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1.
In this protocol we present a reproducible method of preparing DNA probes of high specific activity using Sequenase. The probes
produced by this method had a specific activity of 2.8×109 cpm/μg with 69% of the total radioactivity incorporated into the TCA-precipitable materials. Probes with 5 to 10-fold lower
specific activity were obtained using commercially available kits or using currently empolyed methods. 相似文献
2.
《Gene Analysis Techniques》1987,4(1):9-13
A method for labeling synthetic oligonucleotide probes to high specific activity is described. The method utilizes two partly complementary oligonucleotides that are labeled by a fill-in reaction using the Klenow fragment of DNA polymerase I and four α32P-nucleoside triphosphates. Such probes can, in combination with Southern blot analysis, be used for routine analysis of individual genes in multigene families. 相似文献
3.
Guofan Wu Zhihua Wang Huini Zhang Ning Yang Jie Du Xiaoquan Lu 《Nucleosides, nucleotides & nucleic acids》2013,32(11-12):1051-1067
5′-Thiol-derivatized specific DNA probes were added to the single primer polymerase chain reaction (asymmetric PCR) solution. In the PCR process, the DNA probes extended in the presence of target; the extended probes were then immobilized on a glassy carbon electrode (GCE) via gold nanoparticles. Finally, methylene blue and the extended probes were combined and the electrochemical signals were measured. This signal was higher than that of the GCE modified only by the original probe. When there was no target in PCR solution, the probe did not extend and the signal did not increase. The specific sequences of the β-1,3-glucanase gene were detected successfully from three targets with different length: oligonucleotide, molecule clone vector DNA, and total genome DNA of transgenic capsicum. The detection limits of 2.6 × 10?13, 7.8 × 10?13, and 9.1 × 10?13 moll?1 for oligonucleotide, molecule clone vector DNA, and total transgenic capsicum genome DNA were estimated. 相似文献
4.
I. Mirgain C. Hagnere G. A. Green C. Harf H. Monteil 《World journal of microbiology & biotechnology》1992,8(1):30-38
Mercury-resistance genes were detected byin situ hybridization using new synthetic oligonucleotide probes specific formerA andmerB genes according to the published sequences of the corresponding enzymes. These DNA probes were used for the detection of specific mercury-resistant microorganisms isolated from the Rhine River which had been polluted 3 years previously in 1986. Mercuric reductase and organomercurial lyase genes persist in the bacterial genome even after the disappearance of the pollutant but are absent in axenic amoebae. A total of 49 bacterial isolates showed DNA homologies with the32P-labelled DNA probes and 15 free-living amoebae were selected due to their harboured symbiotic mercury-resistant bacteria. 相似文献
5.
N. V. Borisjuk V. P. Momot Y. Gleba 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1988,76(1):108-112
Summary Behavior of ribosomal RNA genes in the process of somatic hybridization was analyzed using hybrids Nicotiana tabacum + Atropa belladonna. Blothybridization of parental species DNAs to 32P-rDNA specific probes revealed two classes of ribosomal repeats in both tobacco and nightshade; their length was 11.2 kb, 10.4 kb (tobacco) and 9.4 kb, 10.2 kb (night-shade). For analysis of hybrids, labelled 32P rDNA specific probes were hybridized to DNA of parental species and somatic hybrids digested with restriction endonucleases EcoR1, EcoRV and BamH1. A new class of ribosomal DNA repeat, absent in parental species, was found in hybrid line NtAb-1. Possible mechanisms of appearence of a new rDNA class in the process of somatic cell fusion are discussed. 相似文献
6.
Todor Angelov Heidi A. Dahlmann Shana J. Sturla 《Bioorganic & medicinal chemistry》2013,21(20):6212-6216
Oligonucleotide hybridization probes containing nucleoside analogs offer a potential strategy for binding specific DNA sequences that bear pro-mutagenic O6-G alkylation adducts. To optimize O6-Me-G-targeting probes, an understanding of how base pairs with O6-Me-G are stabilized is needed. In this study, we compared the ability of O6-Me-G and G to hydrogen bond with three pyrimidine-like nucleobases (Z, 4-thio-U, and 3-deaza-C) bearing varied hydrogen bond donor and acceptor groups. We found that duplexes containing the pyrimidine analog nucleoside:G pairs were more thermodynamically stable than those containing pyrimidine analog nucleoside:O6-alkyl-G pairs. Thus, hydrogen bonding alone was not sufficient to impart selectivity to probes that target O6-G alkylation adducts in DNA. 相似文献
7.
Upon subfractionation of certain plant seed homogenates on sucrose density gradients, we encountered problems in defining
the location and amount of mitochondria using marker enzymes. In order to overcome the inherent limitations of enzyme assays,
we utilized a heterologous DNA probe specific foratp6 in maize orBrassica tournefortii to detect mitochondria. The samples were treated with SDS, proteinase K, and RNase A followed by agarose gel electrophoresis,
and blotting. The immobilized DNA was detected with [32P]-labelled probes, and quantified using a phosphor imager. The assay is specific, sensitive, and independent of species,
cell type, and developmental stage, thus circumventing the need for expressed protein to assay enzyme activity. 相似文献
8.
L. Fugger N. Morling L. P. Ryder N. Ødum J. Georgsen A. Svejgaard 《Immunogenetics》1989,30(3):208-213
DP gene typing using in vitro DNA amplification combined with sequence-specific oligonucleotide probes has recently been reported.
The resulting DNA amplification was specific for theHLA-DPB locus. Typing for the individualDPB alleles was exclusively dependent on the hybridizations of the probes but hampered by close sequence homology between differentDP alleles yielding complex patterns of reactivity with a panel of probes. We report the combined use of allele-specific DNA
in vitro amplification and allele-specific oligonucleotides in typing forDPB1
*
03 andDPB1
*
06. Complete concordance with PLT typing was observed for theDPB1
*
03 alleles, while in the DPB1*06 group, at least three variantDPB1
*
06 alleles were identified which have not been described previously. 相似文献
9.
G. Mahalakshmi K. N. Vennila B. Selvakumar P. Lakshmana Rao Ruchi Malwade Sunny Deval 《Journal of biomolecular structure & dynamics》2020,38(12):3443-3451
AbstractTwo new 10-methoxydibenzo[b,h][1,6]naphthyridine-2-carboxamide derivatives (R1 and R2) have been synthesized and characterized using different spectral techniques. The binding of these probes with DNA was investigated using spectral (Electronic, fluorescence, 1H NMR and circular dichroism) and molecular docking studies. These probes exhibited a strong fluorescence around 440?nm upon excitation around 380?nm. Electronic and competitive fluorescence titration studies, in HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] buffer/dimethyl sulfoxide (pH 7.4) medium, suggest that these probes bind strongly to DNA, which is substantiated by 1H NMR study. The binding constants are calculated to be 5.3?×?107 and 6.8?×?106 M?1 for R1 and R2, respectively. From the results of spectral studies, it is proposed that the mechanism of binding of these probes with DNA is through minor groove binding mode, which is further confirmed by circular dichroism and molecular docking studies. Initial cell viability screening using MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay shows that normal Vero cells are viable towards these probes at nano molar concentration, which is the concentration range employed in the present study for DNA staining (IC50 in the order of 0.023?mM). The enhancement in fluorescence intensity of these probes upon binding with DNA enables the staining of DNA in agarose gel in gel electrophoresis experiment. The sensitivity of these probes is comparable with that of ethidium bromide and DNA amounts as low as 4 nano gram are detectable.Communicated by Ramaswamy H. Sarma 相似文献
10.
Summary Human chromosome 13 loci homologous to seven recombinant DNA probes were mapped using in situ hybridization of 3H-radiolabeled probes to metaphase chromosomes. Each of these seven probes reveals at least one restriction fragment length
polymorphism, and thus each probe is potentially valuable in a genetic linkage map of this autosome. The data presented in
this paper map the seven loci to specific regions of chromosome 13. This mapping should allow a future comparison of genetic
distance with physical distance on this chromosome, and may permit better utilization of these probes in the clinical diagnosis
of human chromosomal rearrangements involving chromosome 13. 相似文献
11.
Victor Stefanovsky Stephan Dimitrov Valya Russanova Ilvia Pashev 《Molecular biology reports》1990,14(4):231-235
The presence of histones H1 and H4 at the sites of actual DNA synthesis has been studied with Ehrlich ascites tumour cells, pulse labeled for different times with 3H-thymidine and then treated with formaldehyde to crosslink histones to DNA. The fixed chromatin fragments were sonicated to reduce the size of DNA, purified in a CsCl gradient and immunoprecipitated with antibodies to histones H1 and H4. Determination of specific radioactivity in precipitated probes showed that both histones have been associated with nascent DNA even upon 1 min pulse with 3H-thymidine, thus indicating their presence near the replication fork. 相似文献
12.
Direction of DNA replication in mammalian cells 总被引:15,自引:0,他引:15
We have re-examined the direction of DNA synthesis in mammalian cells by means of pulse-labeling with [3H]thymidine and DNA autoradiography. Our results show that, whether or not the cells are treated with 5-fluoro-deoxyuridine, and whether they are labeled first with high specific activity [3H]thymidine and then with low, or vice versa, most (? 90%) of the unambiguous autoradiographic patterns can be explained by bidirectional replication but not by unidirectional replication.We also find that in autoradiographic experiments using two different specific activities of [3H]thymidine, obvious differences in grain density are obtained only when the difference in specific activity is threefold or more. Thus, the apparently contradictory findings of Lark et al. (1971) can be explained by the low difference in specific activity used by those authors. 相似文献
13.
Human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health. Based on multiplex
asymmetrical PCR and coupled with gold labelled silver stain (GLSS), we developed the visual DNA microarray for sensitive
and specific detection of these two viruses. Capturing probes of 5′-end-amino-modified oligonucleotides were immobilized on
glass surface to bind the complement biotinylated target DNA. The Au–streptavidin probe was introduced to the microarray for
specific binding to biotin. Black images of microarray spots which result from the precipitation of silver onto Au–streptavidin
probes, were visualized by naked eyes. In order to improve the efficiency of microarray hybridization, triplex asymmetrical
PCR of HIV-1, HCV and Human enterovirus 71 (EV-71, used as positive control) were performed to prepare abundant biotinylated single-stranded target DNA. The sensitivity of
visual DNA microarray (103 copies/ml) was higher than conventional PCR (104 copies/ml) and was identical to FQ-PCR (103 copies/ml). Total 152 blood samples containing the two viruses were tested using the DNA microarray and fluorescence quantitative
real-time PCR (FQ-PCR). The results were identical (P > 0.05). So this system has high sensitivity and may have potential in clinical applications. 相似文献
14.
Autoradiographic evidence is presented that demonstrates bidirectional DNA replication during a synchronous round of DNA synthesis in a culture of a reversible temperature sensitive DNA initiation mutant of Escherichia coli K12. High specific activity [3H]thymine was incorporated into the origins and termini of chromosomes which were otherwise uniformly labeled with low specific activity [3H]thymine. Autoradiographs of such differentially labeled chromosomes show two regions of high grain density symmetrically disposed on the circular chromosomes. This demonstrates that the origins and termini of replication are not contiguous; therefore replication must have proceeded in two directions. 相似文献
15.
E. V. Dmitrienko I. A. Pyshnaya D. V. Pyshnyi 《Russian Journal of Bioorganic Chemistry》2010,36(5):645-656
The characteristics of the UV-induced immobilization of oligonucleotides on nylon membranes and the efficiency of the enzymatic
labeling of immobilized probes in heterophase identifying specific DNA sequences were studied. Oligonucleotides bound to short
terminal oligothymidylates (up to 10 nt) through a flexible linker based on diethylene glycol phosphodiester are proposed
as probes for immobilization on nylon. The presence of this fragment allows one to enhance the immobilization efficiency and
reduce the UV-dependent degradation of the sequence-specific part of the probe by decreasing the irradiation dose needed for
DNA immobilization. The optimal dose of UV irradiation is evaluated to be ∼0.4 J/cm2 at 254 nm, which provides a high level of the hybridization signal for immobilized probes of various nucleotide sequences.
It was found that nylon amide groups play a key role in the photoinduced fixation of oligonucleotides to the polymer surface,
while its primary amino groups were not as responsible for the covalent binding of DNA as previously thought. Various additives
in the membrane wetting solution were demonstrated to influence both the efficiency of the UV-induced immobilization and the
functional integrity of immobilized probes. Other radical generating systems alternative to UV irradiation are shown to provide
the immobilization of oligonucleotides on nylon membranes. 相似文献
16.
Ohkubo A Kasuya R Sakamoto K Miyata K Taguchi H Nagasawa H Tsukahara T Watanobe T Maki Y Seio K Sekine M 《Nucleic acids research》2008,36(6):1952-1964
We propose a new strategy called the ‘Protected DNA Probes (PDP) method’ in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac4C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac6az8c7A). It was found that PDP containing ac4C and ac6az8c7A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness. 相似文献
17.
Joel A. Krauser 《Journal of inorganic biochemistry》2010,104(8):877-884
Simple and novel nuclease models have been synthesized. These involve metal-binding ligand 1,4,7,10-tetraazlcyclododecane (cyclen) tethered to an acridine ring (a DNA-binding group) by amide linkers of various lengths. Binding of these probes to DNA was studied by monitoring changes in their UV-visible spectra affected by the presence of DNA. Titration of these compounds with increasing amounts of pBR322 DNA caused hypochromic effects and shifted the acridine absorption at 360 nm to a longer wavelength. Under biologically relevant conditions (37 °C and pH 7.4), specific transition metal complexes of these compounds are found to be highly effective catalysts toward the hydrolysis of plasmid DNA. This is demonstrated by their ability to convert the super-coiled DNA (form I) to open-circular DNA (form II). Structure-activity correlation studies show that hydrolytic activity depends on both the structure of ligand (L1 > L2 > L3) and the nature of metal ion cofactor (Co3+ > Zn2+ > Cr2+ > Ni2+ > Cu2+ > Fe3+). 相似文献
18.
Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10. 相似文献
19.
《Mutation Research/Genetic Toxicology》1979,66(4):385-390
Ethidium mono- and diazide analogs have been used as photoaffinity probes to study the mechanism of the ethidium-induced petite mutation in yeast [7,10,14]. The azide moiety when exposed to light is converted to a reactive nitrine intermediate. The diradical nitrene effects a covalent attachment to sites of interaction bound reversibly by the drug. Ethidium azide photoaffinity labeling has been used to verify the prerequisite covalent attachment of ethidium to mitochondrial DNA to bring about the petite mutation in yeast [6]. Bastos has also reported a specific photoattachment of ethidium azide to a polypeptide (subunit 9) of the membrane bound ATPase in yeast mitochondria [1].Isolated DNA from yeast cells treated in vivo with [14C]ethidium monoazide plus light contained covalent adducts on both nuclear and mitochondrial DNA, although the specific radioactivity of mitochondrial DNA was more than 10 times higher than nuclear DNA [11]. Drug distribution studies of [14C]ethidium monoazide have indicated that greater than 50% of the covalent adducts in the nuclear centrifugation fraction (2000 × g) reside on nuclear proteins [3]. Nuclear damage in yeast by the photolysis of ethidium azides is apparent from the increased killing effect in cells treated with ethidium azide derivatives plus light as opposed to dark-treated cells [7,10,11]. This communication reports a concomitant increase in nuclear mutations and genetic activity from the photoactivated covalent attachment of ethidium azides to nuclear components. 相似文献
20.
The specificity and sensitivity of three methods for the preparation and detection of nonradioactive probe DNA (biotin-nick translation, biotin-photolabel, and antigen-chemical linkage) were evaluated and compared with a nick-translated32P-labeled DNA probe in DNA hybridization studies. The DNA probes were prepared from a restriction fragment (HindIII-3) from bacteriophage P1 DNA, and target DNA consisted of purified phage P1 DNA or P1 prophage DNA in lysogens ofEscherichia coli. A probe concentration of 50 ng/ml resulted in clear detection with the three nonradioactiveHindIII-3 DNA probes, whereas the specificity of the32P-HindIII-3 DNA probe was satisfactory at a concentration of 25 ng/ml. However, the detection of false positives was greater with the32P-labeled probe. The sensitivity of the radiolabeled DNA probe was marginally greater than that of the nonradioactive probes in dot blot hybridizations with purified phage P1 DNA. However, when the preparation time, ease of use, safety, duration of storage, and expense were compared for the four methods of labeling, the nonradiolabeled probes were generally superior to the radiolabeled probe. 相似文献