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Competitive Polymerase Chain Reaction (competitive + polymerase_chain_reaction)
Selected AbstractsQuantitative analysis of total mitochondrial DNA: Competitive polymerase chain reaction versus real-time polymerase chain reactionJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2004Hari K. Bhat Abstract An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 ± 1.01 × 104 molecules/ng total genomic DNA using competitive PCR vs 4.90 ± 0.84 × 104 molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:180,186, 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20024 [source] Stable augmentation of activated sludge with foreign catabolic genes harboured by an indigenous dominant bacteriumENVIRONMENTAL MICROBIOLOGY, Issue 10 2002Kazuya Watanabe Summary Comamonas sp. rN7 is a phenol-degrading bacterium that represents the dominant catabolic population in activated sludge. The present study examined the utility of this bacterium for establishing foreign catabolic genes in phenol-digesting activated sludge. The phc genes coding for phenol hydroxylase and its transcriptional regulators of C. testosteroni R5 were integrated into the chromosome of strain rN7. The specific phenol-oxygenating activity of a resultant transformant designated rN7(R503) was three times higher than the activity of strain rN7, and the phc genes were stably inherited by rN7(R503) grown in a non-selective laboratory medium. Inoculation of phenol-acclimatized activated sludge with rN7(R503) resulted in a high phenol-oxygenating activity and improved resistance to phenol-shock loading compared to sludge inoculated with either no cells, rN7 or R5. Quantitative competitive polymerase chain reaction (PCR) showed that the phc genes were retained in the rN7(R503)-inoculated sludge at a density of more than 108 copies per ml of mixed liquor for more than 35 days, whereas those in the R5-inoculated sludge disappeared rapidly. No transfer of the phc genes to other indigenous populations was apparent in the rN7(R503)-harbouring sludge. From these results, we concluded that the phenol treatment of the activated sludge was enhanced by the phc genes harboured by the rN7(R503) population. This study suggests a possible bioaugmentation strategy for stably utilizing foreign catabolic genes in natural ecosystems. [source] Quantitative analysis of total mitochondrial DNA: Competitive polymerase chain reaction versus real-time polymerase chain reactionJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2004Hari K. Bhat Abstract An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 ± 1.01 × 104 molecules/ng total genomic DNA using competitive PCR vs 4.90 ± 0.84 × 104 molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:180,186, 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20024 [source] Study of Cytochrome P4502E1 mRNA Level of Mononuclear Cells in Patients With Alcoholic Liver DiseaseALCOHOLISM, Issue 2001Hirokazu Yano Background: Cytochrome P-4502E1 (CYP2E1) is an important enzyme because of its unique ability to convert many substrates to cytotoxins. The increased production of reactive intermediates by elevated enzyme concentrations leads to various pathological conditions. Therefore, it is important to detect induced CYP2E1 levels in alcoholic individuals to avoid xenobiotic-promoted liver injury. In the present investigation, we detected CYP2E1 mRNA levels of mononuclear cells obtained from 10 ml of blood by using competitive polymerase chain reaction (PCR) method. Methods: Mononuclear cells were obtained from healthy individuals who did and did not drink habitually and patients with alcoholic liver disease (ALD). Complementary DNA synthesis was performed with RNA obtained from mononuclear cells by reverse transcription-PCR. Competitive PCR of CYP2E1 was performed with the sense (5,-CTGCAACGTCATA-GCCGACA-3,) and antisense (5,-TCCATTTCCACGAGCAGGCA-3,) primer and competitor DNA. Competitive PCR of ,-actin also was performed. Electrophoresis was scanned, and each band was digitized. The concentration of CYP2E1 and ,-actin mRNA was calculated from the ratio of competitor DNA. Results: In healthy individuals who did and did not drink habitually, CYP2E1 mRNA levels were 103.3 copies/,l RNA and 101.7 copies/,l RNA, respectively. In actively drinking patients with ALD, CYP2E1 mRNA levels were 103.5 copies/,l RNA, but those levels decreased to 101.7 copies/,l RNA after 4 days of abstinence. No significant difference was observed in CYP2E1 mRNA levels between alcoholic fibrosis and cirrhosis. As control, we measured ,-actin mRNA levels in mononuclear cells in all samples. The mean value of ,-actin mRNA was 104.3 copies/,l RNA in all cases, which included patients with ALD. Conclusions: The results demonstrated that it is possible to measure the CYP2E1 mRNA levels of mononuclear cells in a 10 ml blood sample. The CYP2E1 mRNA level in mononuclear cells increases during drinking and decreases in abstinence for a short period of 3 to 4 days. It is concluded that CYP2E1 mRNA level may be used as an effective marker for alcoholic intake. [source] Three temporal classes of gene expression during the Chlamydia trachomatis developmental cycleMOLECULAR MICROBIOLOGY, Issue 4 2000E. I. Shaw The obligate intracellular bacterium Chlamydia trachomatis has a unique developmental cycle that involves functionally and morphologically distinct cell types adapted for extracellular survival and intracellular multiplication. Infection is initiated by an environmentally resistant cell type called an elementary body (EB). Over the first several hours of infection, EBs differentiate into a larger replicative form, termed the reticulate body (RB). Late in the infectious process, RBs asynchronously begin to differentiate back to EBs, which accumulate within the lumen of the inclusion until released from the host cell for subsequent rounds of infection. In an effort to characterize temporal gene expression in relation to the chlamydial developmental cycle, we have used quantitative,competitive polymerase chain reaction (QC-PCR) and reverse transcription (RT)-PCR techniques. These analyses demonstrate that C. trachomatis double their DNA content every 2,3 h, with synthesis beginning between 2 and 4 h after infection. We determined the onset of transcription of specific temporal classes of developmentally expressed genes. RT-PCR analysis was performed on several genes encoding key enzymes or components of essential biochemical pathways and functions. This comparison encompassed approximately 8% of open reading frames on the C. trachomatis genome. In analysis of total RNA samples harvested at 2, 6, 12 and 20 h after infection, using conditions under which a single chlamydial transcript per infected cell is detected, three major temporal classes of gene expression were resolved. Initiation of transcription appears to occur in three temporal classes which we have operationally defined as: early, which are detected by 2 h after infection during the germination of EBs to RBs; mid-cycle, which appear between 6 and 12 h after infection and represent transcripts expressed during the growth and multiplication of RBs; or late, which appear between 12 and 20 h after infection and represent those genes transcribed during the terminal differentiation of RBs to EBs. Collectively, the data suggest that chlamydial early gene functions are weighted toward initiation of macromolecular synthesis and the establishment of their intracellular niche by modification of the inclusion membrane. Surprisingly, representative enzymes of intermediary metabolism and structural proteins do not appear to be transcribed until 10,12 h after infection; coinciding with the onset of observed binary fission of RBs. Late gene functions appear to be predominately those associated with the terminal differentiation of RBs back to EBs. [source] |