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119643_2 BIOMOLECULAR TECHNIQUES in Biologia sperimentale e bioinformatica LM-6 PROIETTI Silvia
(syllabus)
Theoretical part (32 hours) Introduction to nucleic acids and proteins handling: Methods for DNA, RNA and proteins isolation from biological samples and related analysis. Comparison between protein samples: differential-in-gel electrophoresis (DIGE). Methods for gene expression analysis: Real-time PCR. DNA microarray technology. RNA-Seq. Methods for protein-protein interaction analysis: Far-Western, Pull-down, yeast-two/three hybrid assay, Co-Immunoprecipitation, Tandem Affinity Purification (TAP) system, Phage Display, Bimolecular Fluorescent Complementation (BiFC), FRET. Methods for DNA/RNA-protein interaction analysis: Chromatin Immunoprecipitation assay (ChIP and ChIP-on-chip). Electrophoretic Mobility Shift Assay (EMSA). DNA pull-down. Southwestern. Yeast three-hybrid system. Methods for epigenetic modifications analysis : DNA Methylation analysis: Methylation-Sensitive Amplification Polymorphism (MSAP). Bisulfite (non methylation)-specific PCR and Methylation-specific PCR (MSP). ATAC-Seq. Methods for histone modifications analysis by ChiP. Mutagenesis techniques and genome editing: Site-directed mutagenesis and CRISPR-Cas9 System. Diagnostic application of PCR. Next Generation Sequencing: second and third generation sequencing platforms. Bioinformatics tools will be used for in silico prediction of interaction between biomolecules, or as complementary for the use of the discussed techniques (for input or output analysis).
Practical part (16 hours) Total RNA extraction, RT-qPCR. Genomic DNA extraction. DNA methylation analysis. Total protein isolation and protein-protein interaction assays.
(reference books)
Brown T.A. Gene cloning and DNA analysis: an introduction. 7th ed., 2016, Wiley-Blackwell. Watson J.D., Caudy A.A., Myers R.M., Witkowski J.A. Recombinant DNA, genes and genomes – a short course. 3rd ed., 2007, W.H. Freeman & Co. Lesk A.M. Introduction to Genomics. 3rd ed., 2017, Oxford University press.
Slides are available in the teaching platform. Handouts are provided by the teacher for practical activities. Non-attending students are encouraged to contact the teacher for information about the program, teaching materials, and the examination mode.
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