BCH 5413 EXAM ONE QUESTIONS
WITH VERIFIED ANSWERS
If you want to identify the protein PTEN on a Western blot, what is secondary antibody?
- Answer-Fab can bind to primary antibody and Fc portion can bind to a HRP antibody
so when bound to TMB will light up
Which of the following increases the stringency of Southern Blot? Why? - Answer-
Increased hybridization, more Interactions between target and probe
PCR: primer - Answer-Short ssDNA sequence used in PCR, pair of primers used to
hybridize with the sample DNA and define to region of DNA that will be amplified
PCR: template - Answer-Important role in amplification, DNA must be quantified
because the success of any PCR reaction depends on the quantity and purity of
template DNA
PCR: dNTP - Answer-Added to PCR reaction with the 4 in equal amounts for optimal
base incorporation
PCR: magnesium - Answer-Functions as a cofactor for activity of DNA polymerases by
enabling incorporation of dNTPs during polymerization. Catalyze phosphodiester bond
formation between 3'-OH of primer and phosphate group of dNTP
PCR: DNA polymerase - Answer-Usually taq actually makes DNA
Name one negative and one positive control for PCR. - Answer-Positive is one you
expect to work
Negative shouldn't give you amplicons, will have no template
What value was there in the discovery and then use of Thermus Aquaticus DNA
polymerase in PCR? Why did it make the process more efficient, describe specific steps
in the process? - Answer-It is essential because regular DNA pol can't operate under
high temperature conditions of PCR. Makes it possible to do PCR on DNA samples
without having to add new DNA polymerases after every cycle
What is the main principle behind bisulfite PCR? - Answer-Used to detect DNA
methylation patterns because they are erased during PCR amplification. Current
sequencing and microarray technologies cannot distinguish between methylated and
unmethylated cytosines.
Give two advantages of TaqMan probe over Sybr Green. - Answer-Specific
hybridization between target and probe is required to generate fluorescent signal.
, Probes can be labeled with different, distinguishable report dies which allows
amplification and detection of two distinct sequences in one reaction tube
Post-PCR processing is eliminated which reduces assay labor and material costs.
In QPCR, what does a lower Ct value represent in terms of relative RNA concentration
in the starting sample? Why? - Answer-The lower Ct the larger number of target nucleic
acid in the sample. So lower Ct is more expression. They are inversely promotional.
What is the main advantage of microarray technology over QPCR? - Answer-You can
screen a lot of genes in one step and requires a lot less RNA. qPCR is used to validate
microarray results to see which groups of miRNAs are being expressed.
Choosing an E. coli protein expression system has a lot of advantages such as yield,
cost and ease. Discuss (not just list) two reasons why a researcher might not choose
this type of system. Be specific, what might their goal be and why wouldn't this system
work for them? - Answer-There are no post-translational modifications and limited cDNA
usage.
what is an inducible vector? - Answer-Basically a vector that can be turned on when it
needs to be
Why might a researcher choose an inducible vector? - Answer-To control the level of
protein expressed so that it does not accumulate to toxic levels in the cell
Give a brief summary of how the IPTG induction system works. - Answer-Add IPTG to
sequester lac repressor so that when its ready T7 can transitive RNA
How would I purify a protein if the protein has 'his-tags'? - Answer-Lyse cells and collect
protein lysate and unit over a nickel column. You then run histidine over and the tag will
release so that you can collect it. You can also cleave off the tag using enterkinase.
What is a GST tag? How does it help in protein purification? - Answer-It is a 211 amino
acid protein whose DNA sequence is frequently integrated into expression vectors for
production of recombinant proteins. Enhances solubility of many eukaryotic proteins
expressed in bacteria. It helps in protein purification because proteins can be purified or
detected based on the ability of GST to bind its substrate, glutathione.
What are the steps to be taken if the protein is insoluble? - Answer-Collect the inclusion
bodies and refold the protein
Reduce growth temp
Use low/moderate copy number plasmid vector
Fise a periplasmic targeting sequence to N-terminus
Study a more soluble protein
WITH VERIFIED ANSWERS
If you want to identify the protein PTEN on a Western blot, what is secondary antibody?
- Answer-Fab can bind to primary antibody and Fc portion can bind to a HRP antibody
so when bound to TMB will light up
Which of the following increases the stringency of Southern Blot? Why? - Answer-
Increased hybridization, more Interactions between target and probe
PCR: primer - Answer-Short ssDNA sequence used in PCR, pair of primers used to
hybridize with the sample DNA and define to region of DNA that will be amplified
PCR: template - Answer-Important role in amplification, DNA must be quantified
because the success of any PCR reaction depends on the quantity and purity of
template DNA
PCR: dNTP - Answer-Added to PCR reaction with the 4 in equal amounts for optimal
base incorporation
PCR: magnesium - Answer-Functions as a cofactor for activity of DNA polymerases by
enabling incorporation of dNTPs during polymerization. Catalyze phosphodiester bond
formation between 3'-OH of primer and phosphate group of dNTP
PCR: DNA polymerase - Answer-Usually taq actually makes DNA
Name one negative and one positive control for PCR. - Answer-Positive is one you
expect to work
Negative shouldn't give you amplicons, will have no template
What value was there in the discovery and then use of Thermus Aquaticus DNA
polymerase in PCR? Why did it make the process more efficient, describe specific steps
in the process? - Answer-It is essential because regular DNA pol can't operate under
high temperature conditions of PCR. Makes it possible to do PCR on DNA samples
without having to add new DNA polymerases after every cycle
What is the main principle behind bisulfite PCR? - Answer-Used to detect DNA
methylation patterns because they are erased during PCR amplification. Current
sequencing and microarray technologies cannot distinguish between methylated and
unmethylated cytosines.
Give two advantages of TaqMan probe over Sybr Green. - Answer-Specific
hybridization between target and probe is required to generate fluorescent signal.
, Probes can be labeled with different, distinguishable report dies which allows
amplification and detection of two distinct sequences in one reaction tube
Post-PCR processing is eliminated which reduces assay labor and material costs.
In QPCR, what does a lower Ct value represent in terms of relative RNA concentration
in the starting sample? Why? - Answer-The lower Ct the larger number of target nucleic
acid in the sample. So lower Ct is more expression. They are inversely promotional.
What is the main advantage of microarray technology over QPCR? - Answer-You can
screen a lot of genes in one step and requires a lot less RNA. qPCR is used to validate
microarray results to see which groups of miRNAs are being expressed.
Choosing an E. coli protein expression system has a lot of advantages such as yield,
cost and ease. Discuss (not just list) two reasons why a researcher might not choose
this type of system. Be specific, what might their goal be and why wouldn't this system
work for them? - Answer-There are no post-translational modifications and limited cDNA
usage.
what is an inducible vector? - Answer-Basically a vector that can be turned on when it
needs to be
Why might a researcher choose an inducible vector? - Answer-To control the level of
protein expressed so that it does not accumulate to toxic levels in the cell
Give a brief summary of how the IPTG induction system works. - Answer-Add IPTG to
sequester lac repressor so that when its ready T7 can transitive RNA
How would I purify a protein if the protein has 'his-tags'? - Answer-Lyse cells and collect
protein lysate and unit over a nickel column. You then run histidine over and the tag will
release so that you can collect it. You can also cleave off the tag using enterkinase.
What is a GST tag? How does it help in protein purification? - Answer-It is a 211 amino
acid protein whose DNA sequence is frequently integrated into expression vectors for
production of recombinant proteins. Enhances solubility of many eukaryotic proteins
expressed in bacteria. It helps in protein purification because proteins can be purified or
detected based on the ability of GST to bind its substrate, glutathione.
What are the steps to be taken if the protein is insoluble? - Answer-Collect the inclusion
bodies and refold the protein
Reduce growth temp
Use low/moderate copy number plasmid vector
Fise a periplasmic targeting sequence to N-terminus
Study a more soluble protein
