BCH5413 Exam 1 Questions and Verified
Answers
Question 1
What is the basic difference between a Southern, a northern, and a western blot? (ie.
What does each detect?)
Correct Answer
Southern blot: detects DNA (presence/absence of gene) - uses agarose gel
Northern blot: detects RNA (RNA expression of genes in particular tissue or cell
types) - uses agarose gel containing formaldehyde
Western blot: detects protein (utilizes antibodies to detect specific proteins
extracted from cells in a sample) - uses polyacrylamide gel
Question 2
After isolating intact chromatin from cells, lightly digesting it with micrococcal
nuclease and running it on an agarose gel, you see a banding pattern of repeating
200bp fragments (200, 400, 600 etc). Explain this result.
Correct Answer
The banding patterns of repeating 200bp seen in the agarose gel represents that
amount of DNA base pairs that were associated with the nucleosome after
digestion. The micrococcal nuclease digestion will cut the DNA in areas that are
exposed in between nucleosomes. The digested DNA is then purified leaving only
the amount of DNA associated with the nucleosomes left to run the gel. The
number can vary between 200, 400, 600 etc. if the nucleosomes were protected by
proteins during digestion which did not allow the micrococcal nuclease to cut in
between the nucleosomes or there was not enough time given to allow for full
digestions, leaving multiple nucleosomes worth of DNA together.
Page 1 of 261
,Question 3
In a cloning experiment, how do you get rid of the bacteria that do not take up the
plasmid?
Correct Answer
a. Plasmids can be used that have selectable markers with antibiotic resistance, for
example ampicillin resistance genes. When the cells that take up the plasmid are
grown on media that have that antibiotic (e.g., ampicillin). BActeria that have taken
up the resistance survive, and the bacteria that do not remain susceptible and die.
Question 4
Chargaff's rule: A=G and T=C
Correct Answer
False - 1:1 for pyrimidines and purines (A+G [purines] = C+T [pyrimidines])
Question 5
What is the main principle behind bisulfite PCR?
Correct Answer
a. The main principle behind bisulfite PCR is that this sequencing relies on the
sodium bisulfite reaction. This reaction helps protect the methylcytosines from
conversion, while the unmethylated cytosines residues are converted into uracil.
After PCR, the residues are converted into thymines, whereas the methylated
cytosines will continue to appear as cytosines. The methylcytosines are therefore the
methylated versions of the cytosine bases. It is important to note with this method
of PCR that the DNA being analyzed will be significantly fragmented because the
strands are no longer complementary and are almost completely devoid of cytosine
residues. Advantages of this method include enhanced accuracy and sensitivity to
better understand methylation events and SNPs, as well as a reduction of overall
cost.
Page 2 of 261
,Question 6
a. Give a brief summary of how the IPTG induction system works.
Correct Answer
i. mRNA will not be transcribed unless a high amount of T7 RNA pol is induced. T7
RNA pol is regulated by the lac repressor system, which can be suppressed by
adding IPTG sugar molecules that bind to the lac repressor and result in the
translation of cDNA and subsequent mRNA transcription. The expression of cDNA
of interest will not be induced. So, m RNA will not be transcribed unless the T7 RNA
polymerase is expressed in the cells. The T7 RNA polymerase is under control here
of the Lac repressor system. The sugar IPTG is added once the logarithmic phase of
growth is reached. This will bind the Lac repressor, allowing the T7 RNA polymerase
to be made. Then it will interact with your DNA of interest to drive transcription of
the cDNA. The cells will then translate that cDNA into your protein.
Question 7
Why would you do site directed mutagenesis? (Give 2 reasons)
Correct Answer
a. Site directed mutagenesis (SDM) is a method that introduces mutation(s) into a
specific location in DNA. It can be used 1) to study the relationship between protein
structure and function and 2) to screen for certain mutants. Furthermore, in terms of
application, an example this method can be used in is in combination with
genetic/protein engineering as it relates to gene therapy.
Page 3 of 261
, Question 8
In Southern blot analysis
a. What are the three ways in which you can increase the stringency of a
hybridization?
Correct Answer
i. decreasing the salt concentration [Na+] or the ionic strength.
1. Na+ has that (+) that can interact with the (-) charges in DNA. This makes it easier
for H-bonds between two strands of DNA to stay together. The two strands are less
repelled from each other due to the negative charges. This is something I covered in
my DNA and RNA structure lecture. When you lower the salt, you make it harder for
DNA to H-bond and only the best matches stay together (increased stringency).
ii. increasing the hybridization and wash temperatures
1. When you increase the temperature, it is harder for H-bond to stay together and
only the best matches can stay together (again increased stringency).
iii. increasing the duration of wash
1. The more you wash, the cleaner you get - this applies to hybridization as well.
only the best matches stay together in longer washes.
Question 9
Putting things together. Dr. Gumz covered an example of combining a series of
experiments from her lectures to clone and study the mouse Per1 promoter. For each
experiment, give a brief description of how the experiment works AND describe the
purpose of including the experiment in this example.
a. Subclone to pGL3.
Correct Answer
i. pGL3 contains the reporter gene for this experiment. The experiment took the
Per1 clone from the TOPO vector to the pGL3 (reporter plasmid). pGL3 contains the
reporter gene for the Luciferase enzyme.
Page 4 of 261
Answers
Question 1
What is the basic difference between a Southern, a northern, and a western blot? (ie.
What does each detect?)
Correct Answer
Southern blot: detects DNA (presence/absence of gene) - uses agarose gel
Northern blot: detects RNA (RNA expression of genes in particular tissue or cell
types) - uses agarose gel containing formaldehyde
Western blot: detects protein (utilizes antibodies to detect specific proteins
extracted from cells in a sample) - uses polyacrylamide gel
Question 2
After isolating intact chromatin from cells, lightly digesting it with micrococcal
nuclease and running it on an agarose gel, you see a banding pattern of repeating
200bp fragments (200, 400, 600 etc). Explain this result.
Correct Answer
The banding patterns of repeating 200bp seen in the agarose gel represents that
amount of DNA base pairs that were associated with the nucleosome after
digestion. The micrococcal nuclease digestion will cut the DNA in areas that are
exposed in between nucleosomes. The digested DNA is then purified leaving only
the amount of DNA associated with the nucleosomes left to run the gel. The
number can vary between 200, 400, 600 etc. if the nucleosomes were protected by
proteins during digestion which did not allow the micrococcal nuclease to cut in
between the nucleosomes or there was not enough time given to allow for full
digestions, leaving multiple nucleosomes worth of DNA together.
Page 1 of 261
,Question 3
In a cloning experiment, how do you get rid of the bacteria that do not take up the
plasmid?
Correct Answer
a. Plasmids can be used that have selectable markers with antibiotic resistance, for
example ampicillin resistance genes. When the cells that take up the plasmid are
grown on media that have that antibiotic (e.g., ampicillin). BActeria that have taken
up the resistance survive, and the bacteria that do not remain susceptible and die.
Question 4
Chargaff's rule: A=G and T=C
Correct Answer
False - 1:1 for pyrimidines and purines (A+G [purines] = C+T [pyrimidines])
Question 5
What is the main principle behind bisulfite PCR?
Correct Answer
a. The main principle behind bisulfite PCR is that this sequencing relies on the
sodium bisulfite reaction. This reaction helps protect the methylcytosines from
conversion, while the unmethylated cytosines residues are converted into uracil.
After PCR, the residues are converted into thymines, whereas the methylated
cytosines will continue to appear as cytosines. The methylcytosines are therefore the
methylated versions of the cytosine bases. It is important to note with this method
of PCR that the DNA being analyzed will be significantly fragmented because the
strands are no longer complementary and are almost completely devoid of cytosine
residues. Advantages of this method include enhanced accuracy and sensitivity to
better understand methylation events and SNPs, as well as a reduction of overall
cost.
Page 2 of 261
,Question 6
a. Give a brief summary of how the IPTG induction system works.
Correct Answer
i. mRNA will not be transcribed unless a high amount of T7 RNA pol is induced. T7
RNA pol is regulated by the lac repressor system, which can be suppressed by
adding IPTG sugar molecules that bind to the lac repressor and result in the
translation of cDNA and subsequent mRNA transcription. The expression of cDNA
of interest will not be induced. So, m RNA will not be transcribed unless the T7 RNA
polymerase is expressed in the cells. The T7 RNA polymerase is under control here
of the Lac repressor system. The sugar IPTG is added once the logarithmic phase of
growth is reached. This will bind the Lac repressor, allowing the T7 RNA polymerase
to be made. Then it will interact with your DNA of interest to drive transcription of
the cDNA. The cells will then translate that cDNA into your protein.
Question 7
Why would you do site directed mutagenesis? (Give 2 reasons)
Correct Answer
a. Site directed mutagenesis (SDM) is a method that introduces mutation(s) into a
specific location in DNA. It can be used 1) to study the relationship between protein
structure and function and 2) to screen for certain mutants. Furthermore, in terms of
application, an example this method can be used in is in combination with
genetic/protein engineering as it relates to gene therapy.
Page 3 of 261
, Question 8
In Southern blot analysis
a. What are the three ways in which you can increase the stringency of a
hybridization?
Correct Answer
i. decreasing the salt concentration [Na+] or the ionic strength.
1. Na+ has that (+) that can interact with the (-) charges in DNA. This makes it easier
for H-bonds between two strands of DNA to stay together. The two strands are less
repelled from each other due to the negative charges. This is something I covered in
my DNA and RNA structure lecture. When you lower the salt, you make it harder for
DNA to H-bond and only the best matches stay together (increased stringency).
ii. increasing the hybridization and wash temperatures
1. When you increase the temperature, it is harder for H-bond to stay together and
only the best matches can stay together (again increased stringency).
iii. increasing the duration of wash
1. The more you wash, the cleaner you get - this applies to hybridization as well.
only the best matches stay together in longer washes.
Question 9
Putting things together. Dr. Gumz covered an example of combining a series of
experiments from her lectures to clone and study the mouse Per1 promoter. For each
experiment, give a brief description of how the experiment works AND describe the
purpose of including the experiment in this example.
a. Subclone to pGL3.
Correct Answer
i. pGL3 contains the reporter gene for this experiment. The experiment took the
Per1 clone from the TOPO vector to the pGL3 (reporter plasmid). pGL3 contains the
reporter gene for the Luciferase enzyme.
Page 4 of 261
