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Lecturer(s)
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Sedláček Zdeněk, prof. Ing. DrSc.
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Riegert Bystřická Dagmar, Mgr. Ph.D.
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Vaněček Tomáš, RNDr. Ph.D.
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Course content
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The course will be divided in three blocks. Block I: PCR, qPCR, Sanger sequencing Day 1 Familiarization with the rules in the clinical genetic laboratory and its operation. Studying the laboratory protocol for selected methods (PCR, real-time PCR), which the students will practically implement. Isolation of DNA from buccal swabs. Quality check of the isolated DNA. Real-time PCR, results analysis and evaluation. Elaboration of own protocols. Day 2 End-point PCR and PCR RFLP: Preparation of reaction mixture for PCR reaction, preparation of agarose gel, gel electrophoresis, restriction reaction preparation, analysis of the obtained results and their evaluation. Sample preparation for Sanger sequencing. Elaboration of own protocols. Day3 Sequence analysis and evaluation. Elaboration of own protocols. Short discussion of results obtained by the three different methods performed in the laboratory. Work with scientific articles, clinical results interpretation. Preparation of an own result sheet. Block II: Molecular diagnostics of human disease with panel NGS The main focus of this five-day block will be the analysis of human samples using next-generation sequencing (NGS). The course will also touch other methods of molecular pathology lab, like FISH, fragment analysis, real-time PCR, epigenetic analysis, immunohistochemistry etc Day 1 Lab tour and an introduction to the methods. Excursion to the immunohistochemistry laboratory. Day 2 Receipt and tracking of the clinical sample in the laboratory. Isolation of DNA (RNA) from the formalin-fixed, paraffin-embedded tissue. Quality check of the isolated DNA (RNA). Overview of possible subsequent methodology (case dependent). Day 3 Library preparation for next-generation sequencing. FISH laboratory methodology. Day 4 Sequencing of the NGS library. Other molecular pathology methodology. Molecular microbiology laboratory methodology. Day 5 Evaluation of obtained results. Preparation of the results report. Elaboration of own protocols. Discussion Block III: Advanced NGS approaches applied to diagnosis and research of disease Theoretical part: Comparison of targeted versus whole-genome approaches in the diagnosis of human genetic disorders, single nucleotide polymorphism (SNP) arrays (principle of the method, detection of CNVs, UPD and relatedness, variant interpretation), whole-exome and whole-genome sequencing (WES and WGS; principle of the methods, data processing, variant calling, prioritisation and interpretation). Demonstrations: Examples of patients and families where a diagnosis has been reached using SNP arrays, WES or WGS, with special focus on neurodevelopmental disorders. Practical work by the students: Practical solution of several bioinformatics tasks associated with the use of SNP arrays and WES (computer classroom). Protocols elaborated by the students.
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Learning activities and teaching methods
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unspecified
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Learning outcomes
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In this practical course, students will implement basic DNA diagnostic methods (PCR, qPCR, Sanger sequencing) in clinical genetic setting and learn advanced cytogenetic methods (Fluorescent in-situ hybridization (FISH)) and next generation sequencing (NGS) approaches to diagnosis
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Prerequisites
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Fundamental human genetics (KMB/XXX), Basic methods in molecular biology (KMB/608 or equivalent course from former university).
KMB/932 and KMB/608
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Assessment methods and criteria
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unspecified
Credit: approved protocols from the practicals (max. 3 absences)
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Recommended literature
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Theda, C., Hwang, S.H., Czajko, A. et al. Quantitation of the cellular content of saliva and buccal swab samples. Sci Rep 8, 6944 (2018). https://doi.org/10.1038/s41598-018-25311-0.
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Biolanalytics: Analytical Methods anad Concepts in Biochemistry and Molecular Biology. Friedrich Lottspeich, Joachim W. Engels, Wiley-VCH Verlag GmbH 2018.
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Chromosomal Abnormalities - A Hallmark Manifestation of Genomic Instability. InTech. DOI: 10.5772/67415. Due to the fast evolving character of the next generation sequencing approaches, students will be provided with upto-date literature in form of instructive reveiws regularly published in Lancet, JAMA and NEJM journals at the beginning of the course..
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Kalkan R (2017) The Use of Molecular Cytogenetic Techniques for the Identification of Chromosomal Abnormalities.
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