BIOC 410 EXAM QUESTIONS AND CORRECT ANSWERS
Co-immunoprecipitation - answer 1. Add primary antibody (binds to protein) and beads
with secondary antibody (binds to primary antibody) to cell lysate and incubate
2. Do a spin and wash to isolate the beads and everything bound to the beads
3. Segregate by size on SDS page gel
Immunoblot (Western) - does protein X interact with protein Y?
- Probe gel with antibody for protein suspected to bind to the protein isolated with the
primary antibody
Mass Spec. - What unknown proteins may interact with protein X? (analyze by size)
Chromatin Immunoprecipitation (ChIP) - answer 1. Cross link DNA-protein complexes in
the cells
2. Lyse cells
3. Probe with an antibody designed towards the protein of interest, precipitate
chromatin with the protein-specific antibody
4. Reversal of the crosslinking
5. Purification of DNA and quantitative PCR
Then
PCR - Does protein X interact with a specific DNA region?
Sequence - what unknown sequences does protein X bind?
Can be used to localize where origin binding proteins associate
Can compare the relative signal of this throughout the cell cycle
Can compare binding with (conditional) mutants to see if one protein is necessary for
binding
Conditional Mutants - answer - Inactivate a protein function under specific conditions
- Temperature sensitivity
- Permissive = protein functional, restrictive = not functional
- Auxin-inducible degron added to protein, when auxin is added to the cells it will be
degraded by the ubiquitin proteasome system.
, Lee Hartwell's CDC screen - answer Look for mutations in genes where there is a block
at the same point in the cell cycle, observe what stages there is arrest in S. cerevisiae
Genetic Identification - answer - To test if a specific sequence creates a sufficient origin
- Clone the gene into a plasmid containing a marker (nutritional (His in His knockouts
with no His media), drug resistance)
- If it is an origin, you would expect to see a very high transformation efficiency because
all genes would have the gene expressed if there is an origin
- If there is a few colonies, it is likely that they are a result of the gene integration into the
genome. Always will have a rare few that do this.
Short Nascent Sequencing Detection - answer - Specific origin
- Isolate short DNA sequences with a 5' RNA primer (likely near an origin) measure by
PCR with specific primers
- Extract DNA from cells (can use asynchronous but synch works better)
- Denature the DNA (heat)
- Isolate SS DNA using a sucrose gradient
- Digest the DNA with lambda exonuclease - degrades SS DNA with a 5' phosphate from
the 5' end (DNA with an RNA primer from initiation is protected).
- Do PCR with oligos specific for the origin of interest and it will amplify any RNA primed
region
- Low Ct on qPCR = active origin -can also probe for specific DNA using a southern blot
Mapping Direction of Okazaki Fragments - answer - Direct measure of global replication
- Observe a shift from continuous replication to discontinuous replication near
replication origins
- Shift from left to right
Brewer and Fangman (B&F) - answer - Single origin
Detection of Replication Bubbles/Forks
- 2-D non denaturing gel technique combined with a southern blot. Characterizing
structure of replication intermediates (bubbles and forks). Can demonstrate the
presence of a single origin of replication. Can demonstrate the presence of a single
origin.
1. Extract DNA from asynchronous cells
Co-immunoprecipitation - answer 1. Add primary antibody (binds to protein) and beads
with secondary antibody (binds to primary antibody) to cell lysate and incubate
2. Do a spin and wash to isolate the beads and everything bound to the beads
3. Segregate by size on SDS page gel
Immunoblot (Western) - does protein X interact with protein Y?
- Probe gel with antibody for protein suspected to bind to the protein isolated with the
primary antibody
Mass Spec. - What unknown proteins may interact with protein X? (analyze by size)
Chromatin Immunoprecipitation (ChIP) - answer 1. Cross link DNA-protein complexes in
the cells
2. Lyse cells
3. Probe with an antibody designed towards the protein of interest, precipitate
chromatin with the protein-specific antibody
4. Reversal of the crosslinking
5. Purification of DNA and quantitative PCR
Then
PCR - Does protein X interact with a specific DNA region?
Sequence - what unknown sequences does protein X bind?
Can be used to localize where origin binding proteins associate
Can compare the relative signal of this throughout the cell cycle
Can compare binding with (conditional) mutants to see if one protein is necessary for
binding
Conditional Mutants - answer - Inactivate a protein function under specific conditions
- Temperature sensitivity
- Permissive = protein functional, restrictive = not functional
- Auxin-inducible degron added to protein, when auxin is added to the cells it will be
degraded by the ubiquitin proteasome system.
, Lee Hartwell's CDC screen - answer Look for mutations in genes where there is a block
at the same point in the cell cycle, observe what stages there is arrest in S. cerevisiae
Genetic Identification - answer - To test if a specific sequence creates a sufficient origin
- Clone the gene into a plasmid containing a marker (nutritional (His in His knockouts
with no His media), drug resistance)
- If it is an origin, you would expect to see a very high transformation efficiency because
all genes would have the gene expressed if there is an origin
- If there is a few colonies, it is likely that they are a result of the gene integration into the
genome. Always will have a rare few that do this.
Short Nascent Sequencing Detection - answer - Specific origin
- Isolate short DNA sequences with a 5' RNA primer (likely near an origin) measure by
PCR with specific primers
- Extract DNA from cells (can use asynchronous but synch works better)
- Denature the DNA (heat)
- Isolate SS DNA using a sucrose gradient
- Digest the DNA with lambda exonuclease - degrades SS DNA with a 5' phosphate from
the 5' end (DNA with an RNA primer from initiation is protected).
- Do PCR with oligos specific for the origin of interest and it will amplify any RNA primed
region
- Low Ct on qPCR = active origin -can also probe for specific DNA using a southern blot
Mapping Direction of Okazaki Fragments - answer - Direct measure of global replication
- Observe a shift from continuous replication to discontinuous replication near
replication origins
- Shift from left to right
Brewer and Fangman (B&F) - answer - Single origin
Detection of Replication Bubbles/Forks
- 2-D non denaturing gel technique combined with a southern blot. Characterizing
structure of replication intermediates (bubbles and forks). Can demonstrate the
presence of a single origin of replication. Can demonstrate the presence of a single
origin.
1. Extract DNA from asynchronous cells
