BCH 4101 Lecture Notes - Lecture 15: Synthetic Lethality, Hybridization Probe, Taqman
28 Apr 2018
School
Department
Course
Professor
December 4, 2017
Final Exam Review Session
1. During your Q-PCR experiment, you measure the fluorescence during the extension step of the cycle. Which type
of fluorescent probe are you most likely using?!
Hydrolysis (Taqman probe)!
During annealing, the hydrolysis probe binds to the target sequence!
During extension, the probe is partially displaced and the reporter is cleaved. The free reporter fluoresces!
!
Contrasts Dual Hybridization probe:!
Based on donor and acceptor fluorophore!
Accept the donor, passes its energy to the acceptor, which emits a different wavelet!
Binding occurs during annealing, so you can only measure fluorescence during annealing —> not correct!
2. Shown here are the results of hierarchical clustering of gene expression data for tumours (n = 8) of different
stages of lung cancer (genetic heat map). Can you identify a genetic signature for each stage that would allow
you to rapidly diagnose patients? (each column represents all of the measured genes in the sample)!
Circle change in gene expressions unique to the disease trying to characterize!
Find groups of genes that are coordinately regulated!
This example had a block of red genes unique to each stage (everywhere else was green)!
3. Human synthetic lethality screen (for example using CRISPR-Cas9 to introduce secondary site mutations in the
hunt for cancer therapeutic targets) most commonly use either pairs of “matched” or isogenic cell lines of the
same genetic background to create the double mutants. Why is it so important to consider isogenic cells when
designing these types of studies?!
Synthetic lethal screen: looking for two genes that work in the same or in complementary pathways!
Trying to identify two genetic mutations that would lead to death, suggesting that these two genes work in parallel
pathways!
!
Minimize the impact of genetic variability!
Make sure that the only thing that’s different is the target mutation!
4. Affinity purification-MS is a method used to detect PPIs. Part of the purification protocol involves lysis/rupture of
the cells in order to recover the protein complex. Give reasons why a method that includes cell lysis might hinder
our ability to identify PPIs.!
Used to identify protein complexes (looks at more than just binary interactions)!
Some complexes are held together by very weak interactions - breaking open the cell can make the complex fall
apart!
Protein folding might be affected - might change the conformation of the protein and alter interactions with other
proteins!
Lysing the cell may bring proteins that are normally far apart closer together, allowing them to interact and form a
complex that wouldn’t happen in the cell (since they are usually separated)!
!
ANS:!
Disrupt weak interactions!
Bring proteins together in lysate that might not normally interact!
Expose previously concealed non-native binding sites
1
