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)

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

Determination of the hepatitis C virus (HCV) genotype has become the standard procedure in laboratory diagnostics of HCV infection. Genotype elucidation has prognostic value assignment helps in assessing disease prognosis and promotes establishing appropriate duration of treatment. Now 11 major genotypes and more than 70 subtypes of HCV have been identified using the sequence variability within 5' non-coding region (5' NCR). In Russia the most common subtypes are 1a, 1b, 2a, 3a and more rare - 4 and 5 types. While the "gold standard" for testing is nucleic acid sequencing, a variety of other assays, including the line probe assay or type-specific amplification, has been developed to provide more rapid and cheaper forms of testing. The aim of this study was to determine the type-specific single nucleotide polymorphism (SNP) in 5' NCR HCV by the classical three-step minisequencing method with followed MALDI-TOF mass spectrometry detection The fragments of 5'NCR of HCV genomes were amplified by the nested RT PCR. The removal of excess nucleotides and primers was performed. Three oligonucleotide primers were design to detect two sets of type-specific SNP in 5' NCR HCV. The primer extension reaction was performed using modified thermostable DNA polymerase and in the presence of ddNTP. The molecular weights of primers extension reaction products were analyzed using MALDI-TOF mass spectrometry. The HCV genotype was determined according the presence in analyses sample the molecules with expected molecular weights. The suggested method was used to type HCV from 69 HCV-positive sera. The 1a genotype was determine in 4.5% samples, 1b - 48%, 2a - 4.5% 3a - 29%, 4 - 1.5%. The mixes of two genotypes were found in 13% samples. All data confirmed by direct nucleic acid sequence. Thus, the new method for HCV typing has been developed using the minisequencing reaction and mass spectrometry for the determination of nucleic acid molecular weight.
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PMID:[Using the mass spectrometry analysis for hepatitis C virus typing]. 1585 Feb 17