The functional genome Flashcards
What is WES used to do?
Used to capture the sequence of the coding region of the genome
What does WGS captures?
Captures the whole genome
What is the aim of NGS?
The aim is to identify potential disease causing genetic variants
What are the steps involved in candidate gene filtering using WES?
- We perform WES on a patient and make the assumption that theres an alteration in the protein and not the promoter region
- The first step of filtering is targeted sequencing of exons
- Then we remove synonymous variants
- After that we remove previously identified variants
- Finally we restrict to variants fitting dominant/recessive model of inheritance
What do filtered WES variants not prove and what do we need and how?
Filtered WES variants do not prove causality
- Need more evidence:
- You can take blood tests to see whether protein is present
- Muscle biopsy
- Look in tissue/cell affected
- How does the variant affect protein behavior
- Knockdown or over expression of gene to see its effect on phenotype
What is cell culture technique(In vitro)?
It’s the removal of cells . from animal and subsequent growth in favourable
What do primary cells have and what can be done to stop this in cell culture technique (in vitro)?
Primary cells have finite divisions but can be immobilised to provide a continuous source
Advantages of in vitro cell culture technique
Cheap and rapid
What is (in vitro) cell culture technique a good alternative to?
Good alternative to using animal models as it reduces ethical concerns
For gene knockdown in cell culture, what can we use?
We can use RNAi mediated gene silencing?
What is the shorthairpin RNA method in gene knockdown?
- Modified to include gene of interests complementary sequence
- Packaged in a vector like a plasmid and expression is controlled by a RNA polymerase III promoter
- Drives 50-70 nucleotide DNA which is complementary to gene of interest
- This exits the nucleus via a protein called exportins and cleaved by a nuclease called dicer forming siRNA
- The cleaved segments bind to RNA induced silencing complex(RISC) and direct cleavage and degradation of complementary mRNA
SiRNA method compared to shorthairpin RNA method
SiRNA method similar, however its chemically synthesised, not vector based
How do we find out where the gene of interest encoded protein is localized in protein localisation in cell culture?
Can find out by:
- Antibody staining
- Transfect cells with GFP tagged gene of interest
- Transfect cells with GFP tagged mutant gene of interest
What is the process involved in induced pluripotent stem cell in cell culture?
- You take cells from your skin
- Proliferate and let them grow
- Using certain factors, you de differentiate them into pluripotent stem cells
- Then using factors, you turn the pluripotent stem cells into what cell you want
Why is cell culture not enough?
- Cells behave differently in a petri dish than to how they behave in a whole organism
- Doesn’t stimulate the actual conditions inside an organism i.e signals from other tissues
