BCH 5413 TEST BANK COMBINED
SET QUESTIONS WITH
COMPLETE SOLUTIONS
What does it mean to say that a plasmid is transiently transfected into cells? - Answer-
Foreign DNA is not integrated into genome but it Is expressed.
Look at slide 23/28 from the lecture. The figure represents the effects of dominant
negative Per1 on the expression of αENAC. For each bar on the graph, explain why it
was included and what the overall conclusion based upon the figure. - Answer- Vector-
vehicle was negative control no Per1
Vector-aldo was to look at activity without mutated per1 (positive control )
dnPer1 vehicle showing without hormone still lowered activity
Bottom was to show if that would help activity if hormone was added
Microarray - Answer- Look at global changes in gene expression, compared kidney
tumor tissue to normal tissue. How SFRP was noticed.
cloning - Answer- how gene was integrated
transfestion - Answer- needed to be stable because we are looking at growth over
weeks
protein expression - Answer- to prove that protein is actually there
in vivo effects - Answer- show downstream effects of the protein they were testing
overall conclusion - Answer- SFRP is a part of tumor phenotype
Why would you do site directed mutagenesis? (Give 2 reasons) - Answer- To introduce
or remove restriction enzyme cut sites
To introduce or remove tags for purification or identification
How has laundry detergent been improved by SDM? - Answer- It mutated methionine to
alanine in subtilisin so that the protease always stays active. Bleach can't inactivate
your laundry detergent.
In addition to an appropriate vector, what else do you need to perform SDM? - Answer-
Multiple cloning site
Antibiotic secretion marker
Origin of replication
, Synthetic oligonucleotide (primers)
Describe the basic difference between cassette and oligonucleotide mutagenesis vs.
Quikchange and Inverse PCR mutagenesis. - Answer- Cassette and Pcr utilize
restriction enzymes to remove wild type and replace with mutation
Quick and Inverse are extension of primers that contain desired mutation so the
mutation is actually in your primer
In cassette mutagenesis, what is the function of T4 PNK AND how does it carry out that
function? - Answer- Will add on 5' phosphate on. It carries out this function by having
primers that have a 5' phosphate so T4 DNA ligase can join primers with vector. This
will give you a plasmid with a mutated DNA sequence right in the middle
In Quikchange mutagenesis, what is the purpose of dpnI AND how does it carry out that
purpose? - Answer- You have your original plasmid that is methylated and needs to be
removed. Dpn1 is a nuclease that cuts at methylated sites so it will come in and chew
up you original DNA plasmid so all you have left is the new one.
After any type of mutagenesis is complete, the additional steps of transformation,
selection, miniprep, and sequencing are needed. Explain why each is necessary to
complete the mutagenesis project. - Answer- Transformation is the process where
bacteria uptake genetic material to gain a function
Selection: determine which plasmid have the mutation so plate them
Miniprep: to isolate plasmid
Sequencing: Sanger to screen for mutations and make sure other mutations were not
introduced. Need strong signals to prove you mutation is actually there.
Positive controls. For each of the following, what is the positive control and what will
happen if that step is working properly? - Answer- PCR: control plasmid and primers
can run product on the gel, makes sure you PCR is working
DpnI Digest: control plasmid and primers. blue/white to show ratio of wild type to
mutated
Ligation: cut plasmid with 1 restriction enzyme, should ligate back together
Transformation: undigested plasmid, should get a bunch of colonies
Negative controls. For each of the following, what is the negative control and what will
happen if there is a problem with that step? - Answer- PCR: no template, if PCR product
reagent is contaminated
DpnI digestion: wild type plasmid not degraded fully if colonies form
Ligation: no ligase, if colonies plasmids may not have been digested to completion
Transformation: no plasmid, if colonies form then antibiotic went bad
What is the meaning of the term transgenic? - Answer- An organism with an injected
piece of DNA inserted into its nucleus.
SET QUESTIONS WITH
COMPLETE SOLUTIONS
What does it mean to say that a plasmid is transiently transfected into cells? - Answer-
Foreign DNA is not integrated into genome but it Is expressed.
Look at slide 23/28 from the lecture. The figure represents the effects of dominant
negative Per1 on the expression of αENAC. For each bar on the graph, explain why it
was included and what the overall conclusion based upon the figure. - Answer- Vector-
vehicle was negative control no Per1
Vector-aldo was to look at activity without mutated per1 (positive control )
dnPer1 vehicle showing without hormone still lowered activity
Bottom was to show if that would help activity if hormone was added
Microarray - Answer- Look at global changes in gene expression, compared kidney
tumor tissue to normal tissue. How SFRP was noticed.
cloning - Answer- how gene was integrated
transfestion - Answer- needed to be stable because we are looking at growth over
weeks
protein expression - Answer- to prove that protein is actually there
in vivo effects - Answer- show downstream effects of the protein they were testing
overall conclusion - Answer- SFRP is a part of tumor phenotype
Why would you do site directed mutagenesis? (Give 2 reasons) - Answer- To introduce
or remove restriction enzyme cut sites
To introduce or remove tags for purification or identification
How has laundry detergent been improved by SDM? - Answer- It mutated methionine to
alanine in subtilisin so that the protease always stays active. Bleach can't inactivate
your laundry detergent.
In addition to an appropriate vector, what else do you need to perform SDM? - Answer-
Multiple cloning site
Antibiotic secretion marker
Origin of replication
, Synthetic oligonucleotide (primers)
Describe the basic difference between cassette and oligonucleotide mutagenesis vs.
Quikchange and Inverse PCR mutagenesis. - Answer- Cassette and Pcr utilize
restriction enzymes to remove wild type and replace with mutation
Quick and Inverse are extension of primers that contain desired mutation so the
mutation is actually in your primer
In cassette mutagenesis, what is the function of T4 PNK AND how does it carry out that
function? - Answer- Will add on 5' phosphate on. It carries out this function by having
primers that have a 5' phosphate so T4 DNA ligase can join primers with vector. This
will give you a plasmid with a mutated DNA sequence right in the middle
In Quikchange mutagenesis, what is the purpose of dpnI AND how does it carry out that
purpose? - Answer- You have your original plasmid that is methylated and needs to be
removed. Dpn1 is a nuclease that cuts at methylated sites so it will come in and chew
up you original DNA plasmid so all you have left is the new one.
After any type of mutagenesis is complete, the additional steps of transformation,
selection, miniprep, and sequencing are needed. Explain why each is necessary to
complete the mutagenesis project. - Answer- Transformation is the process where
bacteria uptake genetic material to gain a function
Selection: determine which plasmid have the mutation so plate them
Miniprep: to isolate plasmid
Sequencing: Sanger to screen for mutations and make sure other mutations were not
introduced. Need strong signals to prove you mutation is actually there.
Positive controls. For each of the following, what is the positive control and what will
happen if that step is working properly? - Answer- PCR: control plasmid and primers
can run product on the gel, makes sure you PCR is working
DpnI Digest: control plasmid and primers. blue/white to show ratio of wild type to
mutated
Ligation: cut plasmid with 1 restriction enzyme, should ligate back together
Transformation: undigested plasmid, should get a bunch of colonies
Negative controls. For each of the following, what is the negative control and what will
happen if there is a problem with that step? - Answer- PCR: no template, if PCR product
reagent is contaminated
DpnI digestion: wild type plasmid not degraded fully if colonies form
Ligation: no ligase, if colonies plasmids may not have been digested to completion
Transformation: no plasmid, if colonies form then antibiotic went bad
What is the meaning of the term transgenic? - Answer- An organism with an injected
piece of DNA inserted into its nucleus.
