Anchor Polymerase Chain Reaction Display: A High-Throughput Method to Resolve, Score, and Isolate Dimorphic Genetic Markers Based on Interspersed Repetitive DNA Elements

2000 ◽  
Vol 284 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Srinivas Ayyadevara ◽  
John J Thaden ◽  
Robert J Shmookler Reis
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetic Markers ◽  
Repetitive Dna ◽  
Chain Reaction ◽  
High Throughput Method ◽  
Dna Elements ◽  
Polymerase Chain ◽  
Interspersed Repetitive Dna ◽  
Repetitive Dna Elements ◽  
Anchor Polymerase Chain Reaction

scholarly journals Molecular diagnosis of scabies using a novel probe-based polymerase chain reaction assay targeting high-copy number repetitive sequences in the Sarcoptes scabiei genome

2021 ◽  
Vol 15 (2) ◽  
pp. e0009149
Author(s):  
Lena Chng ◽  
Deborah C. Holt ◽  
Matt Field ◽  
Joshua R. Francis ◽  
Dev Tilakaratne ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Sensitivity And Specificity ◽  
Repetitive Dna ◽  
Base Pair ◽  
Copy Number ◽  
Diagnostic Performance ◽  
Sarcoptes Scabiei ◽  
Chain Reaction ◽  
Non Invasive ◽  
Polymerase Chain

Background The suboptimal sensitivity and specificity of available diagnostic methods for scabies hampers clinical management, trials of new therapies and epidemiologic studies. Additionally, parasitologic diagnosis by microscopic examination of skin scrapings requires sample collection with a sharp scalpel blade, causing discomfort to patients and difficulty in children. Polymerase chain reaction (PCR)-based diagnostic assays, combined with non-invasive sampling methods, represent an attractive approach. In this study, we aimed to develop a real-time probe-based PCR test for scabies, test a non-invasive sampling method and evaluate its diagnostic performance in two clinical settings. Methodology/Principal findings High copy-number repetitive DNA elements were identified in draft Sarcoptes scabiei genome sequences and used as assay targets for diagnostic PCR. Two suitable repetitive DNA sequences, a 375 base pair microsatellite (SSR5) and a 606 base pair long tandem repeat (SSR6), were identified. Diagnostic sensitivity and specificity were tested using relevant positive and negative control materials and compared to a published assay targeting the mitochondrial cox1 gene. Both assays were positive at a 1:100 dilution of DNA from a single mite; no amplification was observed in DNA from samples from 19 patients with other skin conditions nor from house dust, sheep or dog mites, head and body lice or from six common skin bacterial and fungal species. Moderate sensitivity of the assays was achieved in a pilot study, detecting 5/7 (71.4% [95% CI: 29.0% - 96.3%]) of clinically diagnosed untreated scabies patients). Greater sensitivity was observed in samples collected by FLOQ swabs compared to skin scrapings. Conclusions/Significance This newly developed qPCR assay, combined with the use of an alternative non-invasive swab sampling technique offers the possibility of enhanced diagnosis of scabies. Further studies will be required to better define the diagnostic performance of these tests.

scholarly journals DETERMINATION OF MOLECULAR GENETIC MARKERS IN PROGNOSIS OF THE EFFECTIVENESS OF TREATMENT OF MALIGNANT INTRACEREBRAL BRAIN TUMORS

2019 ◽  
Vol 4 ◽  
pp. 25-34
Author(s):  
Oleksandr Glavatskyi ◽  
Irina Vasileva ◽  
Olena Galanta ◽  
Hennadii Khmelnytskyi ◽  
Irina Shuba ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Brain Tumors ◽  
Real Time ◽  
Genetic Markers ◽  
Predictive Value ◽  
Molecular Genetic ◽  
Chain Reaction ◽  
Mgmt Gene ◽  
Polymerase Chain

Intracerebral malignant brain tumors remain one of the most complex problems of neuro-oncology. Today, promising results of the use of targeted drugs have been received, which determine the important diagnostic and predictive value of molecular genetic markers of glial and metastatic brain tumors. Aim: The study of the prevalence of MGMT (O6-methylguanine-DNA methyltransferase) and PTEN (phosphatase and tensin homologue deleted on chromosome 10) gene expression by real time polymerase chain reaction in tumor tissue of gliomas and brain metastases. Materials and methods: From thirty patients were received tumor material (29 cases of glioma III-IV degree of anaplasia and one case of metastatic brain lesion of adenocarcinoma). The normalized expression of MGMT and PTEN genes was determined by real-time polymerase chain reaction. Results: In all 30 (100 %) patients with tumor fragments, we determined normalized expression of MGMT and PTEN genes. In most cases, 53 % of the observations (16 out of 30 patients) showed a low normalized expression of MGMT gene (<40 c. u.) and a low normalized PTEN expression rate of 73 % (22 out of 30 patients) (<40 c. u.). The average expression level of the MGMT gene in the range from 40 to 100 c. u. (6/20 % of patients) was considered prognostic favourable for the response to temozolomide chemotherapy. Conclusions: The study of MGMT gene expression, a chemotherapy marker for temozolomide, indicates a trend toward correlation between expression levels and therapeutic efficacy. The study of the expression of the PTEN gene, the blocker of the PI3K / AKT signal pathway, indicates a different degree of expression of this enzyme in the tumour samples studied. The predictive value of the indicator for target therapy is appropriate in comparison with the EGFR mutation. Further profound analysis of the results is required with increasing number of sampling and observation period.

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