Quantification of the retrotransposon BARE-1 reveals the dynamic nature of the barley genome

Genome ◽  
2006 ◽  
Vol 49 (4) ◽  
pp. 389-396 ◽  
Author(s):  
V D Soleimani ◽  
B R Baum ◽  
D A Johnson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Evolutionary Process ◽  
Pcr Primers ◽  
Hordeum Spontaneum ◽  
Pcr Analysis ◽  
Barley Genome ◽  
Solo Ltrs ◽  
A Genome

We used quantitative real-time PCR analysis to measure the copy number of the BARE-1 retrotransposon in 5 cultivars of barley (Hordeum vulgare), as well as in samples from its wild relative, Hordeum spontaneum. Two sets of PCR primers were used to amplify regions within the long terminal repeat (LTR) and the reverse transcriptase (RT) gene of BARE-1 (GenBank accession Z17327). The LTR primers detected an average of 2.148 × 105 ± 0.012 × 105 copies per haploid genome among barley samples, whereas the RT primers detected an average of 1.588 × 104 ± 0.085 × 104 copies. The average ratio of LTR:RT was estimated to be 13.5:1. This finding indicates that more than 7% of the barley genome is occupied by BARE-1 elements in the form of solo LTRs and another 2.6% of the genome is occupied by the full-length element. Taken together, BARE-1 sequences represent approximately 9.6% of the barley genome among the barley plants used in this study. For the above estimation, a genome size of 5.44 × 103 Mb for H. vulgare and 5.39 × 103 Mb for H. spontaneum were assumed. Our study on quantification results of the BARE-1 for a small group of barley cultivars showed that there are significant differences among cultivars in terms of BARE-1 copy number, providing further evidence that BARE-1 is active and has a major role in shaping the barley genome as a result of breeding and selection. Quantification results also showed that most of the elements (> 90%) are present as truncated copies (solo LTRs). These results show that there is a high level of recombination leading to the formation of truncated elements and a subsequent DNA loss from the genome. Taken together, our study provides a glimpse into a dynamic micro-evolutionary process that is the by-product of genome reshuffling and directional selection in barley breeding programsKey words: BARE-1, genome evolution, quantification, real-time PCR, retrotransposons.

Unreliable Real-Time PCR Analysis of Human Endogenous Retrovirus-W (HERV-W) RNA Expression and DNA Copy Number in Multiple Sclerosis

2009 ◽  
Vol 25 (3) ◽  
pp. 377-378 ◽  
Author(s):  
Jeremy A. Garson ◽  
Jim F. Huggett ◽  
Stephen A. Bustin ◽  
Michael W. Pfaffl ◽  
Vladimir Benes ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Endogenous Retrovirus ◽  
Human Endogenous Retrovirus ◽  
Pcr Analysis ◽  
Rna Expression ◽  
Dna Copy Number

Retrovirus Insertion Site Analysis Following In Vivo Drug Selection in a Phase I Clinical Trial Utilizing G156A MGMT for Enhanced Chemotherapy of Advanced Malignancies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3048-3048
Author(s):  
Colin L. Sweeney ◽  
Karen Lingas ◽  
Jane S. Reese ◽  
Susan Flick ◽  
Stanton L. Gerson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Insertion Site ◽  
Pcr Analysis ◽  
Site Analysis ◽  
Cd34 Cells ◽  
Vector Copy Number ◽  
Post Infusion

Abstract The G156A mutant of the DNA repair gene O6-methylguanine DNA-methyltransferase (MGMT) confers hematopoietic resistance to O6-benzylguanine (BG) combined with DNA-alkylating agents BCNU or temozolomide, and allows for selective in vivo expansion with drug administration of murine hematopoietic progenitors transduced with G156A MGMT retrovirus. Here we report our latest findings on retroviral vector copy number and insertion site analysis following drug treatment from a Phase I clinical trial utilizing MGMT-mediated chemoprotection for enhanced treatment of advanced solid tumors. Seven patients have entered the trial and 6 have completed the cell infusion process. For all patients, autologous CD34+ cells were transduced ex vivo with an MFG retroviral vector containing the G156A MGMT gene (packaged with PG13 by the National Gene Vector Laboratory, Ken Cornetta, Director) in the presence of the fibronectin fragment CH-296 and the cytokines SCF, Tpo, and Flt-3 ligand for 72 hours with three additions of retroviral supernatant. At 72 hours following patient treatment with BG and BCNU, cells were re-infused. Prior to infusion, the average vector copy number by quantitative real-time PCR analysis for six patients was 0.34 copies per genome, with an average of 24% of CFUs transduced by standard PCR for G156A MGMT, and an average of 9% of CD34+ cells expressing the MGMT transgene by flow cytometry. In one patient with metastatic melanoma we have further analysis of insertions. For this patient, the pre-infusion vector copy number of the bulk CD34+ population was 0.54 copies per genome by real-time PCR, with 27% of CFUs transduced and 8% of CD34+ cells expressing the MGMT transgene prior to infusion. Linear amplification-mediated (LAM)-PCR analysis of retroviral insertion sites in pre-infusion CFUs from this patient confirmed a polyclonal population, with an average of 1.6 retroviral insertions per positive CFU. In this patient, BG (120 mg/m2) and BCNU (33 mg/m2) were administered at 6 weeks post-infusion, and temozolomide (300 mg/day for 5 days) was administered at 13 weeks. Peripheral blood (PB) and bone marrow (BM) granulocyte and mononuclear cells (MNCs) were collected at weeks 5, 11, 15, and 16 for DNA and CFU analysis. Vector copy number at all post-infusion time points was below the limit of detection of SYBR Green probe-based real-time PCR (<100 copies of G156A MGMT per 5000 genomes). LAM-PCR detected the vector in post-treatment samples based on an internal vector control band present in BM MNCs at week 11 and in BM granulocytes at week 16, although specific insertion sites were not detected. Standard PCR revealed 1 out of 100 CFUs from week 11 BM MNCs contained the vector, with 2 out of 30 CFUs from week 15 PB MNCs. LAM-PCR in a subset of week 11 CFUs confirmed a single insertion site present in the same PCR-positive CFU. Sequence analysis of clonal vector insertions pre- and post-infusion is ongoing, and thus far a number of sites have been characterized, adding to the emerging database of clinical retroviral insertions. These are the first data to show emergence of transduced mutant MGMT cells after nonmyeloablative conditioning in humans and suggest that despite a low frequency of vector-marked hematopoietic cells, clinical in vivo drug selection can be observed.

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 Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

ID: 316 Real-time PCR analysis of BCL2L12, a novel member of BCL2 family of the apoptosis-related genes, in human leukemia.

2006 ◽  
Vol 4 (s1) ◽  
pp. 82-82
Author(s):  
K. Floros ◽  
H. Thomadaki ◽  
S. Pavlovic ◽  
M. Talieri ◽  
M. Colovic ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Human Leukemia ◽  
Bcl2 Family

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Berta Fidalgo ◽  
Elisa Rubio ◽  
Victor Pastor ◽  
Marta Parera ◽  
Clara Ballesté-Delpierre ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Enteric Pathogens ◽  
Pcr Analysis ◽  
Culture Methods ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
Link Type ◽  
Microbiological Culture ◽  
Micro Organisms

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis. Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination. Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures. Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography. Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis. Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

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