CG080 Methods in genomics and proteomics

Faculty of Science
Spring 2019
Extent and Intensity
2/0/0. 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)
Mgr. Tomáš Klumpler, Ph.D. (lecturer)
RNDr. Hana Konečná (lecturer)
Mgr. Gabriela Lochmanová, Ph.D. (lecturer)
doc. Mgr. Jan Paleček, Dr. rer. nat. (lecturer)
doc. 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.
National Centre for Biomolecular Research – Faculty of Science
Contact Person: prof. RNDr. Jiří Fajkus, CSc.
Supplier department: National Centre for Biomolecular Research – Faculty of Science
Timetable
Mon 18. 2. to Fri 17. 5. 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
major goal of this subject is to acquaint students overall and generally with methods used in genomics and proteomics.
Learning outcomes
at the end of 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, labeling) - enzymes frequently used in molecular biology (restriction endonucleases I, II, III, methylation sensitive enzymes, isoschizomers). 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 labeling 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 labeling 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 glycosylated 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 analysis (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 optimization of protein expression in soluble form; principles for protein purification; basic chromatographic methods; analysis of protein purity; fusion proteins; affinity purification; storage of purified proteins
Literature
    recommended literature
  • Evolutionary genomics and systems biology. Edited by Gustavo Caetano-Anolles. Hoboken, N.J.: Wiley-Blackwell, 2010, xix, 465 p. ISBN 0470195142. info
  • PEVSNER, Jonathan. Bioinformatics and functional genomics. 2nd ed. Hoboken, N.J.: Wiley-Blackwell, 2009, xxviii, 95. ISBN 9780470085851. info
  • BUDINSKA, Eva, Otakar FOJT and Ladislav DUŠEK. Bioinformatics in Genomics and Proteomics Data. 2009. URL info
  • Biotechnology and genomics. Edited by P. K. Gupta. 1st ed. Meerut, India: Rastogi Publications, 2009, 796 p. ISBN 9788171338450. info
  • TWYMAN, R.M. Principles of proteomics. BIOS Scientific Publishers, 2008. ISBN 1 85996 273 4. info
  • EIDHAMMER, Ingvar. Computational methods for mass spectrometry proteomics. Chichester: John Wiley & Sons, 2007, x, 284. ISBN 9780470512975. info
  • CAMPBELL, A. Malcolm and Laurie J. HEYER. Discovering genomics, proteomics, and bioinformatics. 2. ed. San Francisco: CSHL Press, 2007, xv, 447. ISBN 0805382194. info
  • Genomics. Edited by Isidore Rigoutsos - G. Stephanopoulos. New York: Oxford University Press, 2007, xxi, 314 p. ISBN 9780195300819. info
  • Mass spectrometry data analysis in proteomics. Edited by Rune Matthiesen. Totowa, N. J.: Humana Press, 2007, x, 320. ISBN 9781588295637. info
  • THIELLEMENT, Hervé. Plant proteomics : methods and protocols. Totowa, N.J.: Humana Press, 2007, xiii, 399. ISBN 9781597452274. info
  • Functional genomics : methods and protocols. Edited by Michael J. Brownstein - Arkady B. Khodursky. Totowa, N.J.: Humana Press, 2003, xii, 258. ISBN 1588292916. info
  • SIMPSON, Richard J. Proteins and proteomics : a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2003, xiii, 926. ISBN 0879695544. info
  • Posttranslational modifications of proteins : tools for functional proteomics. Edited by Christoph Kannicht. Totowa, N.J.: Humana Press, 2002, xi, 322. ISBN 0896036782. info
  • Proteomics. Edited by Timothy Palzkill. New York: Kluwer Academic Publishers, 2002, viii, 127. ISBN 0-306-46895-6. info
  • Comparative genomics : empirical and analytical approches to gene order dynamics, map alignment and the evolution of gene families. Edited by David Sankoff - J. H. Nadeau. 1st ed. Dordrecht: Kluwer Academic Publishers, 2000, xiii, 557. ISBN 0792365844. info
  • Functional genomics : a practical approach. Edited by Stephen P. Hunt - Rick Livesey. 1st ed. Oxford: Oxford University Press, 2000, xviii, 253. ISBN 9780199637744. info
Teaching methods
The lecture is based on ppt presentations and their explication. Presentations themselves will be available as study materials (black-and-white printable pdf with high resolution and restricted access rights). It is recommended to attend the lecture, because of the explication, which significantly extends the presentation and because there are no available textbooks in Czech language covering certain parts of the subject. Students will test their acquired knowledge by means of "in-rough" tests, which will have similar form and content as the final examination test, they will be corrected to give the feed-back, but not graded.
Assessment methods
written & oral examination; multiple-choice test consists of sixty questions with four choices per question with always at least one correct answer. to pass the test, it is necessary to answer correctly at least 60 % of the questions; test covers complete content of the course. the examination will be finished by oral examination.
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 2013, Spring 2014, Spring 2015, Spring 2016, Spring 2017, spring 2018, Spring 2020, Spring 2021, Spring 2022, Spring 2023, Spring 2024, Spring 2025.
  • Enrolment Statistics (Spring 2019, recent)
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