Real-time PCR-based determination of gene copy numbers inPichia pastoris

2010 ◽  
Vol 5 (4) ◽  
pp. 413-420 ◽  
Author(s):  
Sandra Abad ◽  
Kerstin Kitz ◽  
Astrid Hörmann ◽  
Ulrike Schreiner ◽  
Franz S. Hartner ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gene Copy ◽  
Copy Numbers ◽  
Gene Copy Numbers

scholarly journals Photo-Induced Electron Transfer Real-Time PCR for Detection ofPlasmodium falciparum plasmepsin 2Gene Copy Number

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Samaly Santos Souza ◽  
Mariangela L'Episcopia ◽  
Carlo Severini ◽  
Venkatachalam Udhayakumar ◽  
Naomi W. Lucchi
Keyword(s):  
Electron Transfer ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Gene Copy ◽  
Content Type ◽  
Copy Numbers ◽  
Ofplasmodium Falciparum ◽  
Photo Induced Electron Transfer ◽  
Gene Copy Numbers

ABSTRACTPiperaquine is an important partner drug used in artemisinin-based combination therapies (ACTs). An increase in theplasmepsin 2and3gene copy numbers has been associated with decreased susceptibility ofPlasmodium falciparumto piperaquine in Cambodia. Here, we developed a photo-induced electron transfer real-time PCR (PET-PCR) assay to quantify the copy number of theP. falciparumplasmepsin 2gene (PfPM2) that can be used in countries whereP. falciparumis endemic to enhance molecular surveillance.

scholarly journals Quantitative Detection of the nosZ Gene, Encoding Nitrous Oxide Reductase, and Comparison of the Abundances of 16S rRNA, narG, nirK, and nosZ Genes in Soils

2006 ◽  
Vol 72 (8) ◽  
pp. 5181-5189 ◽  
Author(s):  
S. Henry ◽  
D. Bru ◽  
B. Stres ◽  
S. Hallet ◽  
L. Philippot
Keyword(s):  
Nitrous Oxide ◽  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Gene Copy ◽  
Nitrous Oxide Reductase ◽  
Gene Encoding ◽  
Copy Numbers ◽  
Dry Soil ◽  
Gene Copy Numbers

ABSTRACT Nitrous oxide (N2O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N2O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 101 and 102 target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 105 to 107 target copies g−1 of dry soil, whereas genes for 16S rRNA were found at 108 to 109 target copies g−1 of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils.

scholarly journals Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes

2003 ◽  
Vol 69 (12) ◽  
pp. 7289-7297 ◽  
Author(s):  
Jaana Vaitomaa ◽  
Anne Rantala ◽  
Katrianna Halinen ◽  
Leo Rouhiainen ◽  
Petra Tallberg ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Methods ◽  
Gene Copy ◽  
Quantitative Real Time Pcr ◽  
Microcystin Synthetase ◽  
Copy Numbers ◽  
Restoration Strategies ◽  
Cell Densities ◽  
Gene Copy Numbers

ABSTRACT Cyanobacterial mass occurrences in freshwater lakes are generally formed by Anabaena, Microcystis, and Planktothrix, which may produce cyclic heptapeptide hepatotoxins, microcystins. Thus far, identification of the most potent microcystin producer in a lake has not been possible due to a lack of quantitative methods. The aim of this study was to identify the microcystin-producing genera and to determine the copy numbers of microcystin synthetase gene E (mcyE) in Lake Tuusulanjärvi and Lake Hiidenvesi in Finland by quantitative real-time PCR. The microcystin concentrations and cyanobacterial cell densities of these lakes were also determined. The microcystin concentrations correlated positively with the sum of Microcystis and Anabaena mcyE copy numbers from both Lake Tuusulanjärvi and Lake Hiidenvesi, indicating that mcyE gene copy numbers can be used as surrogates for hepatotoxic Microcystis and Anabaena. The main microcystin producer in Lake Tuusulanjärvi was Microcystis spp., since average Microcystis mcyE copy numbers were >30 times more abundant than those of Anabaena. Lake Hiidenvesi seemed to contain both nontoxic and toxic Anabaena as well as toxic Microcystis strains. Identifying the most potent microcystin producer in a lake could be valuable for designing lake restoration strategies, among other uses.

Possible gene dosage effect of glutathione-S-transferases on atopic asthma: Using real-time PCR for quantification of GSTM1 and GSTT1 gene copy numbers

2004 ◽  
Vol 24 (3) ◽  
pp. 208-214 ◽  
Author(s):  
Charlotte Brasch-Andersen ◽  
Lene Christiansen ◽  
Qihua Tan ◽  
Annette Haagerup ◽  
J�rgen Vestbo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gene Dosage ◽  
Atopic Asthma ◽  
Gene Copy ◽  
Gene Dosage Effect ◽  
Gstt1 Gene ◽  
Copy Numbers ◽  
Glutathione S Transferases ◽  
Gene Copy Numbers

scholarly journals Comparison of Different Approaches To QuantifyStaphylococcus aureus Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese

2001 ◽  
Vol 67 (7) ◽  
pp. 3122-3126 ◽  
Author(s):  
Ingeborg Hein ◽  
Angelika Lehner ◽  
Petra Rieck ◽  
Kurt Klein ◽  
Ernst Brandl ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Taqman Probe ◽  
Gene Copy ◽  
Real Time Quantitative Pcr ◽  
Copy Numbers ◽  
Gene Copies ◽  
Different Types ◽  
Pure Cultures ◽  
Gene Copy Numbers

ABSTRACT Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures ofS. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60nuc gene copies/μl) than using a fluorigenic TaqMan probe (6 nuc gene copies/μl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 � 102 to 6.4 � 102 copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.

scholarly journals Assessment of bacteria and archaea levels in Çakalburnu Lagoon (İzmir) sediments by real-time PCR

2021 ◽  
Vol 38 (2) ◽  
pp. 147-154
Author(s):  
Burcu Omuzbüken ◽  
Aslı Kaçar
Keyword(s):  
Real Time ◽  
Coastal Lagoons ◽  
Methanogenic Archaea ◽  
Gene Copy ◽  
Rrna Gene ◽  
Sulfate Reducing ◽  
Copy Numbers ◽  
Lagoon Sediments ◽  
Microbial Groups ◽  
Gene Copy Numbers

Coastal lagoons are shallow water masses, discredited from the marines as a barrier that permits water to change through one or more inputs. These fragile ecosystems have a specific type of sediments with their own characteristics. Biogeochemical processes, mostly intervened by the benthic microbial loop, are significant for understanding the relationships among the lagoon and the contiguous coastal partition. This study was conducted in the Çakalburnu Lagoon (İzmir) area, which is located at the Bay of İzmir and the area covers 67 hectares. The aim of the present study is to constitute of determining the number of different microbial communities in the lagoon sediments. We collected from lagoon sediments samples at 7 stations and we applied a Real-time qPCR assay to determine levels of archaea (ARC), methanogenic archaea (MCRA), anaerobic methane oxidation archaea (ANME 1, ANME 2a, ANME 2c), bacteria (BAC) and sulfate-reducing bacteria (SRB2) in the study. The amount of maximum abundance of archaeal and bacterial 16S rRNA gene in sediments are 2,66x1010 gene copy numbers/g and 3,89x107 gene copy numbers/g, respectively. So, it was established that the archaeal abundance was intense in the lagoon sediments. The characterization of microbial diversity is significant for the comprehension of the biological fundamentals of the ecosystem. The data presented in our study contributes to the studies on preserving ecological and microbiological balance and determining biogeochemical cycles in sensitive ecosystems such as lagoons. The research will be conducted on studies to determine the abundance levels of seasonal and annual microbial groups in the future.

scholarly journals Relationship between amount of esterase and gene copy number in insecticide-resistant Myzus persicae (Sulzer)

1999 ◽  
Vol 339 (3) ◽  
pp. 737-742 ◽  
Author(s):  
Linda M. FIELD ◽  
Roger L. BLACKMAN ◽  
Chris TYLER-SMITH ◽  
Alan L. DEVONSHIRE
Keyword(s):  
Gene Amplification ◽  
Myzus Persicae ◽  
Copy Number ◽  
Transcriptional Control ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Copy Numbers ◽  
And Function ◽  
Gene Copy Numbers

Overproduction of the insecticide-degrading esterases, E4 and FE4, in peach-potato aphids, Myzus persicae (Sulzer), depends on both gene amplification and transcriptional control, the latter being associated with changes in DNA methylation. The structure and function of the aphid esterase genes have been studied but the determination of their copy number has proved difficult, a common problem with gene amplification. We have now used a combination of pulsed-field gel electrophoresis and quantitative competitive PCR to determine relative esterase gene copy numbers in aphid clones with different levels of insecticide resistance (R1, R2 and R3). There are approx. 4-fold increases between susceptible, R1, R2 and R3 aphids, reaching a maximum of approx. 80 times more genes in R3; this gives proportionate increases in esterase protein relative to susceptible aphids. Thus there is no overexpression of the amplified genes, in contrast with what was thought previously. For E4 genes, the loss of 5-methylcytosine is correlated with a loss of expression, greatly decreasing the amount of enzyme relative to the copy number.

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