300394 VO Introduction to molecular techniques (2024W)

Combining morphology and molecular biology techniques is vital for thorough studies of diverse biological processes. Recently, genome sequencing provided us with new sets of phylogenetic information and allowed investigation of how the activity of individual genes is regulated. Different analyses based on switching particular genes on or off in particular places in the organism or in cultured cells gave crucial insights into their function. At present, biology is able not only to describe a structure or a process, but also to understand how this structure came to be and what genes were regulating its development.
The goal of this course is to give an overview of the routine molecular methods used in the modern zoology and developmental biology studies. The course is primarily aimed for the Bachelor students of all curricular directions and for the Master students specializing in non-molecular aspects of zoology.

Lecture 1: DNA chemistry “light version”
1. Structure of the DNA chain, base pairing, DNA synthesis
2. Enzymes cutting, polymerizing and modifying DNA
3. Polymerase chain reaction and its applications (RACE PCR, degenerate primer PCR etc.)
4. Vectors for cloning DNA pieces
5. Gel electrophoresis

Lecture 2: Genome sequencing
1. Old and new methods of genome sequencing
2. Analysis of the genome data
3. Transcriptome analysis
4. Examples of the use of the genome data

Lecture 3: Gene expression analysis (Part 1)
1. Basics of transcription, splicing and translation
2. Reverse transcription
3. Quantifying the amount of transcripts
4. Localizing RNA in the tissue by in situ hybridization
5. Polyclonal and monoclonal antibodies
6. Localizing proteins in the tissue and in vitro

Lecture 4: Gene expression analysis (Part 2)
1. Structure of a gene
2. Regulatory elements of the gene: promoters, enhancers, insulators
3. Reporter constructs
4. Ways to search for regulatory elements: deletion mapping, phylogenetic footprinting, DNase footprinting, X-ChIP, reporter assays

Lecture 5: Gene expression analysis (Part 3)
1. Testing gene regulatory elements
2. Histone and DNA modifications: role of chromatin state in the regulation of transcription
3. Ways to analyze chromatin state
4. The use of chromatin state data for prediction of the regulatory elements

Lecture 6: Gene function analysis (Part 1)
1. RNA interference
2. Morpholino knockdown and mRNA rescue experiments
3. Generation of genetic knockouts and knockins
4. Conditional knockouts

Lecture 7: Gene function analysis (Part 2)
1. Developmental signaling pathways and ways to analyze them
2. Generating dominant negative and constitutively active forms of proteins
3. Assaying protein-protein interactions (two-hybrid screen, BiFC)