This 69 page document includes lecture notes/summary of all lectures given in the course Advanced Molecular Biology (Radboud University bachelor 2nd year/pre-master) FWI-BB017C 6EC. My grade = 8,0.
It includes a table of contents where you can easily find the 4 themes with all different topics i...
Advanced Molecular Biology (2nd year)
Molecular Cell Biology 9th edition (Achieve has extra tests)
Lectures, Self-study (reading relevant parts of book), Q&A, Tutorials (discuss questions related to the
subjects of the module so prepare) and Team based learning for cancer (test and synthetise your
knowledge about entire course. Virtual practical online experiments. Exam min 5.5. Check learning
outcomes for detailed list of subjects covered via cirrus. Practice exam. Exam mainly open questions.
Everything in the exam comes from the lectures.
Journal clubs is only 1 day morning or afternoon (to present). Journal club is 20% of grade. Make
Journal club in your own time. It focuses of scientific insight 60%, presentation 20% and participation
10% (e.g. asking questions). You have a nature or science paper. Read other papers or reviews to
understand. Identify a key message of the paper and select key experiments and associated figures.
Critically evaluate whether these experiments are properly performed/controlled and whether they
support the message of the authors. Identify questions you cant answer yourself to ask in the
response college. Prepare and practice your presentation. 15 min in English (save as powerpoint).
Introduction of 3 min with an introductory figure e.g. out the book (link paper to lectures). Present
result and key experiments in 10 min understandable for other students. Conclusion and discussion
in 2 min. All students in the group have to present a part of the presentation (5 min). After 5 min
from questions. Short powerpoint presentation (about 15 slides).
Inhoudsopgave
Theme 1 .................................................................................................................................................. 2
Techniques in Molecular Biology ........................................................................................................ 2
Measure amount of DNA/RNA or protein ...................................................................................... 3
Define spatial and temporal distribution of DNA/RNA/Protein ..................................................... 6
Analyses of molecular interactions ................................................................................................. 8
Investigate the consequence of functional perturbations ............................................................. 9
Transcriptional control of gene expression 1 ................................................................................... 13
Basics of transcriptional regulation (prokaryotes and eukaryotes) .............................................. 13
RNA polymerases .......................................................................................................................... 16
Transcriptional control of gene expression 2 ................................................................................... 18
Eukaryotic gene control elements and associated transcription factors ..................................... 18
Transcriptional control of gene expression 3 ................................................................................... 25
Regulation of gene expression ...................................................................................................... 25
Chromatin and gene regulation .................................................................................................... 27
Theme 2 ................................................................................................................................................ 31
Post-transcriptional gene control 1 .................................................................................................. 31
Post-transcriptional gene control 2 .................................................................................................. 37
Post-transcriptional gene control 3 .................................................................................................. 41
Theme 3 ................................................................................................................................................ 47
1
, DNA metabolism ............................................................................................................................... 47
Cell Cycle ........................................................................................................................................... 57
Theme 4 ................................................................................................................................................ 66
Cancer ............................................................................................................................................... 66
Theme 1
Techniques in Molecular Biology
Overview of techniques. Details and Links/references in the slides. In the exam you need some
understanding of the techniques not in detail. You don’t need to explain the techniques but you
need to be able to interpret the results. E.g. you get a gel and you need to explain what the result is.
Model organisms (like yeast, fruit fly, and mouse) are highly useful in understanding human
diseases. Your findings can be confirmed in humans (e.g. liver = hepatocyte) in three ways:
- Primary hepatocyte culture tissue cells: have a limited life span, difficult to genetically
manipulate and quickly looses cellular identity
- Immortalized cell line: able to manipulate, they are cancerous cells with little resemblance to
hepatocytes (liver cells)
- Induced pluripotent stem cells (iPS) and differentiate them to hepatocytes with cytokines:
can be manipulated, can mimic in vivo situation better than cell lines
Cell aggregates in an artificial matrix self-organize into tissue-like structures. Instead of 2D in Petri
dish now cells clump/self-organize into 3D organoids.
Absolute: how many molecules are present in cell/samples
Relative: how much more or less molecules are present in different cells/samples
2
,Absolute is harder to measure than relative measurement (e.g.protein difference in a gel) because it
requires standards with known quantities/concentrations.
Accuracy: how close a measurement is to the true or accepted value
Precision: how close measurement is the same item are to each other
Absolute measurement should be both accurate and precise. Relative measurement do not
necessarily need to be accurate as far as the control is suffering from the same bias/inaccuracy.
Measure amount of DNA/RNA or protein
Quantitative PCR (qPCR)
Measure sample dsDNA/RNA concentration via incorporation of dsDNA-specific fluorescent dye
(SYBR green). Number of cycles needed to reach threshold is proportional to the number of starting
molecules present in the sample. Melting curves enables testing if the product amplified is the
desired product. mRNAs need to be converted to cDNA by RT-qPCR first. Standard curve is used to
determine amount of DNA present.
Southern and Northern blots
Southern blot detects specific DNA sequences in sample (restriction enzymes to digest the DNA and
separated on agarose gel). Northern blot detects messenger or micro RNA in samples separated on
agarose gel. The DNA/RNA is then transferred to nitrocellulose membrane by blotting and detected
by radioactive labelled probes.
Western blot
3
, Detect specific proteins of sample (separated on mass/size) on polyacrylamide gel, under denaturing
condition (SDS-PAGE). Proteins transfered via electrophoresis from gel to membrane. Blocked and
incubated with antibodies. Define size amount of protein.
Microarray
Measure all mRNA/cDNA in a sample. RNAs are labelled with green or red fluorescent dye. Only
when complementary probe is present on the array the RNAs bind and can be analyzed.
NGS (next generation sequencing)
Enables sequencing of millions of DNA/cDNA fragments, so quantification of all DNA/mRNA present
in a sample. Illumina requires adaptor sequences to each fragment, used for phase amplification of
separate clusters (identical sequences). Using ddNTps the clusters are sequenced and imaged after
incorporation of each nucleotide.
4
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