CG080 Methods in genomics and proteomics

Faculty of Science
Spring 2013
Extent and Intensity
2/0. 2 credit(s) (plus 2 credits for an exam). Type of Completion: zk (examination).
Teacher(s)
Mgr. Radka Dopitová, Ph.D. (lecturer)
prof. RNDr. Jiří Fajkus, CSc. (lecturer)
doc. Mgr. Miloslava Fojtová, CSc. (lecturer)
doc. Mgr. Jan Havliš, Dr. (lecturer)
doc. RNDr. Jan Hejátko, Ph.D. (lecturer)
doc. RNDr. Jaromír Marek, Ph.D. (lecturer)
doc. Mgr. Jan Paleček, Dr. rer. nat. (lecturer)
Mgr. Petra Procházková Schrumpfová, Ph.D. (lecturer)
prof. RNDr. Zbyněk Zdráhal, Dr. (lecturer)
Mgr. Markéta Žďárská, Ph.D. (lecturer)
Guaranteed by
prof. RNDr. Jiří Fajkus, CSc.
Department of Biochemistry – Chemistry Section – Faculty of Science
Contact Person: prof. RNDr. Jiří Fajkus, CSc.
Supplier department: Department of Biochemistry – Chemistry Section – Faculty of Science
Timetable
Tue 9:00–10:50 C02/211
Prerequisites
basic knowledge of analytical chemistry, biochemistry and molecular biology
Course Enrolment Limitations
The course is also offered to the students of the fields other than those the course is directly associated with.
fields of study / plans the course is directly associated with
Course objectives
At the end ot the course students should be able to: explain principles of basic methods of genomic and proteomic analysis; to propose appropriate methods for analysis of given sample type with respect to purpose of analysis
Syllabus
  • Genomics
  • Isolation of DNA and RNA. Electrophoresis of nucleic acids.
  • DNA manipulation techniques (digestion, cloning, labelling) - enzymes frequently used in molecular biology (restriction endonucleases I, II, III, methylation sensitive enzymes, isoschizomeres). Cloning vectors (bacteriophages, plasmids, cosmids, bacterial artificial chromosomes, yeast artificial chromosomes). Cloning into plasmid vectors (ligation and transformation, screening of colonies). Radioactive and non-radioactive labelling of nucleic acids.
  • Techniques based on DNA renaturation (Southern blot, northern blot, PCR, FISH, microarrays); handling with RNA. Design of the experiment - digestion of DNA by methylation sensitive enzymes, electrophoresis, transfer of DNA fragments to the membrane, radioactive labelling of DNA probe, hybridization, interpretation of results. PCR - principles, history, DNA polymerases. PCR optimization, temperature gradient. FISH - principles, examples, probes. Fibre FISH, multiplex FISH, RNA fish, analysis of repetitive sequences, application in diagnostics. Microarrays - principle, advantages for complex analysis of gene expression, interpretation of results.
  • Sequencing, analysis of gene expression - principles of the chemical and Sanger sequencing, automatic capilar sequencing machines, next-generation sequencing). Analysis of gene expression at the RNA and protein levels, in vitro and in vivo approaches. Alternative splicing, analysis. Quantitative RT-PCR, cDNA microarrays.
  • Mutants and their use in genomics: Basic approaches of functional genomics, reverse and forward genetics. Insertional mutagenesis in forward and reverse genetics, EMS mutagenesis, identification of mutated loci via map-based cloning and next-gen sequencing. EMS mutagenesis in reverse genetics – tilling.
  • Proteomics
  • Preparation of protein isolates and fractionation/separation of proteins and peptides - basic methods of protein isolation from various sample types; electrophoretic separation techniques (IEF, SDS-PAGE, 2-D gel electrophoresis, DIGE, etc.);liquid chromatography; separation procedures for analysis of phosphoproteins and glycosyled proteins; multidimensional procedures for analysis of complex protein samples
  • Mass spectrometry of proteins - basic types of ionization techniques (ESI and MALDI) and MS instrumentation (TOF, ion trap, FTMS, hybrid instruments); protein identification methods; characterization of protein modifications; methods of protein quantification (relative and absolute quantification techniques)
  • Protein complex analyis (isolation of complexes, cross-linking, analysis, verification); sequential and parallel analyses, methods of protein-protein interaction study (Y2H; BiFC; mbSUS; MeRA; SEAM – tagging myc, TAP, FLAG, His; ion mobility utilisation); stoichiometry of complexes - protein and peptide quantification by MS; structure of complexes - molecular cross-linking, methods (FTICR MS)
  • Methods of macromolecular structural analysis - crystallography, NMR, circular dichroism - advantages end disadvantages. Basic principles and methods of protein crystallography - macromolecular crystallization techniques, diffraction experiment, the phase problem, methods of solving the phase problem in proteins, electron density maps, structural model, model refinement.
  • Recombinant protein preparation – selection of host organism for recombinant protein expression; E.coli expression system; structure of expression vector; selection of E.coli host strain with respect to possible problems such as protein toxicity, codon usage; protein stability; targeting of protein expression; inclusion bodies and optimalization of protein expression in soluble form; principles for protein purification; basic chromatografic methods; analysis of protein purity; fusion proteins; afinity purification; storage of purified proteins
Literature
  • Real-time PCR. Edited by M. Tevfik Dorak. New York, N.Y.: Taylor & Francis, 2006, xxvi, 333. ISBN 041537734X. URL info
  • ŠMAJS, David, S.J. NORRIS and G.M. WEINSTOCK. Construction of Treponema pallidum BAC library for functional and genomic apllications. In: Zhao,S. et al. (eds): Methods in Molecular Biology Series. Bacterial Artificial Chromosomes: Methods and Protocols. Washington, D.C. USA, 2002. info
Teaching methods
lectures
Assessment methods
final written test
Language of instruction
Czech
Further Comments
Study Materials
The course is taught annually.
The course is also listed under the following terms Spring 2011 - only for the accreditation, Spring 2014, Spring 2015, Spring 2016, Spring 2017, spring 2018, Spring 2019, Spring 2020, Spring 2021, Spring 2022, Spring 2023, Spring 2024, Spring 2025.
  • Enrolment Statistics (Spring 2013, recent)
  • Permalink: https://is.muni.cz/course/sci/spring2013/CG080