The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR

2006 ◽  
Vol 65 (3) ◽  
pp. 476-487 ◽  
Author(s):  
Miguel A. Providenti ◽  
Jason M. O'Brien ◽  
Robyn J. Ewing ◽  
E. Suzanne Paterson ◽  
Myron L. Smith
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Sybr Green ◽  
Quantitative Real Time Pcr ◽  
Genetic Elements

Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

2008 ◽  
Vol 375 (1) ◽  
pp. 150-152 ◽  
Author(s):  
Cheng Xin Yi ◽  
Jun Zhang ◽  
Ka Man Chan ◽  
Xiao Kun Liu ◽  
Yan Hong
Keyword(s):  
Gossypium Hirsutum ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Transgene Copy Number

scholarly journals Rapid and reliable detection of α-globin copy number variations by quantitative real-time PCR

2014 ◽  
Vol 14 (1) ◽  
Author(s):  
Runa M Grimholt ◽  
Petter Urdal ◽  
Olav Klingenberg ◽  
Armin P Piehler
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Globin Gene ◽  
Copy Number Variations ◽  
Globin Genes ◽  
Quantitative Real Time Pcr ◽  
Human Genetic Disease ◽  
Large Deletions ◽  
Wide Range

Abstract Background Alpha-thalassemia is the most common human genetic disease worldwide. Copy number variations in the form of deletions of α-globin genes lead to α-thalassemia while duplications of α-globin genes can cause a severe phenotype in β-thalassemia carriers due to accentuation of globin chain imbalance. It is important to have simple and reliable methods to identify unknown or rare deletions and duplications in cases in which thalassemia is suspected but cannot be confirmed by multiplex gap-PCR. Here we describe a copy number variation assay to detect deletions and duplications in the α-globin gene cluster (HBA-CNV). Results Quantitative real-time PCR was performed using four TaqMan® assays which specifically amplify target sequences representing both the α-globin genes, the –α3.7 deletion and the HS-40 region. The copy number for each target was determined by the 2-ΔΔCq method. To validate our method, we compared the HBA-CNV method with traditional gap-PCR in 108 samples from patients referred to our laboratory for hemoglobinopathy evaluation. To determine the robustness of the four assays, we analyzed samples with and without deletions diluted to obtain different DNA concentrations. The HBA-CNV method identified the correct copy numbers in all 108 samples. All four assays showed the correct copy number within a wide range of DNA concentrations (3.2-100 ng/μL), showing that it is a robust and reliable method. By using the method in routine diagnostics of hemoglobinopathies we have also identified several deletions and duplications that are not detected with conventional gap-PCR. Conclusions HBA-CNV is able to detect all known large deletions and duplications affecting the α-globin genes, providing a flexible and simple workflow with rapid and reliable results.

Identification and copy number profiling of sex chromosome specific gene fragments in crossbred pigs with numerically normal karyotype

2020 ◽  
Vol 60 (9) ◽  
pp. 1145
Author(s):  
Vandana Yadav ◽  
Nihar Ranjan Sahoo ◽  
Pushpendra Kumar ◽  
G. K. Gaur ◽  
A. P. Sahoo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Sex Chromosome ◽  
Normal Karyotype ◽  
Autosomal Gene ◽  
Metastasis Suppressor ◽  
Quantitative Real Time Pcr ◽  
Relative Copy Number ◽  
Crossbred Pigs

We examined the copy number profile of sex chromosome specific genes in crossbred pigs with numerically normal karyotype. A total of 30 (15M + 15F) Landlly (Landrace crossbred) pigs from a research farm were cyto-screened by karyotyping to determine the number of chromosomes per diploid cell using short-term peripheral lymphocyte culture technique. All pigs had numerically normal karyotype with 38, XX (female) and 38, XY (male). Genomic DNA was extracted from cyto-screened pigs of both sexes. A set of sex chromosome (X and Y) specific single copy gene fragments along with an autosomal gene fragment were selected out of 12 pairs of primers on the basis of male specific PCR amplification, PCR and qPCR specificity. Quantitative real-time PCR was performed to study the relative copy number change of selected Phosphate repressible alkaline phosphatase X linked (PHOX) gene, using the Eukaryotic Translation Initiation Factor 1A Y-Linked (EIF1AY) gene as control and Breast Cancer Metastasis-Suppressor 1-Like (BRMS1 L- autosomal gene) as the reference gene. The relative copy number of the PHOX gene in females was found to be 1.873 times higher compared with the EIF1AY gene in males. The present study indicates that the real-time quantitative real-time PCR based copy number analysis can be helpful for the detection of sex chromosome ratio in pigs to aid as a preliminary screening for numerical sex chromosomal aneuploidies, adding to the rate of throughput of traditional cyto-screening.

Detection of BCR-ABL Transcript by Quantitative Real-Time PCR and Amp-CML (Transcript Mediated Amplification) during Imatinib Therapy for Chronic Myeloid Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4791-4791
Author(s):  
Koichi Miyamura ◽  
Fumiharu Yagasaki ◽  
Taiichi Kyo ◽  
Kazunori Ohnishi ◽  
Tomoki Naoe ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Progression Free Survival ◽  
Median Number ◽  
Myelogenous Leukemia ◽  
Small Range ◽  
Quantitative Real Time Pcr ◽  
Number Of Patients ◽  
The Government

Abstract Imatinib induces a complete cytogenetic response in the majority of patients and a reduction of BCR-ABL to 0.1% of the level at diagnosis is associated with longer progression free survival. However, the methods of BCR-ABL detection differ from study to study. Mostly, quantitative real-time PCR assay (RQ-PCR) has been used however, the control gene and expression of results have not been standardized to date. In Japan, only Amp-CML, a commercial kit composed of TMA (transcription mediated amplification) and HPA (hybridization protection assay) has been approved by the government. BCR-ABL was expressed by copy number per microgram RNA. The range of detection is between 50 to 2500 copies per assay. If 0.05 ug of RNA is used for the assay, the detection ranges between 1000 to 50000 copies per microgram RNA. However, the accuracy and sensitivity of Amp-CML in comparison with RQ-PCR are not well known. Thus we compared Amp-CML with RQ-PCR using a large number of patients enrolled in the Japan Adult Leukemia Study Group (JALSG). In the JALSG clinical trial, imatinib was used as initial therapy for 171 patients with chronic myelogenous leukemia. We measured levels of BCR-ABL transcripts in the blood of all patients in this trial before and 1, 2, 3, 6 and 12 months after the treatment using RQ-PCR and Amp-CML. In both assays the results expressed the copy numbers of BCR-ABL per microgram of RNA. The median number of BCR-ABL transcripts detected by RQ-PCR was 98,964 at diagnosis and 63,442, 33182, 3342, 331 and 98 at 0, 1, 2, 3, 6, 12 months, respectively, after treatment. The median number of BCR-ABL transcripts detected by Amp-CML was >50000 at diagnosis and >50000, 20284, 2180, <1000 and <1000 at 0, 1, 2, 3, 6, 12 months, respectively, of treatment. These results suggested that results of Amp-CML and RQ-PCR were well correlated (R=0.67), although the range of Amp-CML was restricted. In the next step, we attempted to improve the sensitivity by increasing the amount of RNA used for Amp-CML. Using 0.5 ug of RNA, the range of detection by Amp-CML is between 100 and 5000 copies, and 50 additional samples were analyzed. In this range, BCR-ABL copy number was also well correlated. (R=0.968) Using 2ug of RNA, we are investigating more sensitive methods expecting range of detection by Amp-CML is between 25 and 1250 copies. Tentatively 13 samples which showed 10–100 copy/ug by RQ-PCR was reevaluated by both methods. Ratio of Amp-AML to RQ-PCR is 1.4 in median and range is 0.3-2.1. Thus Amp-CML may have accuracy even the very small range. Thus, Amp-CML can be used for the monitoring BCR-ABL transcripts during treatment with imatinib.

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