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)

Thermostable DNA polymerases can catalyze nontemplated addition of a nucleotide to the 3' end of amplification products. This presents a potential source of error in genotyping studies employing Taq DNA polymerase to amplify microsatellite loci. Although the activity is marker specific, experimental variation is often seen in the degree of modification. Consequently, for a given microsatellite marker, an allele may be inconsistently identified as either the unmodified or modified amplification product. Full automation of high-throughput genotyping has been hampered by the need for manual editing of data because of this source of allele misidentification. In this study we estimate a 1% to 3% error rate attributable to nontemplated nucleotide addition in the ABI PRISM genotyping system. We present a PCR-based strategy to minimize this source of error.
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PMID:Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase. 859 17

The Applied Biosystems PRISM fluorescence-based genotyping system as well as the Invitrogen TA Cloning vector system are influenced by the tendency of Taq DNA polymerase to add an adenine nucleotide to the 3' end of PCR products after extension. Incomplete addition of adenine to a majority of PCR product strands creates problems in allele-calling during genotyping and potentially diminishes the cloning efficiency of such products. Experiments reported here show that certain terminal nucleotides can either inhibit or enhance adenine addition by Taq and that PCR primer design can be used to modulate this activity. The methods we propose can substantially improve allele-calling for problematic microsatellite markers when using GENOTYPER software.
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PMID:Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning. 889 Dec 24

Quantitative competitive RT-PCR techniques have been developed to detect BCR-ABL fusion transcripts in CML but they are hardly reproducible. In this work, we have developed BCR-ABL quantification by real time RT-PCR using the ABI PRISM 7700 (Perkin Elmer), a new technique which allows simple and rapid quantification of a target sequence during the extension phase of PCR amplifications. A fluorogenic probe labeled with both a reporter dye at the 5' end and a quencher-dye at the 3' end hybridizes to the target sequence on the third exon of the ABL gene. The exonuclease activity of the Taq DNA polymerase cleaves the probe and releases the reporter dye, resulting in an increase in the fluorescence signal. The absolute copy number of the target sequence (BCR-ABL) or a control gene (ABL) in an unknown sample can then be calculated using a calibration curve prepared from a set of BCR-ABL RNA standards, and results are expressed as a BCR-ABL/ABL ratio. In our hands, the sensitivity of a serial dilution of total RNA from a positive cell line (K562) in a negative cell line (HL60) was 10(-4). Fifteen CML patients in cytogenetic CR, including 11 allografted patients, two autografted patients and two patients treated by IFN were studied sequentially by this new real time quantitative RT-PCR technique in parallel with conventional qualitative two round nested RT-PCR. The two autografted patients showed high BCR-ABL/ABL ratio in all samples. The two patients treated by IFN showed a progressive decrease in the ratio. In the 11 allografted patients, four were sequentially studied 2 years or more after allo-BMT, and all ratios were below 10(-4). The four patients remained in clinical and cytogenetic CR. The seven other allografted patients were studied immediately after the procedure. Three of them showed a progressive decrease in the BCR-ABL/ABL ratio which reached 10(-4) 7 months after allo-BMT. The three patients remained in hematologic and cytogenetic CR. The remaining four allografted patients had progressive increase of BCR-ABL ratio; three developed cytogenetic relapse 9, 11, 28 months after allo-BMT, and the last patient remained in cytogenetic CR in the bone marrow but developed granulocytic sarcoma. Results of real-time quantitative RT-PCR were in agreement with those of qualitative two round nested PCR. However, evolution changes in the results of real-time quantitative RT-PCR often preceded those of the conventional technique: a decrease of the BCR-ABL/ABL ratio preceded progression from first round to second round positivity and then negativity with the classical technique; conversely, an increase in the ratio preceded evolution with the classical technique. Thus, real-time quantitative RT-PCR may show better correlation with clinical and cytogenetic evolution than conventional qualitative techniques and may help in making early therapeutic decisions in CML, especially after molecular relapse.
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PMID:Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using a 'real time' quantitative RT-PCR assay. 1036 Mar 86

A real time quantitative PCR-based simian virus 40 (SV40) detection and quantification method has been developed. This method takes advantage of the 5' to 3'-exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for direct monitoring of PCR product accumulation through a dual-labelled fluorogenic probe. This method provides accurate, precise and reproducible quantification of SV40 DNA over a linear dynamic range of at least 100,000-fold with a minimum detection level of 6.4 copy equivalents/microL of SV40 viral particle in test samples. The sample preparation procedure employed allows for efficient and consistent recovery of SV40 DNA from test samples. High concentrations of protein and cellular DNA presenting in test samples have been demonstrated to have no impact on SV40 quantification. This method offers significant advantages over other PCR methods and cell-based infectivity assays currently available for SV40 detection and quantification. The availability of this method should greatly facilitate the pathogenic investigation of SV40, as well as viral clearance evaluations required for the development of new biological products.
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PMID:A real time quantitative PCR-based method for the detection and quantification of simian virus 40. 1065 79

Continuous cell lines used for pharmaceutical protein manufacturing have the potential to be contaminated by viruses. To ensure the safety of pharmaceutical proteins derived from continuous cell lines, validation of the ability of the manufacturing process to clear potential contaminating viruses is required for product registration. In this paper, a real time quantitative PCR method has been applied to the evaluation of simian virus 40 (SV40) removal during chromatography and filtration procedures. This method takes advantage of the 5'-3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for automated SV40 DNA quantification through a dual-labeled fluorogenic probe. This method provides accurate and reproducible quantification of SV40 DNA. The SV40 clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. This method offers significant advantages over cell-based infectivity assays, such as higher sensitivity, greater reliability, higher sample throughput and lower cost. This method can be potentially used to evaluate the clearance of all model viruses during chromatography and filtration procedures. This method can be used to substitute cell-based infectivity assays for process validation of viral removal procedures and the availability of this method should greatly facilitate and reduce the cost of viral clearance evaluations required for new biologic product development.
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PMID:Real time quantitative PCR as a method to evaluate simian virus 40 removal during pharmaceutical protein purification. 1065 80

Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1x10(6) particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3-6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5'-->3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell-based production system.
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PMID:Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture. 1096 40

To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms.
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PMID:Quantitative analysis of laminin 5 gene expression in human keratinocytes. 1585 26

The standard DNA sequencing methods for use in conjunction with commercially available sequencing kits are effective in sequencing a majority of templates. However, templates rich in dinucleotide and tetranucleotide repeats and a telomeric DNA containing tandem repeats are difficult to sequence adequately using these methods. Base compression artifacts due to formation of secondary structure on the nascent strands and slippage of the DNA polymerase accompanied by premature chain termination in homopolymer and short tandem repeat regions of DNA are commonly encountered problems in sequencing core laboratories. In an attempt to sequence such repeat regions of telomeric DNA templates using dye terminator chemistry, we investigated the effect of increasing the annealing time and temperature in combination with the use of denaturing conditions. Specifically, we compared the commonly used ABI PRISM BigDye, dGTP BigDye, and DYEnamic ET terminator chemistries for sequencing telomeric DNA templates rich in CA- and AACCCC-type repeats and for sequencing a template rich in dinucleotide (GT and CT) and tetranucleotide repeats. The routine reaction protocol was modified by adding either 1 M 1-carboxy-N,N,N-trimethylmethanaminium inner salt (betaine) or 5% dimethyl sulfoxide (DMSO) as denaturants in the reaction mixture. In addition, the annealing and denaturation times were increased to allow successful primer extension for linear growth of sequencing reaction product. Many of the artifacts in sequencing are known to be due to reduced stability of the hybrid formed between the template and the nascent strand. The effects of using denaturants to break secondary structures in the nascent chain and of increasing the denaturation and annealing times are discussed.We were able to sequence DNA templates with tandem repeats that failed to sequence under routine reaction conditions.
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PMID:Sequencing telomeric DNA template with short tandem repeats using dye terminator cycle sequencing. 1949 47

The high genetic diversity in hepatitis B and C viruses (HBV and HCV, respectively), and the presence of differences in pathogenicity and response to therapy, necessitates continous monitorization of the genotypes and antiviral resistance patterns. The aims of this study were to investigate the genotype distribution of HBV and HCV in chronically infected patients and to detect the resistance patterns of HBV strains in treatment-naive patients and those under lamivudine (LAM) treatment. A total of 44 chronic hepatitis B (CHB) patients (mean age: 42 +/- 13.6 years; 31 were male) and 30 chronic hepatitis C (CHC) patients (mean age: 47 +/- 12.4 years; 8 were male) were included to this study which was carried on between September 2007-March 2008. Mutational analysis and antiviral drug resistance studies were performed by sequence analysis (ABI PRISM 310 Genetic Analyzer; Applied Biosystems, USA), with the use of the region between 80-260 codons in HBV DNA polymerase gene, and the genotype determining sequences in 5' non-coding region in HCV genome. As a result, all of the 44 CHB patients (100%) were found to be infected with HBV genotype D, while genotype 1 (26/30; 86.6%) was found to be the predominant HCV genotype in CHC patients. The frequency of HCV genotypes were as follows; 63.3% (n = 19) genotype 1b, 20% (n = 6) 1a, 13.3% (n = 4) 4a and 3.3% (n = 1) 1c. Various mutations were detected in 34.1% (15/44) of CHB patients and these were identified as M2041 (n = 6), Q215S (n = 2), L801 + M2041 (n = 1), L80V + M2041 (n = 1), Q215S + M2041 (n = 1) and M2041 + L180M (n = 1) mutations responsible for LAM resistance; V214A (n = 1) and A181T + N236T (n = 1) mutations responsible for adefovir (ADV) resistance, and V84M + V173L (n = 1) mutation responsible for ADV + LAM resistance. In 12% (3/25) of the treatment-naive CHB patients LAM resistance was detected. However, total antiviral resistance rate was found 63.2% (12/19) for CHB patients (LAM resistance in 9, ADV resistance in 2, and LAM + ADV resistance in 1 patient) who were currently under LAM treatment. Accordingly, total LAM resistance in our CHB cases was 27.3% (6.8% primary and 20.5% secondary resistance), ADV resistance was 4.5% and multidrug (LAM + ADV) resistance was 2.3%. It was concluded that, our data would be helpful in the diagnosis and management of patients with chronic liver diseases due to viral hepatitis in our region.
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PMID:[Genotype distribution of chronic hepatitis B and hepatitis C patients and investigation of the resistance patterns in hepatitis B cases]. 2054 58

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