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)

We developed a novel single-step reverse transcription-polymerase chain reaction (RT-PCR), which is equal in sensitivity and specificity to RT-nested PCR, based on both reverse transcriptase and Taq DNA polymerase working efficiently under single buffer reaction conditions. Using in vitro synthesized hepatitis C virus (HCV) RNA, it was demonstrated that 10-100 copies of HCV RNA could be detected with a set of primers that amplify a 144 base-pair sequence unique to the 5'-noncoding region of HCV RNA. Furthermore, this method was successfully performed on serum and liver biopsy specimens obtained from patients with chronic hepatitis C. In addition, HCV RNA from in vitro HCV-infected MT-2C cells, which supported HCV replication, was also detected by this method. The method is anticipated to improve the detection of small amounts of RNA, such as that of HCV, promoting both labor savings and the prevention of carry-over contamination.
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PMID:Single-step reverse transcription-polymerase chain reaction for the detection of hepatitis C virus RNA. 977 96

The extent of population diversity among GB virus C (GBV-C)/hepatitis G virus (HGV) within a persistently infected individual (Iw) was investigated by sequence analysis of multiple clones generated from polymerase chain reaction (PCR)-amplified products of cDNA analogous to fragments of 5' non-coding region (5'NC), envelope region 1/2 (E1/E2) and non-structural region 3 (NS3) of viral genome. Although nucleotide substitutions were more common in coding regions than in the 5'NC region, there was no region corresponding to the hypervariable region of hepatitis C virus in the E1/E2 region. Transition substitution exceeded transversion by 7 to 12-fold, and 79.4% of substitutions were synonymous. This bias against substitutions producing amino acid replacements and the use of Pfu DNA polymerase with an error rate 10 times lower than the observed frequency of substitution, suggests that most substitutions were not artefactual. This data suggests that individual genomes of HGV within an infected individual may differ from each other at 0.23-0.84% nucleotide position and at 0.42-0.61% amino acid position.
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PMID:Diversity of hepatitis G virus within a single infected individual. 1112 38

The hepatitis C virus (HCV) is known to circulate in vivo as a quasispecies, a population of closely related, but genetically nonidentical virions. HCV reverse transcriptase (RT)-(nested) polymerase chain reaction (PCR) strategies are used to study quasispecies diversity at certain important viral genetic loci, predominantly at hypervariable region 1 (HVR 1) of the E2 envelope gene, and the interferon sensitivity determining region (ISDR) of the nonstructural 5a (NS5a) gene. We have found that the choice of DNA polymerase employed in viral PCR has effects on the inferred viral diversity at two distinct loci on the HCV genome. Nested HVR 1 and ISDR PCR was performed with both proofreading (Pwo) and nonproofreading (Taq) DNA polymerases on identical cDNA derived from three separate HCV-positive sera. Amplicons were cloned and sequences determined for 18-20 individual clones per sample. Quasispecies diversity determined from HVR 1 and ISDR PCR products showed that there was a marked effect on the inferred diversity depending on which DNA polymerase was employed in the PCR. The deduced amino acid sequences of the major variants within each specimen were identical for both Taq and Pwo DNA polymerase-mediated PCRs. However, a greater number of minor variants were observed in the Taq-generated amplicons, 80% of which were not observed in the Pwo-generated amplicons. Primer editing in the Pwo-generated amplicons was observed in 19% (20/104) of clones examined. Single-strand conformational polymorphism analysis of multiple replicates of each amplicon revealed good intra-PCR reproducibility in terms of genetic heterogeneity, and that as such the observations were not due to poor PCR reproducibility. The use of nonproofreading DNA polymerases to assess viral diversity can yield an incorrect quasispecies spectrum and affect RT-PCR assay performance. The contribution of Taq-induced errors and lack of adaptability of primers to potentially heterologous template-binding sites indicate that proofreading DNA polymerases should be the enzyme of choice in these systems.
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PMID:Inferred hepatitis C virus quasispecies diversity is influenced by choice of DNA polymerase in reverse transcriptase-polymerase chain reactions. 1116 7

Single-stranded conformation polymorphism (SSCP) is a technique used widely for the detection of differences in DNA sequence based on PCR technology. Developed by geneticists for the detection of mutations causing disease, it has been adopted more recently for the analysis of the quasi-species of viral genomes, such as hepatitis C virus (HCV). The rigorous standardisation and determination of the limit of detection of the technique has rarely been shown. Variants within the quasi-species of the hypervariable region of HCV were cloned into pUC119 and the resulting plasmids quantitated and used as templates to optimise SSCP. Variables studied included the number of variants detected, the sensitivity of detection of variants in the minority, the electrophoresis temperature, methods of generation of single-stranded DNA, effect of numbers of cycles of PCR and use of DNA polymerase with proof-reading ability. It was demonstrated that the optimised method could detect at least five variants within a quasi-species and that variants could be detected down to a level of 2% of the quasi-species. Electrophoresis at room temperature for 18 h was highly reproducible. Generation of single-stranded DNA using a single primer with Taq polymerase for 20 cycles gave an accurate reflection of the quasi-species make-up and use of Pfu polymerase reduced sensitivity of detection of minor bands. This SSCP method provides an accurate tool to evaluate HCV quasi-species.
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PMID:Optimisation and sensitivity of single-stranded conformation polymorphism for the detection of hepatitis C virus quasi-species. 1122 60

The term 'genotyping' describes the genetic characterization of a genome. The genotype analysis is performed to identify mutations that differentiate one individual or strain from another. The mutations may confer resistance to specific antiviral drugs or they may simply allow classification of a strain as to 'type' and 'subtype'. There are four human viruses for which genotype information is clinically useful. Hepatitis B virus (HBV) infections are being treated with antiretroviral drugs and resistance after prolonged treatment is common. Since HBV cannot be cultured, the only method of detecting resistance-conferring mutations in the genome is a genotypic analysis. Hepatitis C virus (HCV) infection can be cured by treatment with the combination of interferon and ribavirin but certain strains of virus are more resistant to treatment than others. The current recommendations are that all HCV type 1 infections be treated for 12 months whereas other types may be successfully treated in 6 months. Since interferon treatment may have significant side effects, the determination of HCV genotype is an important aspect of this therapeutic regimen. Treatment of cytomegalovirus (CMV) disease with nucleoside analogues occasionally results in resistant virus with mutations in the phosphotransferase gene (UL97) and/or the DNA polymerase gene (UL54) that can be tested with phenotypic or genotypic assays. Since CMV grows very slowly, it may be more clinically useful to perform a rapid genotypic assay although only the UL97 gene can be efficiently genotyped. Finally, the virus for which genotyping has become the standard of care, human immunodeficiency virus type 1 (HIV-1) can now be genotyped routinely by many clinical virology labs experienced with molecular amplification methods and automated DNA sequencing technology. All currently-available antiretroviral drugs are directed against either the protease or reverse transcriptase genes of HIV-1 and the mutations within these genes that confer resistance have been well described. Sequence-based genotyping methods are not necessarily the best approach for routine genotyping of these four viruses, but sequencing is the gold standard from which other methods are developed and against which they are compared.
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PMID:Clinically relevant sequence-based genotyping of HBV, HCV, CMV, and HIV. 1141 49

We have shown that highly proofreading DNA polymerase is required for the polymerase chain reaction in the genetic analysis of hepatitis C virus (HCV). To clarify the status of HCV quasispecies in hepatic tissue using proofreading DNA polymerase, we performed a genetic analysis of the HCV core protein-encoding region in cancerous and noncancerous lesions derived from 4 patients with hepatocellular carcinoma. In contrast to the previously published data, we observed neither deletions nor stop codons in the analyzed region and no significant difference in the complexity of HCV quasispecies between cancerous and noncancerous lesions. This result suggests that the HCV core gene is never structurally defective in hepatic tissues, including cancerous lesions. However, in 3 of the patients, the consensus HCV species differed between cancerous and noncancerous lesions, suggesting that the predominant replicating HCV species differs between these 2 types of lesions. Moreover, during the course of the study, we obtained several interesting variants possessing a substitution at codon 9 of the core gene, whose substitution has been shown to induce the production of the F protein synthesized by a - 2/+1 ribosomal frameshift.
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PMID:Hepatitis C virus quasispecies in cancerous and noncancerous hepatic lesions: the core protein-encoding region. 1210 85

We established a cell culture system for the replication of hepatitis C virus (HCV) by using human T and B leukemia cell lines. These 2 cell lines were infected in vitro by using HCV-positive pooled patient serum samples. HCV RNA was extracted from infected cell lines at different times after infection, and a sequence of the virus 5' untranslated region was analyzed. Hepatitis C minus-strand RNA was detected in the infected cell lines by highly strand-specific rTth (recombinant Thermus thermophilus DNA polymerase)-based reverse transcription followed by a novel, highly sensitive, single-tube nested polymerase chain reaction (PCR) method. PCR products were analyzed by direct DNA sequencing. These results indicate that the HCV can replicate in T and B lymphocytes. This model should represent a valuable tool for the detailed study of the initial steps of the HCV replication cycle and for the evaluation of antiviral molecules.
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PMID:Detection of extrahepatic hepatitis C virus replication by a novel, highly sensitive, single-tube nested polymerase chain reaction. 1252 Jul 3

RNA viruses are characterized by their high rates of genetic variation. Their genetic diversity is generally studied by reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification and nucleotide (nt) sequence determination. The misinterpretation of viral diversity due to copy errors introduced by the enzymes used in this two-step protocol has not yet been assessed systematically. In order to investigate the impact of such errors, we sought to bypass the intrinsic viral heterogeneity by starting from a homogeneous cDNA template. With this in mind, the hepatitis C virus (HCV) 5' non-coding region (5'NCR) was amplified either by PCR starting from a homopolymeric cDNA template or by RT-PCR starting from the in vitro RNA transcript derived from the same original cDNA template. Amplicons were cloned and the 17-20 individual clones were sequenced in each assay. Different quasispecies patterns were obtained with various commercially available DNA polymerases, resulting in different computed error rates. The non-proofreading Taq DNA polymerase provided the highest error rate which was seven times higher than that obtained with the most reliable of the proofreading polymerases tested. We, therefore, emphasize that the misleading interpretation of the observed heterogeneity for a given viral sample could be due to ignorance of the fidelity of the polymerase used for viral genome amplification, and thus that proofreading DNA polymerases should be preferred for the investigation of natural genetic diversity of RNA viruses.
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PMID:From RNA to quasispecies: a DNA polymerase with proofreading activity is highly recommended for accurate assessment of viral diversity. 1271 Oct 59

This review covers the non-HIV antiviral patent literature from December 2001 to April 2002. Most of the patent applications describe new compounds for the treatment of hepatitis C virus (HCV) by inhibition of the NS3 serine protease. Several examples of both nucleoside and non-nucleoside inhibitors of the HCV polymerase NS5B have been reported. Hepatitis B virus (HBV) therapy continues to be dominated by nucleoside analogs, but several non-nucleoside HBV polymerase inhibitors have also been reported. In addition, a number of patents describing non-nucleoside inhibitors of the human cytomegalovirus (HCMV), the herpes simplex virus (HSV-1 and HSV-2) and the varicella zoster virus (VZV) DNA polymerase are also reviewed. A number of patents that appeared in 2002 hold promise for the treatment of respiratory syncytial virus (RSV) with small molecule inhibitors. Various approaches to the treatment of hepatitis D virus (HDV), picornaviruses and the human papilloma virus (HPV) are also described.
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PMID:Non-HIV antivirals - a review of the recent patent literature. 1280 98

Patients who will receive chemotherapy require careful assessment of liver function prior to treatment to determine which drugs are not appropriate, and which drugs need dose modification. However, if the hepatic parenchymal abnormalities are caused by an underlying neoplasm and the neoplasm is sensitive to the drugs, it may not be necessary to reduce the dose. Clearly, this is an area where clinical judgment must be used to assess the risk/benefit ratio. Treatment of chronic hepatitis B virus (HBV) involves either the nucleoside analogue lamivudine or interferon alpha. The advantage of lamivudine includes limited adverse effects and the fact that histological improvement has been documented in the majority of patients. Primary prophylaxis with lamivudine may be a well tolerated and effective method to reduce the frequency of chemotherapy-induced HBV reactivation in chronic HbsAg carriers. HbsAg screening is necessary before beginning chemotherapy for non Hodgkin's lymphoma patients. However, the main problem with long-term lamivudine therapy is the emergence of genotypic resistance because of base pair substitution at specific sites within the YMDD locus of the DNA polymerase gene. Significant hepatic dysfunction is uncommon among hepatitis C virus (HCV) infected patients treated with chemotherapy for hematological malignancies. However, infection with elevated AST levels is a significant risk factor for veno-occlusive disease after hematopoietic stem cell transplantation. Clinical judgment and a high index of suspicion remain critical tools in preventing and treating hepatic manifestations of cancer chemotherapy.
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PMID:[Hepatotoxicity of chemotherapy]. 1285 43


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