scholarly journals Determination of Cytochrome P450 2D6 (CYP2D6) Gene Copy Number by Real-Time Quantitative PCR

2005 ◽  
Vol 2005 (3) ◽  
pp. 248-253 ◽  
Author(s):  
Laurent Bodin ◽  
Philippe H. Beaune ◽  
Marie-Anne Loriot
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Copy Number ◽  
Gene Dosage ◽  
Gene Copy Number ◽  
Rapid Identification ◽  
Gene Copy ◽  
Cyp2d6 Gene ◽  
Real Time Quantitative Pcr ◽  
Gene Copies

Gene dosage by real-time quantitative PCR has proved to be accurate for measuring gene copy number. The aim of this study was to apply this approach to the CYP2D6 gene to allow for rapid identification of poor and ultrarapid metabolizers (0, 1, or more than 2 gene copy number). Using the2−ΔΔCtcalculation method and a duplex reaction, the number of CYP2D6 gene copies was determined. Quantitative PCR was performed on 43 samples previously analyzed by Southern blotting and long PCR including 20 samples with a heterozygous deletion, 11 with normal copy number (2 copies), and 12 samples with duplicated genes. The average ratio ranged from1.02to1.28,1.85to2.21, and2.55to3.30, respectively, for the samples with 1 copy, 2 copies, and 3 copies. This study shows that this method is sensitive enough to detect either a heterozygous gene deletion or duplication.

scholarly journals Quantification of MYCN, DDX1, and NAG Gene Copy Number in Neuroblastoma Using a Real-Time Quantitative PCR Assay

2002 ◽  
Vol 15 (2) ◽  
pp. 159-166 ◽  
Author(s):  
Katleen De Preter ◽  
Frank Speleman ◽  
Valérie Combaret ◽  
John Lunec ◽  
Geneviève Laureys ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Pcr Assay ◽  
Real Time Quantitative Pcr ◽  
Quantitative Pcr Assay

scholarly journals CYP2D6 Genotyping for Functional-Gene Dosage Analysis by Allele Copy Number Detection

2009 ◽  
Vol 55 (8) ◽  
pp. 1546-1554 ◽  
Author(s):  
Naoya Hosono ◽  
Mamoru Kato ◽  
Kazuma Kiyotani ◽  
Taisei Mushiroda ◽  
Sadaaki Takata ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Real Time ◽  
Copy Number ◽  
Gene Dosage ◽  
Functional Gene ◽  
Gene Copy ◽  
Cyp2d6 Gene ◽  
Allele Ratio ◽  
Pharmacogenetic Study ◽  
Gene Copies

Abstract Background: Cytochrome P450 2D6 (CYP2D6), one of the most important drug-metabolizing enzymes, has been reported to possess variation in the encoding CYP2D6 gene (cytochrome P450, family 2, subfamily D, polypeptide 6) that affects enzymatic activity. For the pharmacogenetic study of CYP2D6, accurate measurement of the dosage of the functional gene is essential; however, current genotyping techniques are insufficient because of their inability to provide the exact copy number of functional CYP2D6 genes. Methods: We developed 3 quantitative real-time PCR (qPCR) assays for estimating the total copy number of the CYP2D6 gene, as well as 24-multiplex PCR-based real-time Invader assays (mPCR-RETINAs) for estimating the allele ratio at each variation locus. After determining the allele copy number at each locus, we estimated the frequencies of CYP2D6 alleles in a population and the diplotype in each individual by a CNVphaser (copy number variation phaser). The qPCR assays and RETINAs used for HapMap Japanese and Chinese samples were applied to 455 Japanese individuals. Results: Forty-two individuals (9.2%) had one CYP2D6 gene copy, 207 (45.5%) had 2 copies, 161 (35.4%) had 3 copies, 40 (8.8%) had 4 copies, and 5 (1.1%) had 5 copies of the CYP2D6 gene. We found 16 different CYP2D6 alleles, with frequencies similar to those described in previous reports. In the diplotype analysis, we observed that CYP2D6*1/*1 and *1/*10-*36 were the most common diplotypes (approximately 20%) in our population. Conclusions: Our method is the first to determine the exact number of functional CYP2D6 gene copies. We believe our method will facilitate and accelerate the detailed pharmacogenetic analysis of CYP2D6.

TaqMan real-time PCR quantification strategy of CYP2D6 gene copy number for the LightCycler 2.0

2009 ◽  
Vol 403 (1-2) ◽  
pp. 207-211 ◽  
Author(s):  
Duc L. Nguyen ◽  
Julia Staeker ◽  
Barbara Laika ◽  
Werner Steimer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Cyp2d6 Gene

scholarly journals Differential quantification of CYP2D6 gene copy number by four different quantitative real-time PCR assays

2010 ◽  
pp. 1 ◽  
Author(s):  
Anuradha Ramamoorthy ◽  
David A. Flockhart ◽  
Naoya Hosono ◽  
Michiaki Kubo ◽  
Yusuke Nakamura ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Quantitative Real Time Pcr ◽  
Cyp2d6 Gene ◽  
Pcr Assays

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 Estimation of total bacteria by real-time PCR in patients with periodontal disease

2016 ◽  
Vol 144 (1-2) ◽  
pp. 10-14 ◽  
Author(s):  
Gavrilo Brajovic ◽  
Branka Popovic ◽  
Miljan Puletic ◽  
Marija Kostic ◽  
Jelena Milasin
Keyword(s):  
Periodontal Disease ◽  
Real Time ◽  
Copy Number ◽  
Quantitative Estimation ◽  
Periodontal Diseases ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Additional Tool ◽  
Significant Difference ◽  
Total Bacteria

Introduction. Periodontal diseases are associated with the presence of elevated levels of bacteria within the gingival crevice. Objective. The aim of this study was to evaluate a total amount of bacteria in subgingival plaque samples in patients with a periodontal disease. Methods. A quantitative evaluation of total bacteria amount using quantitative real-time polymerase chain reaction (qRT-PCR) was performed on 20 samples of patients with ulceronecrotic periodontitis and on 10 samples of healthy subjects. The estimation of total bacterial amount was based on gene copy number for 16S rRNA that was determined by comparing to Ct values / gene copy number of the standard curve. Results. A statistically significant difference between average gene copy number of total bacteria in periodontal patients (2.55.107) and healthy control (2.37.106) was found (p=0.01). Also, a trend of higher numbers of the gene copy in deeper periodontal lesions (>7 mm) was confirmed by a positive value of coefficient of correlation (r=0.073). Conclusion. The quantitative estimation of total bacteria based on gene copy number could be an important additional tool in diagnosing periodontitis.

scholarly journals Nosocomial Outbreak of Carbapenemase-Producing Proteus mirabilis With Two Novel Salmonella Genomic Island 1 Variants Carrying Different blaNDM–1 Gene Copies in China

2022 ◽  
Vol 12 ◽  
Author(s):  
Lang Yang ◽  
Hong He ◽  
Qichao Chen ◽  
Kaiying Wang ◽  
Yanfeng Lin ◽  
...  
Keyword(s):  
Proteus Mirabilis ◽  
Copy Number ◽  
Genomic Island ◽  
Gene Copy Number ◽  
Carbapenem Resistance ◽  
Multidrug Resistant ◽  
Gene Copy ◽  
Nosocomial Outbreak ◽  
Gene Copy Number Variation ◽  
Gene Copies

NDM-1-producing multidrug-resistant Proteus mirabilis brings formidable clinical challenges. We report a nosocomial outbreak of carbapenem-resistant P. mirabilis in China. Six P. mirabilis strains collected in the same ward showed close phylogenetic relatedness, indicating clonal expansion. Illumina and MinION sequencing revealed that three isolates harbored a novel Salmonella genomic island 1 carrying a blaNDM–1 gene (SGI1-1NDM), while three other isolates showed elevated carbapenem resistance and carried a similar SGI1 but with two blaNDM–1 gene copies (SGI1-2NDM). Four new single nucleotide mutations were present in the genomes of the two-blaNDM–1-harboring isolates, indicating later emergence of the SGI1-2NDM structure. Passage experiments indicated that both SGI variants were stably persistent in this clone without blaNDM–1 copy number changes. This study characterizes two novel blaNDM–1-harboring SGI1 variants in P. mirabilis and provides a new insight into resistance gene copy number variation in bacteria.

scholarly journals Effect of EPSPS Gene Copy Number and Glyphosate Selection on Fitness of Glyphosate-Resistant Bassia Scoparia in The Field

2021 ◽  
Author(s):  
Charlemagne Ajoc Lim ◽  
Prashant Jha ◽  
Vipan Kumar ◽  
Alan T. Dyer
Keyword(s):  
Sugar Beet ◽  
Seed Production ◽  
Great Plains ◽  
Copy Number ◽  
Gene Copy Number ◽  
Reproductive Traits ◽  
Gene Copy ◽  
Epsps Gene ◽  
Gene Copies

Abstract The widespread evolution of glyphosate-resistant (GR) Bassia scoparia in the U.S. Great Plains poses a serious threat to the long-term sustainability of GR sugar beet. Glyphosate resistance in B. scoparia is due to an increase in the EPSPS (5-enolpyruvyl-shikimate-3-phosphate) gene copy number. The variation in EPSPS gene copies among individuals from within a single GR B. scoparia population indicated a differential response to glyphosate selection. We tested the hypothesis of reduced GR B. scoparia fitness (reproductive traits) to increasing glyphosate rates (applied as single or sequential applications) potentially experienced within a GR sugar beet field. The variation in EPSPS gene copy number and total glyphosate rate (single or sequential applications) did not influence any of the reproductive traits of GR B. scoparia, except seed production. Sequential applications of glyphosate with a total rate of 2,214 g ae ha− 1 or higher prevented seed production in B. scoparia plants with 2–4 (low levels of resistance) and 5–6 (moderate levels of resistance) EPSPS gene copies. Timely sequential applications of glyphosate (full recommended rates) can potentially slow down the evolution of GR B. scoparia with low to moderate levels of resistance (2–6 EPSPS gene copies), but any survivors (highly-resistant individuals with ≥ 8 EPSPS gene copies) need to be mechanically removed before flowering from GR sugar beet fields. This research warrants the need to adopt ecologically based, multi-tactic strategies to reduce exposure of B. scoparia to glyphosate in GR sugar beet.

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