Modern biotechnological methods

Subject description

Proteins: Purification: protein sources; homogenization; centrifugation; ultrafiltration; dialysis; principles of liquid chromatography (gel filtration, hydrophobic, ion-exchange, affinity …) and modes of their implementation (classical, FPLC, HPLC …); electrophoretic methods (native and SDS PAGE, isoelectric focussing, Western blotting …); isolation of membrane proteins (detergents). Methods for detection, quantification and characterization of proteins: in solution, gels, biological membranes and on synthetic membranes (immunological, radioisotopic and spectroscopic methods, dyes). Structural characterization of proteins: determination of amino acid composition and sequence, post-translational modifications. Proteomics: 2D PAGE; mass spectrometry, MudPIT technology.

Nucleic acids (NA): Isolation and purification (biological sources/tissues – storage and homogenization; protection provisions; NA precipitation and centrifugation; electrophoresis and isolation of NA; chromatography; subtraction). Construction of gene library/bank (restriction enzymes, cutting and joining DNA molecules, partial digestion, PCR methods; vectors and DNA transfer, DNA cloning in various host cells, selection of recombinant clones; genomic and cDNA libraries). Gene library screening (gene specific probes, colony hybridization, expression libraries; RFLP, positional cloning, chromosome walking/jumping). DNA sequencing (Sanger method, post-genomic approaches – automatization). Characterization of NA (restriction analysis, Southern and Northern blotting; comparative sequence analysis; genetic polymorphisms and mutation analysis). Mutagenesis (random and site-specific mutagenesis; protein engineering). Heterologous gene expression (fusion proteins, secretion; mRNA analysis, RT-PCR, in situ hybridization, FISH; DNA fingerprinting; yeast two-hybrid system; differential screening, phage display, qPCR, DNA microarrays (biochips)). Transgenesis (methods; gene silencing). Genome editing (CRISPR/Cas, gene therapy, immunotherapy/CAR-T). Genome analysis (genome maping and sequencing, comparative genomics; transcriptomics). Bioinformatics, databases and Internet.

The subject is taught in programs

Objectives and competences

The main educational goal is to familiarize students with the contemporary methods and techniques of biochemistry and molecular biology especially emphasising those used in the so-called “modern biotechnology”. The course directs students to autonomous theoretical (analysis of literature, problem solving, formulation of conclusions) and experimental work (organization and planning of the work).

Teaching and learning methods

Lectures, moderated discussions, problem-based learning, laboratory demonstrations, consultations.

Expected study results

Student learns or deepens the knowledge about methods and techniques for analysis of proteins and nucleic acids. The course leads student towards application of the acquired knowledge in his research or developmental work. It teaches students to devise their analytical procedures and to resolve their research problems by organizing and designing their experimental work autonomously.

Basic sources and literature

WILLSON, K., WALKER, J. (Eds.), 2005. Principles and Techniques of Biochemistry and Molecular Biology; 6th Edition. Cambridge University Press, Cambridge (U.K.), 783 str., ISBN 0-521-82889-9.

S. B. Primrose and R. M. Twyman (2006): Principles of Gene Manipulation and Genomics; 7th Edition. Blackwell Publishing, Malden (U.S.A.), Oxford (U.K.), Carlton (Austr.), 390 str., ISBN: 1-4051-3544-1.

Zapiski predavanj, revijalni članki s področja, tekoča periodika, druga učna gradiva.