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)

A Polymerase Chain Reaction (PCR) of a DNA sequence containing a CA repeat produces a main band but also several shadow bands that differ by 2 base pairs below the main band. In the experiments described in this paper, these shadow bands were excised from a DNA sequencing gel and directly sequenced. It was found that the sequence in the CA repeat was ambiguous. However, the sequence 5' and 3' to the CA repeat was clear and unambiguous. It is proposed that the shadow bands are generated by 2 base pair random deletions in the CA repeat region. During this process the sequence becomes 'scrambled' only in the CA repeat region. The shadow bands were shown to occur during the PCR since the genomic DNA template did not contain the shadow bands. It is probable that the shadow bands arise by slippage during the PCR. It is predicted that a thermostable DNA polymerase with a high processivity would greatly reduce the occurrence of shadow bands.
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PMID:The determination of the sequences present in the shadow bands of a dinucleotide repeat PCR. 850 34

We have successfully amplified D17S74 (CMM86) alleles by a long-distance polymerase chain reaction (PCR) using TaKaRa Ex Taq (a Taq DNA polymerase with a 3'-exonuclease activity) and Perfect Match Polymerase Enhancer (a special polymerase enhancer). We adopted a hot-start technique with TaqStart antibody. Because of the high guanine content (60%) in D17S74 alleles, removal of K+ from the buffers was quite effective. The use of K(+)-free buffers reduces premature chain termination in G-rich regions, thereby facilitating amplification of targets containing such sequences. The 17 alleles amplified from DNA samples of 72 unrelated Japanese subjects ranged from 1.05 to 3.5 kb, with a heterozygosity of 92%. PCR amplification of D17S74 alleles makes their detection simpler than by conventional Southern blotting, and increases the practical utility of the locus.
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PMID:Long-distance PCR of VNTR at the D17S74 (CMM86) locus. 875 89

The recently developed ultrasensitive reverse transcriptase (RT) test involving a PCR step can detect minute amounts of RT in single retroviral particles and is 10(6) times more sensitive than conventional RT assays. We have found that different DNA-dependent DNA polymerases like DNA Polymerase I from Escherichia coli and eukaryotic enzymes like DNA polymerase alpha and gamma exhibit RT-like activities in this assay, whereas DNA polymerase beta and Klenow enzyme show only minor activities. To discriminate false-positive signals caused by DNA-dependent DNA polymerases in the RT-PCR assay, we have included increasing amounts of activated DNA in the RT reactions. This modification of the assay leads to complete inhibition of aberrant but not authentic RT activities. This improvement specifies the RT-PCR assay as the most sensitive tool for the detection of even very rare replication-competent retroviruses and of related enzymatic activities indigenous, for example, for products of endogenous retrovirus-like RT genes in cell extracts.
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PMID:Specific suppression of false-positive signals in the product-enhanced reverse transcriptase assay. 882 50

PCR (Polymerase Chain Reaction) has become a standard and essential tool used in disciplines such as molecular genetics, genome mapping and sequencing, and forensics. It has proven to be easier, quicker, and less costly than traditional techniques of molecular cloning. While its use in these fielde has already contributed to great advancements and revolutionary changes, current PCR using Taq DNA polymerase is usually limited to amplification up to 5kb. Recently, "long" PCR conditions have been identified which allow the amplification of DNA templates up to 40kb with higher fidelity. In this report, we describe the amplification of longer human genomic fragments (up to 27kb) using LA PCR technology.
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PMID:[Long and accurate PCR (LA PCR)]. 892 Jun 50

Skin from acute and healed herpes simplex virus or herpes simplex virus-associated erythema multiforme (HAEM) lesions was examined by polymerase chain reaction with primers for DNA polymerase, ICP8, thymidine kinase (5' end of herpes simplex virus genome), and ICP27 (3' end of herpes simplex virus genome). The primers were herpes simplex virus specific and equally sensitive. The four herpes simplex virus genes were seen in acute herpes simplex virus lesions, but except for one patient, only polymerase (or polymerase and ICP8) were seen in 7-d healed lesional skin. Herpes simplex virus DNA was not seen 1-1.5 mo after healing. HAEM skins from 18 of 24 patients (75%) were positive for polymerase DNA and four of 24 (17%) were also positive for ICP8 or thymidine kinase DNA. Only one tissue (4%) was positive for polymerase, ICP8, and ICP27 DNA. Skin from healed HAEM lesions was still polymerase DNA positive 1-3 mo after lesion resolution. The polymerase DNA signal was in the basal and spinous cell layers of the epidermis and in the outer root sheath of the hair follicle. Polymerase RNA was identified by reverse transcriptase polymerase chain reaction in skin from acute, but not healed polymerase DNA positive HAEM lesions, suggesting that polymerase expression is associated with HAEM lesion development.
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PMID:Expression of herpes simplex virus DNA fragments located in epidermal keratinocytes and germinative cells is associated with the development of erythema multiforme lesions. 932 89

Taq DNA polymerase is an enzyme essential in performing Polymerase Chain Reaction (PCR) which has recently become a basic technology in research and diagnostic laboratories. In order to reduce the cost of research work in Thailand, recombinant Taq DNA polymerase was locally produced from pTaq cloned in E. coli. The enzyme was characterized and evaluated in comparison with the commercial Taq DNA polymerase produced by Perkin Elmer Cetus, U.S.A. The yield of enzyme was 6.72 mg/ml and the activity of 9,524 units/mg protein with the total of 448,000 units/litre of the bacterial culture. The preparation was free of DNase based upon its ability to degrade Lambda DNA evaluated by gel electrophoresis. Although the enzyme produced gave a high DNA polymerase activity, the preparation was not as pure as the enzyme produced by Perkin Elmer Cetus. Immunoblot analysis indicated that the enzyme preparation contained the products of enzyme degradation obtained during preparation and bacterial protein contaminations. In spite of the existence of bacterial proteins in the preparation, the Taq enzyme produced was proved to be applicable in performing PCR such as the PCR-SSP (Sequence Specific Primers) typing for HLA-DR. The cost of enzyme preparation was about 256 times less than that of the commercial enzyme. Economically, the locally produced Taq DNA polymerase can be used efficiently in the research laboratories performing PCR based typing of the HLA genes.
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PMID:Production and evaluation of Taq DNA polymerase. 934 60

Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma c-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq DNA Polymerase, with and without manual hot-start procedure.
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PMID:Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR. 945 68

The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems. The known DNA adducts S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S-[2-(O6-guanyl)ethyl]GSH were individually placed at a site in a single oligonucleotide. Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events. Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-[2-(N7-guanyl)ethyl]GSH using all of these polymerases. dCTP was the most preferred nucleotide incorporated opposite S-[2-(N7-guanyl)ethyl]GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-[2-(N7-guanyl)ethyl]GSH with pol II-. Both S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH strongly blocked replication by all polymerases. Only dATP and dGTP were incorporated opposite S-[2-(N2-guanyl)ethyl]GSH by both Kf- and pol II-. S-[2-(O6-Guanyl)ethyl]GSH was shown to strongly code for dATP incorporation by Kf-. With pol II-, dTTP was incorporated opposite S-[2-(O6-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding.
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PMID:Polymerase blockage and misincorporation of dNTPs opposite the ethylene dibromide-derived DNA adducts S-[2-(N7-guanyl)ethyl]glutathione, S-[2-(N2-guanyl)ethyl]glutathione, and S-[2-(O6-guanyl)ethyl]glutathione. 954 1

Oxidative damage to DNA deoxyribose generates oxidized abasic sites (OAS) that may constitute one-third of ionizing radiation damage. The antitumor drug bleomycin produces exclusively OAS in the form of C-4-keto-C-1-aldehydes in unbroken DNA strands and 3'-phosphoglycolate esters terminating strand breaks. We investigated whether two human DNA repair enzymes can mediate OAS excision in vitro: Ape1 protein (the main human abasic endonuclease (also called Hap1, Apex, or Ref1)) and DNA polymerase beta, which carries out both the abasic excision and the resynthesis steps. We used a duplex oligonucleotide substrate with one main target for bleomycin-induced damage. Ape1 catalyzed effective incision at the C-4-keto-C-1-aldehyde sites at a rate that may be only a few-fold lower than incision of hydrolytic abasic sites at the same location. Consistent with several previous studies, Ape1 hydrolyzed 3'-phosphoglycolates 25-fold more slowly than C-4-keto-C-1-aldehydes. DNA polymerase beta excised the 5'-terminal OAS formed by Ape1 incision at a rate similar to its removal of unmodified abasic residues. Polymerase beta-mediated excision of 5'-terminal OAS was stimulated by Ape1 as it is for unmodified abasic sites. Escherichia coli Fpg (MutM) protein also excised 5'-terminal OAS, but in our hands, the RecJ protein did not. These observations help define mammalian pathways of OAS repair, point to interactions that might coordinate functional steps, and suggest that still unknown factors may contribute to removal of 3'-phosphoglycolate esters.
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PMID:Excision of C-4'-oxidized deoxyribose lesions from double-stranded DNA by human apurinic/apyrimidinic endonuclease (Ape1 protein) and DNA polymerase beta. 978 84

Inadequate yields of PCR product and the generation of nonspecific PCR products can complicate genotyping studies, particularly when the DNA template is of inferior quality and/or has a low-copy number. In this study, the ability of AmpliTaq Gold DNA Polymerase to enhance the specificity and yield of amplification was evaluated in a quadruplex short tandem repeat (STR) system in which a nonspecific PCR product and poor yield had been previously observed with AmpliTaq DNA Polymerase usage. Because AmpliTaq Gold is inactive until heated during the PCR before thermal cycling, effects similar to those achieved with "hot-start" PCR were attained in a fast, simple and practical fashion. A significant enhancement in yield at the four STR loci and improved balance of alleles resulted with the use of AmpliTaq Gold. Furthermore, a non-specific PCR product, the result of mispriming, was effectively eliminated. The consistency of quality results was improved, thereby promoting successful typing of suboptimal DNA samples and enhancing the accuracy of genotyping. Since PCR product yield is elevated with AmpliTaq Gold usage, and consistent performance and low background are achieved with higher amounts of AmpliTaq Gold compared with AmpliTaq, AmpliTaq Gold can be used to augment measures taken to counteract the effects of some PCR/Taq DNA polymerase inhibitors, such as those found in blood and some forensic specimens. Studies showed that pH affects either the activity or the activation of the polymerase. AmpliTaq Gold was found to be compatible with pH 8.3 buffers, such as GeneAmp PCR Buffer and AmpFlSTR PCR Reaction Mix but not compatible with pH 9.0 buffers, such as GenePrint STR 10 x Buffer (however, conditions for the usage of AmpliTaq Gold with the GenePrint CTTv system are provided). AmpliTaq Gold is useful for the development and optimization of multiplex amplification systems, particularly those in which the primers are not well designed and/or the reaction conditions are not optimal. Finally, because AmpliTaq Gold is initially inactive, preparation of reactions at ambient temperature and automation of the PCR are facilitated. Therefore throughput can be expanded significantly with the use of AmpliTaq Gold DNA Polymerase.
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PMID:Enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA polymerase. 979 57


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