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1.
A new 5′-nuclease polymerase chain reaction (PCR) system for the detection and quantification of Citrobacter freundii and C. braakii was developed with primers and the probe oriented to a specific region of the cfa gene encoding a cyclopropane fatty acid synthase. The qualitative variant of the method consisted of a conventional PCR with
end-point fluorimetry or agarose gel electrophoresis, and the quantitative variant used kinetic real-time PCR measurement.
The PCR system was specific for C. freundii and C. braakii, detecting neither other Citrobacter spp. nor other enteric bacteria (Escherichia coli, Salmonella enterica, and others). The detection limit of the qualitative variant of the method was 103 cfu/mL when the amplification was followed by fluorimetry and 104 cfu/mL when the amplification was followed by gel electrophoresis. The real-time PCR variant of the method facilitated quantification
over a range of concentrations from 102 to 108 cfu/mL, with Escherichia coli (106 cfu/mL) and Salmonella enterica (106 cfu/mL) having no effect on the quantification. 相似文献
2.
Naoki Shinoda Tomotaro Yoshida Toyoko Kusama Masami Takagi Toshiaki Hayakawa Takashi Onodera 《Nucleosides, nucleotides & nucleic acids》2013,32(4):324-330
Many studies have suggested that regulation of the polymerase chain reaction (PCR) is influenced by several factors. However, the understanding of reaction efficiency factors is not sufficient. Here we propose that high GC contents of primer 5′-end increases reaction efficiency in PCR. Using 71 primers (45 pairs), we analyzed factors that affect reaction efficiency, and statistically tested the correlation between the amplification signals and several factors. As a result, there were significant correlations between the amplification signals and the GC contents in the first 1~3 bps of primer 5′-end. 相似文献
3.
Attachment of proteins to the 3′ end of DNA increases stability of the DNA in serum and retards clearance of DNA by major organs, thereby enhancing in vivo half-life and therapeutic potential of DNA. Unfortunately, the length of DNA molecules that can be produced with 3 ′ modifications by solid-phase synthesis for protein attachment is limited to 45–60 nucleotides due to uncertainties about sequence fidelity for longer oligonucleotides. Here we describe selective covalent coupling of proteins or other molecules to the 3′-adenine overhang of unlabeled and fluorophore-labeled double-stranded polymerase chain reaction products putatively at the N6 position of adenine using 2.5% glutaraldehyde at pH 6.0 and 4°C for at least 16 h. Gel mobility shift analyses and fluorescence analyses of the shifted bands supported conjugate formation between double-stranded polymerase chain reaction products and β2-microglobulin. In addition, blunt-ended DNA ladder fragments treated with glutaraldehyde at 4°C showed no evidence of DNA–DNA or DNA–protein conjugate formation. With the present cold glutaraldehyde technique, longer DNA–3′-protein conjugates might be easily mass-produced. The protein portion of a DNA–3′-protein conjugate could possess functionality as well, such as receptor binding for cell entry, cytotoxicity, or opsonization. 相似文献
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José Fiandor María Teresa García-López Federico G. De las Heras 《Nucleosides, nucleotides & nucleic acids》2013,32(7):1325-1334
Abstract Reaction of 3-N-benzyl-5′-deoxy-5′-haloderivatives of uridine with the carbanions derived from diethyl malonate, ethyl acetoacetate, ethyl cyanoacetate and malondinitrile afforded the corresponding highly functionalized 5′-C-chain-extended uridines. 相似文献
6.
We rapidly and efficiently isolated the 5′-region of cDNA encoding the N-terminal region of human centromere antigen B (CENP-B) including an ATG methionine codon by polymerase chain reactions (PCR). The unknown 5′-flanking sequence of the cDNA was amplified using an adaptor-sequence ligated to the 5′ end as a universal primer sequence. To locate the target fragments, we did an additional PCR with another set of two internal primers using samples of the size-fractionated products as templates, rather than using the conventional hybridization procedure. This approach can further be applied to the analysis of other unknown flanking sequences of cDNA or genomic DNA. 相似文献
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Real-Time Imaging and Quantification of Amyloid-β Peptide Aggregates by Novel Quantum-Dot Nanoprobes
Background
Protein aggregation plays a major role in the pathogenesis of neurodegenerative disorders, such as Alzheimer''s disease. However, direct real-time imaging of protein aggregation, including oligomerization and fibrillization, has never been achieved. Here we demonstrate the preparation of fluorescent semiconductor nanocrystal (quantum dot; QD)-labeled amyloid-β peptide (QDAβ) and its advanced applications.Methodology/Principal Findings
The QDAβ construct retained Aβ oligomer-forming ability, and the sizes of these oligomers could be estimated from the relative fluorescence intensities of the imaged spots. Both QDAβ coaggregation with intact Aβ42 and insertion into fibrils were detected by fluorescence microscopy. The coaggregation process was observed by real-time 3D imaging using slit-scanning confocal microscopy, which showed a typical sigmoid curve with 1.5 h in the lag-time and 12 h until saturation. Inhibition of coaggregation using an anti-Aβ antibody can be observed as 3D images on a microscopic scale. Microglia ingested monomeric QDAβ more significantly than oligomeric QDAβ, and the ingested QDAβ was mainly accumulated in the lysosome.Conclusions/Significance
These data demonstrate that QDAβ is a novel nanoprobe for studying Aβ oligomerization and fibrillization in multiple modalities and may be applicable for high-throughput drug screening systems. 相似文献9.
Peng Zhou Weiyi Xu Xueling Peng Zhenhua Luo Qinghe Xing Xulin Chen Chengqian Hou Weihong Liang Jianwen Zhou Xiaoyan Wu Zhou Songyang Songshan Jiang 《PloS one》2013,8(7)
Animal microRNA (miRNA) target prediction is still a challenge, although many prediction programs have been exploited. MiRNAs exert their function through partially binding the messenger RNAs (mRNAs; likely at 3′ untranslated regions [3′UTRs]), which makes it possible to detect the miRNA-mRNA interactions in vitro by co-transfection of miRNA and a luciferase reporter gene containing the target mRNA fragment into mammalian cells under a dual-luciferase assay system. Here, we constructed a human miRNA expression library and used a dual-luciferase assay system to perform large-scale screens of interactions between miRNAs and the 3′UTRs of seven genes, which included more than 3,000 interactions with triplicate experiments for each interaction. The screening results showed that the 3′UTR of one gene can be targeted by multiple miRNAs. Among the prediction algorithms, a Bayesian phylogenetic miRNA target identification algorithm and a support vector machine (SVM) presented a relatively better performance (27% for EIMMo and 24.7% for miRDB) against the average precision (17.3%) of the nine prediction programs used here. Additionally, we noticed that a relatively high conservation level was shown at the miRNA 3′ end targeted regions, as well as the 5′ end (seed region) binding sites. 相似文献
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Reyad A. Elbarbary Hiroaki Takaku Naoto Uchiumi Hiroko Tamiya Mayumi Abe Masayuki Takahashi Hiroshi Nishida Masayuki Nashimoto 《PloS one》2009,4(6)
A long form (tRNase ZL) of tRNA 3′ processing endoribonuclease (tRNase Z, or 3′ tRNase) can cleave any target RNA at any desired site under the direction of artificial small guide RNA (sgRNA) that mimics a 5′-half portion of tRNA. Based on this enzymatic property, a gene silencing technology has been developed, in which a specific mRNA level can be downregulated by introducing into cells a synthetic 5′-half-tRNA that is designed to form a pre-tRNA-like complex with a part of the mRNA. Recently 5′-half-tRNA fragments have been reported to exist stably in various types of cells, although little is know about their physiological roles. We were curious to know if endogenous 5′-half-tRNA works as sgRNA for tRNase ZL in the cells. Here we show that human cytosolic tRNase ZL modulates gene expression through 5′-half-tRNA. We found that 5′-half-tRNAGlu, which co-immunoprecipitates with tRNase ZL, exists predominantly in the cytoplasm, functions as sgRNA in vitro, and downregulates the level of a luciferase mRNA containing its target sequence in human kidney 293 cells. We also demonstrated that the PPM1F mRNA is one of the genuine targets of tRNase ZL guided by 5′-half-tRNAGlu. Furthermore, the DNA microarray data suggested that tRNase ZL is likely to be involved in the p53 signaling pathway and apoptosis. 相似文献
12.
D. P. Bayley V. Florian A. Klein K. F. Jarrell 《Molecular genetics and genomics : MGG》1998,258(6):639-645
The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (Mr= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins. 相似文献
13.
《Journal of Fermentation Technology》1988,66(5):585-589
The production of inosine by microbial conversion of 5′-inosinic acid (IMP) was investigated. Among the various strains of Streptomyces and Bacillus tested, Streptomyces aureus NCIB 9803 was selected for the microbial conversion process due to its high IMP-degrading activity. A maximum conversion yield of 0.43 (86% of the theoretical value) was obtained when IMP was added to the culture medium at 24 h. Kinetic studies with [8-14C] IMP showed that the difference from the theoretical values mainly attributable to the uptake of inosine by S. aureus. 相似文献
14.
Kôichi Ogata Akira Imada Yoshio Nakao 《Bioscience, biotechnology, and biochemistry》2013,77(9):586-623
A Bacillus accumulated 5′-nucleotides in the culture fluid during cultivation. These nucleotides consisted mainly of ribonucleoside 5′-monophosphates and of traces of nucleoside 5′-diphosphates. The composition of the accumulated nucleotides resembled that of the nucleotides of intracellular RNA. Therefore an intimate relationship between the accumulation of nucleotides and the metabolism of endogenous RNA was suggested.The excretion of 5′-nucleotides into the culture fluid took place in parallel with the reduction of intracellular RNA, and it was presumed that the accumulated nucleotides were products of degradation of intracellular RNA. 相似文献
15.
Yuji Nagano Hirotoshi Samejima Shukuo Kinoshita 《Bioscience, biotechnology, and biochemistry》2013,77(1):83-88
An enzyme system which could convert orotic acid to uridine-5′-monophosphate (5′-UMP) was found in cell-free extract of a threonine-requiring auxotroph of Micrococcus glutamicus (Syn. Corynebacterium glutamicum) 534 Co-147. This reaction required 5-phosphoribosylpyrophosphate (PRPP) and magnesium ion as essential components. The product of the enzyme reaction was separated by ion exchange resin chromatography and identified to be uridine-5′-monopbosphate. From the stoichiometric studies and other characteristics, it became evident that this enzyme reaction proceeded according to the following equation and was assumed to be catalyzed by orotidine-5′-monophosphate pyrophosphorylase and orotidine-5′-monophosphate decarboxylase. 相似文献
16.
María-José Camarasa Federico G. De las Heras María Jesús Pérez-Pérez 《Nucleosides, nucleotides & nucleic acids》2013,32(4):533-546
Abstract Reaction of 3′-0-(t-butyldimethylsilyl)-2′-deoxythymidine-5′-carboxaldehyde and 2′,3′-dideoxythymidine-5′-carboxaldehyde with acetone afforded a 3:2 mixture of the two (5′R)- and (5′S)-5′-acetonylthymidine derivatives. 相似文献
17.
I. A. Mikhailopulo G. V. Zaitseva E. V. Vaaks H. Rosemeyer F. Seela 《Nucleosides, nucleotides & nucleic acids》2013,32(2-4):273-278
Abstract Reaction of 02,3′-anhydro-5′-0-trityl-2′-deoxycytidine (1) with LiN3s in DMF resulted in the formation of 1-(3-azido-2,3-dideoxy-5-0-trityl-β-D-erythro-pentofuranosyl) cytosine (2) and 3-0-(4-amino-1,3-pyrimidin-2-yl)-5-0-trityl-2-deoxy-α-D-threo-pentofuranosyl azide (3) (2:3 = 1:1) in 88% yield. Compound 3 was deprotected with 80% aqueous AcOH yielding 4 相似文献
18.
Freshly isolated gonococci upon subculture are readily lysed by normal human serum although a few strains remain inherently resistant to the complement activity. The sensitive gonococci can be converted to serum resistance by incubation with a host derived factor referred to as cytidine 5-monophospho-N-acetylneuraminic acid (CMP-NANA). These gonococci resist complement mediated killing due to their sialylation of an epitope structure on a component of lipo-oligosaccharide (LOS). In the present study, the kinetics of conversion to serum resistance by the action of sialyltransferase (STase) inNeisseria gonorrhoeae was followed with very low concentrations of CMP-NANA. This conversion could not be perceived at 2×10–3 nmol.ml–1 but was fully attainable from 8×10–3 to 2×10–2 nmol.ml–1 CMP-NANA. When pretreated up to 100 min in presence of the very low concentration of 2×10–3 nmol.ml–1, a potentiating effect on the conversion of gonococci by 2×10–2 nmol.ml–1 was observed in relation to the time of preincubation. This action was abolished after exposure to a subinhibitory concentration of chloramphenicol (0.5 µg.ml–1). The gonococci recovered their ability to convert to serum resistance following adequate washing. The potential for increase in STase activity should be of interest for understanding the conversion from a serum sensitive to a serum resistance state. 相似文献
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Shashi Sharma Paban Kumar Dash S. R. Santhosh Jyoti Shukla Manmohan Parida P. V. Lakshmana Rao 《Molecular biotechnology》2010,45(1):49-55
Chikungunya is one of the most important emerging arboviral infections of public health significance. Due to lack of a licensed
vaccine, rapid diagnosis plays an important role in early management of patients. In this study, a QC-RT–PCR assay was developed
to quantify Chikungunya virus (CHIKV) RNA by targeting the conserved region of E1 gene. A competitor molecule containing an
internal insertion was generated, which provided a stringent control of the quantification process. The introduction of 10-fold
serially diluted competitor in each reaction was further used to determine sensitivity. The applicability of this assay for
quantification of CHIKV RNA was evaluated with human clinical samples, and the results were compared with real-time quantitative
RT–PCR. The sensitivity of this assay was estimated to be 100 RNA copies per reaction with a dynamic detection range of 102 to 1010 copies. Specificity was confirmed using closely related alpha and flaviviruses. The comparison of QC-RT–PCR result with real-time
RT–PCR revealed 100% concordance for the detection of CHIKV in clinical samples. These findings demonstrated that the reported
assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous
detection and quantification of CHIKV in acute-phase serum samples. 相似文献