87594 - METODOLOGIE MOLECOLARI NELLA DIAGNOSTICA E NELLA RICERCA

Course Unit Page

Academic Year 2018/2019

Learning outcomes

The student will gain in depth knowledge of the most important methodologies commonly used in diagnostics as well as in molecular biology research. In particular, at the end of the course, the student will be familiar with the major methods used to analyse and study the most important biological macromolecules (DNA, RNA and proteins) and will be able to plan experimental approaches to study a biological problem.

Course contents

1) In-class lessons (4 CFU)

 

Methods for DNA manipulation, amplification and sequencing. Starting from the theoretical aspects of the methodologies, several lessons will be dedicated to the techniques used in molecular biology for DNA amplification (Polymerase Chain Reaction), DNA analysis and manipulation (DNA extraction from biological samples, quantification, electrophoretic separation, restriction and modification) and DNA sequencing (from the basic techniques to the modern NGS protocols).

Methods for RNA and gene expression analysis. This part of the program deals with techniques used for RNA quantification and analysis (both in vitro and in vivo). Applications for “gene-by-gene” analysis (Northern Blot analysis, Primer Extension, Real Time PCR) and for “genome-wide” approaches (micro/macro-arrays, RNA-sequencing) will be presented.

Methods for protein analysis. Methods for proteins separation and analysis (SDS-PAGE, 2D gel electrophoresis, Western Blot), as well as methodologies for the (over)expression and purification of recombinant proteins.

CRISPR/Cas9 system for genome-editing. The last part of the course will be dedicated to the detailed analysis of the CRISPR/Cas9 system, a brand new strategy for genome-editing. A comparison will be made with other tools currently used for genome editing like Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).

 

2) Laboratory part (2 CFU)

 

The wet-lab part consists of practical application of several techniques discussed during in-class lessons, including:

  • Extraction of plasmid DNA from bacterial cells and digestion with restriction endonucleases; analysis of restriction profile on agarose gel;
  • Overexpression of a recombinant protein in Escherichia coli cells;
  • Recombinant protein purification through affinity chromatography and analysis of the purified samples through SDS-PAGE
  • Protein quantification through a colorimetric assay;
  • Functional assay with the purified protein.

Readings/Bibliography

Reviews and seminal papers.

Teaching methods

Lessons, analysis and in class discussion of seminal research papers. Summarizing PowerPoint presentations.

Each student is required to carry out the experimental laboratory activities individually.

Assessment methods

The final exam will verify the acquisition of the main learning outcomes. In particular, the student will be able to:

1) analyse and discuss in details the main molecular methods used both in research and diagnostics;

2) understand, analyse and discuss research papers;

3) plan experimental approaches to study a biological problem.

The final exam consists of an oral exam (assessment of learning outcomes 1 and 3).

Teaching tools

Articles and reviews indicated by the teacher.

PC, videoprojector, board.

Wetlab course in a molecular biology lab with single workstations.

Office hours

See the website of Davide Roncarati