Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The CTG trinucleotide repeat expansions that are associated with myotonic dystrophy can be up to several thousand repeat units in length. We have developed a PCR protocol that has the potential to amplify mutant alleles with very large numbers of CTG repeats. The amplification uses the rTth DNA polymerase, XL system for long PCR targets together with primers which do not closely flank the repeat region and partial substitution of 7-deaza-dGTP for dGTP. Alleles containing up to approximately 800 CTG repeats were detected directly in agarose gels stained with ethidium bromide. Larger CTG repeat expansions required Southern blot transfer and detection with a repeat sequence probe; using this method, alleles containing up to approximately 2700 CTG repeats were detected. The PCR-based method described here was comparable to previous Southern blots of EcoRI-restriction digested genomic DNA in both the approximate size and heterogeneity of mutant alleles detected, but provided more precise sizes of the CTG repeat expansions than the restriction digest approach. This PCR protocol could potentially simplify current mutation detection protocols in the molecular diagnosis of myotonic dystrophy, and facilitate molecular studies of the disease.
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PMID:Characterization of large CTG repeat expansions in myotonic dystrophy alleles using PCR. 872 79

A reverse transcriptase activity has been detected in potato mitochondria using special RNAs as templates: a bacterial RNA coding for neomycin phosphotransferase (neo pa RNA) and a Neurospora crassa mitochondrial RNA (184 nt RNA). Surprisingly, no exogenous primer addition was required. These RNA templates share a primary and secondary structure similar to the T psi CG loop of tRNAs that could constitute the recognition site for the enzyme. Reverse transcriptase activity was inhibited by ddTTP, ethidium bromide and aphidicolin, while potato mitochondrial DNA polymerase was not inhibited by aphidicolin indicating that these activities correspond to distinct enzymes. A conserved sequence of reverse transcriptases was detected in potato mitochondrial DNA suggesting that this enzyme could be mitochondrially encoded.
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PMID:A reverse transcriptase activity in potato mitochondria. 875 99

DNA polymerase photoprobes 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1), 2-[(4-azidophenylsulfenyl)thio]-2'-deoxyadenosine 5'-triphosphate (2), and 2-[(4-azido-2-nitrophenyl)-thio]-2'-deoxyadenosine 5'-triphosphate (3) were designed from a thermodynamic model of DNA polymerase 1-substrate interactions such that the triphosphate would anchor the inhibitor and allow the phenyl azide to interact with the complementary template binding site. Photoprobes 1-3 were synthesized by condensation of 2-thio-2'-deoxyadenosine or its phosphate with p-azidophenacyl bromide, N-(4-azidophenylsulfenyl)phthalimide, and 4-azido-1-fluoro-2-nitrobenzene, respectively, and characterized as reversible and photoinduced irreversible inhibitors of the DNA polymerase I Klenow fragment and HIV I reverse transcriptase. The aryl azides decomposed with irradiation at 300 and 350 nm with half-lives ranging from 0.98 to 2.33 min and 2.15 to 5.38 min, respectively, with quantum efficiencies ranging from 0.29 to 0.55 and no apparent photodecomposition of the 2-thio-2'-deoxyadenosine nucleotide. Photoprobes 1-3 showed mixed noncompetitive inhibition of the Klenow fragment polymerase activity versus poly(dA).(T)10 as variable substrate with apparent competitive inhibition constants of 2.1, 36, and 29 microM, respectively, evidence suggesting that these photoprobes bind to both the free enzyme form and the enzyme-template-primer binary complex. Of the three photoprobes, only nucleotide 1 photoinactivates the Klenow fragment; in the presence of a 200-fold excess of nitrene scavenger, photoprobe 1 inactivates 92% of the Klenow fragment polymerase activity with saturation observed at 9.7 microM and an IC50 of about 2 microM. This evidence demonstrates that photoprobe 1 does bind to the Klenow fragment in the absence of template-primer and that it is an efficient photoprobe.
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PMID:Deoxyadenosine-based DNA polymerase photoprobes: design, synthesis, and characterization as inhibitors of the Escherichia coli DNA polymerase I Klenow fragment. 879 43

The polymerase chain reaction (PCR) is an extremely sensitive assay that has many uses in retroviral-mediated gene transfer protocols. Because the majority of retroviral vectors used in current gene transfer protocols are based on the Moloney-murine leukemia virus (MMLV), we have designed primers which amplify a region of the psi packaging sequence from all MMLV retroviruses tested. This assay detects gene transfer by all MMLV-based vectors and is especially useful for the laboratory that routinely screens a number of different retroviruses for their gene transfer efficiency. Furthermore, we present here a novel technique for harvesting single colonies derived from hematopoietic stem/progenitor cells growing in methylcellulose medium that expedites and substantially improves the resulting quantitative estimates of retroviral transduction frequencies. This technique utilizes a conventional 96-well format and, when coupled with a fluorescence-based post-PCR detection system, makes it unnecessary to run agarose gels to visualize the PCR product. This system of PCR product detection, which uses the 5'-->3' exonuclease activity of Taq DNA polymerase to cleave a fluorescently labeled probe during each round of PCR amplification, is fast, convenient, and at least as sensitive as an ethidium bromide-based detection system when used in conjunction with our universal PCR assay.
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PMID:A rapid and quantitative assay to estimate gene transfer into retrovirally transduced hematopoietic stem/progenitor cells using a 96-well format PCR and fluorescent detection system universal for MMLV-based proviruses. 883 21

High frequency transfer and elimination of drug resistance may indicate an extrachromosomal inheritance of genetic determinants. This study shows the cure and transfer of a small plasmid and tetracycline resistance in Staphylococcus aureus 1030 (55)Tet strains. Several methods are available for plasmid elimination. We used ethidium bromide, an agent that binds to DNA, and thus inhibits DNA polymerase. This caused a high frequency of loss of the small plasmid and resistance to tetracycline. Transfer of tetracycline resistance was done in a mixed culture at a frequency of 10(-6). This type of study is very important to physicians and epidemiology investigators and provides better knowledge on antibiotic-resistance mechanisms that may occur in vivo in a hospital environment.
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PMID:A genetic study of a Staphylococus aureus plasmid involving cure and transference. 898 81

At present, PCR (polymerase chain reaction), is the method most often used in molecular genetics. The aimed amplification of the short DNA chain is reached by three cyclically repeated steps using different temperatures. The reaction demands the presence of DNA polymerase, buffer, dNTP as basic elements of DNA and a pair of short oligonucleotides, which makes the reaction specific. The product of reaction is usually analysed using electrophoresis in agarose gel or polyacrylamide gel and visualised by ethidium bromide.
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PMID:[PCR--polymerase chain reaction. Principles and applications in research and medical practice]. 899 2

A simplified technique for the detection of transcripts from a defined promoter is described. After reverse transcription, a PCR target sequence is selectively added to the 3' end of cDNA strands by DNA polymerase extension directed by an oligonucleotide template. Those cDNA molecules that do not have ends within a few nucleotides of the promoter start site are not extended and thus are excluded from subsequent amplification. Even when amplified products are visualized by ethidium bromide staining of agarose gels, this method requires only 1% of the RNA usually needed for detection of mRNA by standard RNase protection utilizing radiolabeled probes. In contrast to direct detection of cDNA by PCR, this procedure restricts amplification to a narrow subset of transcripts even when other overlapping colinear transcripts are present. We call this detection procedure specific amplification of cDNA ends (SPACE).
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PMID:Simple method for selective amplification of cDNA from a defined promoter. 904 2

A fluorogenic 5' nuclease PCR assay was evaluated for its ability to specifically detect and differentiate DNA of two Orthopoxvirus species. A pair of consensus primers that target a DNA segment of the Orthopoxvirus haemagglutinin gene, and two oligonucleotide probes; each labelled with a different fluorescent reporter dye and the same quencher dye, were used in a single-tube assay. The assay is based on the 5'-->3' nuclease activity of AmpliTaq DNA polymerase that cleaves a fluorescein-labelled hybridized probe. Probe cleavage generates specific fluorescent signals whose intensity can be quantified by fluorometry. After evaluating the effects of various annealing temperatures and probe concentrations and normalizing the emission intensities of the reporter dyes, it was possible to detect and differentiate monkeypox and vaccinia virus DNAs on the basis of a single-base polymorphism. The sensitivity of the 5' nuclease PCR assay is comparable to the sensitivity of ethidium bromide-stained gels, but the assay provides higher specificity and virtually eliminates the need for laborious post-PCR processing.
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PMID:The potential of 5' nuclease PCR for detecting a single-base polymorphism in Orthopoxvirus. 916 Mar 29

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.
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PMID:KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells. 922 20

We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50 microliters assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, and IU of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37 degrees C for RT followed by a variable amount of cycles of two-step PCR amplification (92 degrees C for 60 sec, 53 degrees C for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 10(2.8) TCID 50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique.
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PMID:A simple reverse transcription-polymerase chain reaction for dengue type 2 virus identification. 933 7


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