Biotechnology Information’s (NCBI) GenBank.1 It is now possible for most genetics laboratories to identify the species from which a tissue sample was taken without sequencing all the possible taxa it might represent.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

Amostras de DNA são encontradas em fragmentos, obtidos em vestígios de uma cena de crime, ou coletados de amostras de cabelo ou sangue, para testes genéticos ou de paternidade. Para identificar se esse fragmento pertence ou não a uma sequência de DNA, é necessário compará-los com uma sequência determinada, que pode estar armazenada em um banco de dados para, por exemplo, apontar um suspeito. Para tal, é preciso uma ferramenta eficiente para realizar o alinhamento da sequência de DNA encontrada com a armazenada no banco de dados. O alinhamento de sequências de DNA, em inglês DNA matching, é o campo da bioinformática que tenta entender a relação entre as sequências genéticas e suas relações funcionais e parentais. Essa tarefa é frequentemente realizada através de softwares que varrem clusters de base de dados, demandando alto poder computacional, o que encarece o custo de um projeto de alinhamento de sequências de DNA. Esta dissertação apresenta uma arquitetura de hardware paralela, para o algoritmo BLAST, que permite o alinhamento de um par de sequências de DNA. O algoritmo BLAST é um método heurístico e atualmente é o mais rápido. A estratégia do BLAST é dividir as sequências originais em subsequências menores de tamanho w. Após realizar as comparações nessas pequenas subsequências, as etapas do BLAST analisam apenas as subsequências que forem idênticas. Com isso, o algoritmo diminui o número de testes e combinações necessárias para realizar o alinhamento. Para cada sequência idêntica há três etapas, a serem realizadas pelo algoritmo: semeadura, extensão e avaliação. A solução proposta se inspira nas características do algoritmo para implementar um hardware totalmente paralelo e com pipeline entre as etapas básicas do BLAST. A arquitetura de hardware proposta foi implementada em FPGA e os resultados obtidos mostram a comparação entre área ocupada, número de ciclos e máxima frequência de operação permitida, em função dos parâmetros de alinhamento. O resultado é uma arquitetura de hardware em lógica reconfigurável, escalável, eficiente e de baixo custo, capaz de alinhar pares de sequências utilizando o algoritmo BLAST.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

低能离子束的诱变效应首先由我国科学家发现并将其广泛应用于育种实践,但是离子注入诱导DNA变异的研究结果主要是以微生物离体质粒DNA为材料获得的,以活体高等生物为材料的研究尚未见报道。 我们以30 keV N+(注入剂量80×1015 ions/cm2)注入拟南芥后获得的稳定突变体T80II为实验材料,对突变体植株进行了RAPD标记,并将T80II和对照部分RAPD特异条带进行克隆测序和DNA序列分析。结果显示,在可分辨的总计397个RAPD条带中,T80II株系中有52个条带表现出差异,包括条带的缺失和增加,条带变异率为13.1%;克隆的T80II序列中,平均每16.8个碱基出现一个碱基变异位点,表现出较高频率的碱基突变。碱基突变的类型包括碱基的颠换、转换、缺失、插入等。在检测到的275个碱基突变中,主要是单碱基置换(97.09%),碱基缺失或者插入的比例较小(2.91%)。在碱基置换中,转换的频率(66.55%)高于颠换的频率((30.55%)。此外,构成DNA的四种碱基均可以被离子束辐照诱发变异,而且每一种碱基都可以被其它三种碱基所替换,但是胸腺嘧啶(T)的辐射敏感性要高于其它三种碱基。通过分析突变碱基周边序列,对低能N+离子注入拟南芥突变体引发的碱基突变热点进行了讨论。 另外,低能离子注入诱变获得的突变体特异表达基因的克隆方面也没有报道。我们以突变体T80II作为实验材料,用PCR增效的减法杂交技术构建了T80II特异表达的cDNA减法文库,克隆特异表达的cDNA片段,并对其中1个与14-3-3 protein GF14 nu (GRF7) gene有部分同源性、长712 bp的cDNA片段进行了讨论。我们的研究证明通过减法杂交技术克隆低能离子诱发的突变体特异表达的cDNA是可能的,这为低能离子注入技术在分子生物学上的应用开辟了一个新思路。

Relevância:

70.00% 70.00%

Publicador:

Resumo:

采用PCR技术获得了中国鮡科褶鮡属(Pseudecheneis)5种鱼类及外群种类巨魾[Bagarius yarrelli(Sykes)]和红河纹胸鮡(Glyptothorax fukiensis honghensis Li)的线粒体部分基因序列.序列分析结果表明:间褶鮡(Pseudecheneis intermedius Chu)与平吻褶鮡(P.paviei Vaillant)在Cyt b基因片段上完全无差异,形成单倍型,支持间褶鮡应为平吻褶鮡的同物异名.凭Cyt b基因片段构建了它们的NJ,MP和ML分子树,3棵分子树基本一致,均支持褶鮡属构成一单系群;怒江和伊洛瓦底江"黄斑褶鮡"不同样品分别聚在一起,但二水系的样品未能形成一单系;其他各种的不同样品均能分别聚在一起形成单系.怒江和伊洛瓦底江"黄斑褶鮡"的分类地位值得今后进一步研究.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

Partial (DNA) sequences were examined for one nuclear (28S rRNA gene) and one mitochondrial (16S rRNA) locus for nine species of pomatiopsid snail (Gastropoda: Rissooidea: Pomatiopsidae) from south-east Asia and south-west China. Fresh field samples were

Relevância:

70.00% 70.00%

Publicador:

Resumo:

Phylogenetic relationships among representative species of the subfamily Raninae were investigated using approximately 2000 base pairs of DNA sequences from two mitochondrial (12S rRNA, 16S rRNA) and two nuclear (tyrosinase, rhodopsin) genes. Phylogenetic

Relevância:

70.00% 70.00%

Publicador:

Resumo:

Muntjac deer (Muntiacinae, Cervidae) are of great interest in evolutionary studies because of their dramatic chromosome variations and recent discoveries of several new species. In this paper, we analyze the evolution of karyotypes of muntjac deer in the context of a phylogeny which is based on 1,844-bp mitochondrial DNA sequences of seven generally recognized species in the muntjac subfamily. The phylogenetic results support the hypothesis that karyotypic evolution in muntjac deer has proceeded via reduction in diploid number. However, the reduction in number is not always linear, i.e., not strictly following the order: 46-->14/13-->8/9-->6/7. For example, Muntiacus muntjak (2n = 6/7) shares a common ancestor with Muntiacus feae (2n = 13/14), which indicates that its karyotype was derived in parallel with M. feae's from an ancestral karyotype of 2n greater than or equal to 13/14. The newly discovered giant muntjac (Muntiacus vuquangensis) may represent another pa;allel reduction lineage from the ancestral 2n = 46 karyotype. Our phylogenetic results indicate that the giant muntjac is relatively closer to Muntiacus reevesi than to other muntjacs and may be placed in the genus Muntiacus. Analyses of sequence divergence reveal that the rate of change in chromosome number in muntjac deer is one of the fastest in vertebrates. Within the muntjac subfamily, the fastest evolutionary rate is found in the Fea's lineage, in which two species with different karyotypes diverged in around 0.5 Myr.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

Phylogenetic relationships among all described species (total of 12 taxa) of the decapoda, were examined with nucleotide sequence data from portions of mitochondrial gene and cytochrome oxidase subunit I (COI). The previous works on phylogeny proved that the mitochondrial COI gene in crustacean is a good discriminative marker at both inter- and intra-specific levels. We provide COI barcode sequences of commertial decapoda of Oman Sea, Persian Gulf, Iran. Industrial activities, ecologic considerations, and goals of the decapoda Barcode of Life campaign make it crucial that species of the south costal be identified. The reconstruction of evolut phylogeny of these species are crucial for revealing stock identity that can be used for the management of fisheries industries in Iran. Mitochondrial DNA sequences were used to reconstruct the phylogeny of the Penaeus species of marine shrimp. For this purpose, DNA was extracted using phenol- chloroform well as CTAB method. The evolutionary relationships among 12 species of the decapoda were examined using 610 bp of mitochondrial (mt) DNA from the cytochrome oxidase subunit I gene. Finally the cladograms were compared and the resulted phylogenetic trees confirmed that the Iran's species origin is Indo-west pacific species. Iran's species, which were not grouped with the other decapoda taxa seem to always form a sister clade with Indo-west pacific species with strong bootstrap support 100%. The result completely agrees with the previously defined species using morphological characters.However, we still lack any comprehensive and clear understanding of phylogenetic relationships in this group.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

对贵州5种蝙蝠科蝙蝠的部分线粒体细胞色素氧化酶亚基Ⅰ DNA序列进行了测定,并结合从Genbank获得的爪哇伏翼的相应序列,以Pteropus dasymallus,P.scapulatus,Rousettus aegyptiacus为外群,运用贝叶斯法(Bayesian),最大似然法(Maximum Likelihood,ML)分析了这6种蝙蝠科蝙蝠的分子系统进化关系.结果表明:在贝叶斯,ML树中,这6种蝙蝠科的蝙蝠可分为3个分支:亚洲长翼蝠是第1个独立出来的分支;白腹管鼻蝠是继亚洲长翼蝠之后第2个分离出来的分支;第3个分支又分为两支,一支由大鼠耳蝠和小鼠耳蝠组成,另一支由南蝠和爪哇伏翼组成,支持将这4种蝙蝠同归于蝙蝠亚科的结论,从一定程度上否定了鼠耳蝠属和管鼻蝠亚科之间的姐妹类群关系,也不支持将鼠耳属提升为亚科.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

Fourier spectra of 120 short coding sequences (<1 200 bp) show that not all coding sequences are characterized by 3-base periodicity. Statistical analysis suggests that whether a coding sequence has 3-base periodicity may be related to the composition and distribution of bases, the usage and the order of the amino acids of the encoded protein as well as the synonymous codon usage. Generally, the content of A+U is higher than that of G+C in non-period-3 sequences, inversely in period-3 sequences. In the three codon positions, the base distribution in the non-periodic-3 sequences is more uniform than in the periodic-3 sequences. The usage biases of the amino acids and the codons in non-period-3 sequences are weaker than that in period-3 sequences. All of these phenomena should be considered sufficiently in predicting the genes and exons of DNA sequences by Fourier analysis method.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

Aim: To test a vicariant speciation hypothesis derived from geological evidence of large-scale changes in drainage patterns in the late Miocene that affected the drainages in the south-eastern Tibetan Plateau. Location: The Tibetan Plateau and adjacent areas. Methods: The cytochrome b DNA sequences of 30 species of the genus Schizothorax from nine different river systems were analysed. These DNA sequences were analysed using parsimony, maximum likelihood and Bayesian methods. The approximately unbiased and Shimodaira-Hasegawa tests were applied to evaluate the statistical significance of the shortest trees relative to alternative hypotheses. Dates of divergences between lineages were estimated using the nonparametric rate smoothing method, and confidence intervals of dates were obtained by parametric bootstrapping. Results: The phylogenetic relationships recovered from molecular data were inconsistent with traditional taxonomy, but apparently reflected geographical associations with rivers. Within the genus Schizothorax, we observed a divergence between the lineages from the Irrawaddy-Lhuit and Tsangpo-Parlung rivers, and tentatively dated this vicariant event back to the late Miocene (7.3-6.8 Ma). We also observed approximately simultaneous geographical splits within drainages of the south-eastern Tibetan Plateau, the Irrawaddy, the Yangtze and the Mekong-Salween rivers in the late Miocene (7.1-6.2 Ma). Main conclusions: Our molecular evidence tentatively highlights the importance of palaeoriver connections and the uplift of the Tibetan Plateau in understanding the evolution of the genus Schizothorax. Molecular estimates of divergence times allowed us to date these vicariant scenarios back to the late Miocene, which agrees with geological suggestions for the separation of these drainages caused by tectonic uplift in south-eastern Tibet. Our results indicated the substantial role of vicariant-based speciation in shaping the current distribution pattern of the genus Schizothorax.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

The sequencing analysis of the mitochondrial DNA control region (mtCR DNA) was performed to assess the genetic divergence and population structure of the Chinese sucker Myxocyprinus asiaticus (Cypriniformes Catostomidae) using four sample lots from natural populations of the Yangtze River. The mtCR DNA sequences of approximately 920 base pairs were obtained. A total of 223 nucleotide positions were polymorphic, and these defined 39 haplotypes. Of the 39 haplotypes, 37 (90%) were not shared, and among the populations as a whole there was little sharing of haplotypes. The average haplotype diversity (0.958) and the average nucleotide diversity (0.052) indicated a higher level of genetic diversity of Chinese sucker through the river. Analysis of molecular variation (AMOVA) of data revealed significant partitioning of variance (P<0.001) among populations (60.29%), and within populations (39.71%). The topology according to the neighbor joining and maximum parsimony methods showed mosaic composition of the 39 haplotypes, suggesting that the populations wore not completely divergent. The pairwise F statistic values, however, indicated that the population structuring existed to some extent among the geographic populations. There was a positive relationship between the aquatic distance and the genetic distance (Fst) among the populations (P<0.05). Based on our data, it is suggested that genetic drift, gene flow, and stochastic events are the possible factors influencing the population structure and genetic variation.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

1140 bp of cytochrome b gene were amplified and sequenced from 14 species of primitive cyprinid fishes in East Asia. Aligned with other ten cytochrome b gene sequences of cyprinid fish from Europe and North America retrieved from Gene bank, we obtained a matrix of 24 DNA sequences. A cladogram was generated by the method of Maximum likelihood for the primitive cyprinid fishes. The result indicated that subfamily Leuciscinae and Danioninae do not form a monophyletic group. In the subfamily Danioninae, Opsariichthys biden and Zacco platypus are very primitive and form a natural group and located at the root. But the genera in subfamily Danioninae are included in different groups and have not direct relationship. Among them, Aphyocypris chinensis and Yaoshanicus arcus form a monophyletic group. Tanichthys albonubes and Gobiocypris rarus have a close relation to Gobioninae. The genus Danio is far from other genera in Danioninae, In our cladogram, the genera in Leuciscinae were divided into two groups that have no direct relationship. The genera in Leuciscinae distributed in Europe, Sibera and North America, including Leuciscus, Rutilus, Phoxinus, N. crysole, Opsopoeodus emilae, form a monophyletic group. And the Leuciscinae in southern China including Ctenopharyngodon idellus, Mylopharyngodon piceus, Squalibarbus and Ochetobius elongatus have a common origination.

Relevância:

70.00% 70.00%

Publicador:

Resumo:

DNA是重要的生物大分子,也是主要的抗癌药物靶分子。小分子与DNA之间的相互作用是以DNA为靶分子的各种物质生物效应的基础,它们之间的特异性结合导致了癌变、突变及细胞的死亡。能够与DNA特异性结合的小分子很多都是临床上广泛应用的抗癌药物。因此,小分子与DNA之间的相互作用不论是对阐述抗癌、抗病毒药物的作用,还是对致癌机理的研究,尤其对抗癌药物的体内筛选都有重要意义。近十年来,开发新型的抗癌药物小分子,使它们具有对DNA序列特异性的识别能力己成为国内外研究的热点。斓系配合物由于具有广泛的光学、磁学和电学等特性使得有可能成为新的DNA特异性识别分子。然而,斓系离子在中性条件下极其容易水解的特性又极大的阻碍了斓系配合物对于DNA的识别性研究。本文在中性条件下合成了铜系氨基酸配合物,并成功的获得了这些配合物的晶体结构,利用这些有确定结构的I系氨基酸配合物与特定的DNA序列相作用,通过多种生物物理方法研究了它们对DNA序列特异性识别。主要的结果如下:1.在近中性条件下,合成了Eu-Val([Eus_8(L-HVal)_(16)(H_2O)_(32)]Cl_(24)·12.5H_2O),Eu-Asp([Eu_4(μ3-OH)_4(L-Asp)_2(L-HAsp)_3(H_2O)_7]Cl·11.5H_2O)和Tb-Cys(「Th_2(DL-Cys)_4(H_2O)_8]Cl_2)三种铜系氨基酸配合物,这些配合物的结构由于合成条件(温度,反离子及合成比例)上的差异与已报到的类似配合物的结构具有明显的不同。三种配合物的结构各具特色,从而在与DNA作用时将表现出各自特有的识别性能。2.Eu-Val配合物在与单链DNA作用时,配合物能键合到DNA碱基所在的疏水区,在与富含dC和dT碱基的序列相结合时,发生显著的能量传递,从而极大的增强了配合物中Eu的发射光谱。配合物结合DNA的化学计量比随序列中dC含量的降低而降低。配合物同样能够与处于DNA疏水区的富含dA和dG序列相结合,这种结合不能够产生能量传递,但使得DNA的紫外-可见光谱出现明显的减色和红移现象。此外,这一配合物还能够诱导单链DNApoly(dA)及p01y(rA)产生自身的二级结构,形成双链结构。这为进一步认识斓系离子的生物学效应奠定了基础,斓系氨基酸配合物可以诱导单链poly(dA)及poly(rA)形成自身结构尚无文献报道。3.Eu一AsP配合物能够选择性的稳定非B一构象的Poly(dA)Poly(dT),而使B一构象的印oly(dAdT)]2和[Poly(dGdC)]2变得不稳定。如在1:2比例时,该配合物可使Poly(dA)Polv(dT)的融化温度提高4℃,而使印oly(dAdT)}2的融化温度降低6℃,[Poly(dGdC)]2则出现了两个转变温度。进一步的圆二色实验结果充分表明Eu一Asp配合物对于富含Poly(dA)Poly(dT)和[Poly(dAdT)]2的双链DNA没有构象上的改变,而对于[Poly(dGdC)]2的双链DNA则产生了显著的构象上的改变,很有可能正是这一改变使得[Poly(dGdC)]2变得极其的不稳定。配合物对[Poly(dGdC)]2的这种不稳定影响随着配合物浓度的逐步升高而越来越明显。变温实验结果清楚的表明,在37℃时,Eu-AsP使[Poly(dGdC)JZ发生了构象转化,并且这种转化是可逆的。4.Tb-Cys配合物与单链(除了poly(dA))和双链DNA都能发生能量传递,从而使得配合物的荧光显著增强。不同的DNA表现出不同的增强效果,表明TbCys配合物对DNA的序列存在选择性,单链要强于双链,富含Poly(dAdG)的序列增强效果最好。单链和双链能量传递的差异表明了配合物能够区分DNA的单链和双链,配合物在与单链作用时结合更强。TbCys配合物在与DNA作用时存在不同的结合位点,而且单链和双链的结合位点明显不同。TbCys配合物能够引起富含dC和dT的单链DNA发生减色效应,而能够引起富含poly(dA)、poly(dAdG)的单链DNA和富含poly(dA)poly(dT)、[Poly(dAdT)]2和[poly(dGdC)]2的双链DNA发生明显的红移。此外,这一配合物同样能够影响双链DNA的稳定性,使得富含poly(dA)Poly(dT)序列变得稳定,而使得富含印oly(dAdT)]2和富含印oly(dGdC)]2的序列变得不稳定。这些结果都表明了配合物对DNA存在选择性。比较不同配合物的差别后能够发现DNA对于不同的配合物同样具有选II择性,这种选择性能够用来区分配合物。一这些钢系氨基酸配合物由于选择了天然的氨基酸作为配体,从而大大的降低了对人体的毒性,进一步表现出的对不同的DNA序列的选择型则使其有望成为新型的抗癌诊疗试剂。