BCH 5413- EXAM 1 QUESTIONS
WITH CORRECT DETAILED
ANSWERS
Negative control - Answer- tests to see if NO signal when required reagent is missing
Western: using secondary antibody only
Equal loading controls - Answer- did you really put the same amount of sample into
each lane?
probe for GAPDH or actin
Cross-reactions - Answer- Could one of your reagents be detecting something else as
well?
use BLAST (NCIB website)
Thermes Aquatic - Answer- heat stable polymerase (Taq polymerase)
PCR Denaturing - Answer- At 95 degrees C for 30 seconds
PCR annealing primers - Answer- between 30-50 degree C for 30 seconds
PCR extending primers - Answer- at 72 degree C for 1 minute
PCR reaction components - Answer- 1. Template DNA
2. Primers (forward and reverse)
3. dNTP (G, A, C, T)
4. Taq polymerase
5. Buffer + magnesium
6. Water
Uses of PCR - Answer- 1. Cloning
2. Genotyping (Triplex PCR)
3. Analyze DNA methylation patterns (Bisulfite PCR)
4. Characterize DNA and protein interactions (footprinting)
5. amplify rare footprint (Ligation-mediated PCR)
DNA Footprinting - Answer- used to analyze sequence-specific DNA-protein interactions
•DNA footprinting occurs when digestion of a specific region of DNA is prevented by
binding of a protein. The protection can be either to digestion by DNases, or to chemical
modification. The second process gives greater resolution.
, ***The footprint represents the region of
DNA protected by the protein, and therefore tells where
the protein binds
Ligation-mediated PCR - Answer- used to AMPLIFY rare footprinted fragment by a PCR
step followed by blotting and probing
Taqman - Answer- 2 primers, 1 probe
allows amplification of two sequences but is expensive and labor intensive
SYBR - Answer- dsDNA binding dye
monitors amplification of any ds DNA (no probe required), cheap, easy to design but
nonspeicific and may generate false positive
CT: - Answer- when the cycle number crosses a threshold, lower CT means more
product is present
Restriction endonucleases - Answer- Cut dsDNA into inverted palindromes
HindII sequence recognition - Answer- GTPyPuAC
CAPuPyTG
HindIII sequence recognition - Answer- A ↓ A G C T T
EcoR1 recognition sequence - Answer- G ↓ A A T T C
BamH1 recognition sequence - Answer- G ↓ G A T C C
hemimethylated state of DNA - Answer- protected against cleavage by restriction
endonuclease
first artificial plasmid - Answer- pBR322
Components of Plasmid - Answer- 1. OriC (origin of replication)
2. Selectable marker either Ap ^R (ampicillin resistance) or Tc ^R (tetracycline
resistance)
3. polylinker (MSC)
4. Lac Z
2nd generation plasmid - Answer- pUC19 (oric, lacZ, Ap^R)
polylinker - Answer- multiple cloning site (MSC) containing restriction sites
pBluescript - Answer- Contains: OriC, Amp R, F1 origin, MSC containing promoter (T7
phage promoter/21 restriction sites/T3 phage promoter)
WITH CORRECT DETAILED
ANSWERS
Negative control - Answer- tests to see if NO signal when required reagent is missing
Western: using secondary antibody only
Equal loading controls - Answer- did you really put the same amount of sample into
each lane?
probe for GAPDH or actin
Cross-reactions - Answer- Could one of your reagents be detecting something else as
well?
use BLAST (NCIB website)
Thermes Aquatic - Answer- heat stable polymerase (Taq polymerase)
PCR Denaturing - Answer- At 95 degrees C for 30 seconds
PCR annealing primers - Answer- between 30-50 degree C for 30 seconds
PCR extending primers - Answer- at 72 degree C for 1 minute
PCR reaction components - Answer- 1. Template DNA
2. Primers (forward and reverse)
3. dNTP (G, A, C, T)
4. Taq polymerase
5. Buffer + magnesium
6. Water
Uses of PCR - Answer- 1. Cloning
2. Genotyping (Triplex PCR)
3. Analyze DNA methylation patterns (Bisulfite PCR)
4. Characterize DNA and protein interactions (footprinting)
5. amplify rare footprint (Ligation-mediated PCR)
DNA Footprinting - Answer- used to analyze sequence-specific DNA-protein interactions
•DNA footprinting occurs when digestion of a specific region of DNA is prevented by
binding of a protein. The protection can be either to digestion by DNases, or to chemical
modification. The second process gives greater resolution.
, ***The footprint represents the region of
DNA protected by the protein, and therefore tells where
the protein binds
Ligation-mediated PCR - Answer- used to AMPLIFY rare footprinted fragment by a PCR
step followed by blotting and probing
Taqman - Answer- 2 primers, 1 probe
allows amplification of two sequences but is expensive and labor intensive
SYBR - Answer- dsDNA binding dye
monitors amplification of any ds DNA (no probe required), cheap, easy to design but
nonspeicific and may generate false positive
CT: - Answer- when the cycle number crosses a threshold, lower CT means more
product is present
Restriction endonucleases - Answer- Cut dsDNA into inverted palindromes
HindII sequence recognition - Answer- GTPyPuAC
CAPuPyTG
HindIII sequence recognition - Answer- A ↓ A G C T T
EcoR1 recognition sequence - Answer- G ↓ A A T T C
BamH1 recognition sequence - Answer- G ↓ G A T C C
hemimethylated state of DNA - Answer- protected against cleavage by restriction
endonuclease
first artificial plasmid - Answer- pBR322
Components of Plasmid - Answer- 1. OriC (origin of replication)
2. Selectable marker either Ap ^R (ampicillin resistance) or Tc ^R (tetracycline
resistance)
3. polylinker (MSC)
4. Lac Z
2nd generation plasmid - Answer- pUC19 (oric, lacZ, Ap^R)
polylinker - Answer- multiple cloning site (MSC) containing restriction sites
pBluescript - Answer- Contains: OriC, Amp R, F1 origin, MSC containing promoter (T7
phage promoter/21 restriction sites/T3 phage promoter)
