Bio 22 Lab Exam Questions

SDS electrophoresis: What is correct about SDS?

- It enables proteins to move in the electrical field - It disrupts non-covalent interactions between amino acids - It is a detergent - It denatures proteins

Molecular Cloning: In molecular cloning, ____ can be used as vectors. Select all correct options.

- circular DNA molecules that replicate separately from chromosomal DNA - DNA molecules capable to deliver a DNA fragment of interest to a host cell and enable its replication - plasmids and viruses

Mendelian Genetics: If your mother and father both have cystic fibrosis, which is caused by a recessive allele, the odds of you inheriting only one allele for the disorder are ____.

0 (zero)

Corn Genetics: The purple color of flowers is dominant over white color in pea plants. You should use a chi-square test to determine whether the F2 generation of a monohybrid cross between true breeding plants with purple and white flowers fit the expected genetic ratio. Which value for degrees of freedom will you choose to determine the probability associated with the calculated chi-square value?

1

Molecular Cloning: In which situation would gene cloning occur? Select all correct answers.

?- a DNA fragment is inserted directly into a bacterium and is replicated - a cDNA coding for protein X is amplified by PCR - a DNA fragment is inserted into a plasmid, which is then introduced to a bacterium where the plasmid replicates

PCR: How is cDNA amplified from mRNA?

Amplification of cDNA from mRNA using reverse transcriptase and PCR is called RT-PCR

Restriction enzymes and DNA gel electrophoresis: Answer the question using the accompanying figure. Of the fragments labeled A-D, which is closest to the negative electrode?

B Look for lane with fragment closest to TOP (negative electrode) (disregard the DNA ladder)

mRNA Extraction: You want to determine whether a new drug under development modifies a promoter sequence of the gene coding for protein X. You will incubate cultured cells in the presence or in the absence of the drug for 48 hours, then lyse the cells and isolate _____ from experimental and control cells for further analysis.

DNA

Restriction enzymes and DNA gel electrophoresis: What is a DNA ladder? In 2-3 sentences, explain how to build and use a standard curve to determine the length of DNA fragments in experimental samples.

DNA ladder refers to the shape of the standard curve made after using the HindIII restriction enzyme. There is an inverse proportion relationship between the length of a DNA fragment and its migration rate (or distance traveled through gel), this relationship is characterized by log10. The longer a DNA fragment, the lower the migration rate and the smaller the DNA fragment, the higher the migration rate (or distance traveled through the gel). We can then use the standard curve to determine the amount of base pairs in a DNA fragment by the migration rate it has. It should also be possible to determine the migration rate of a DNA fragment by the number of base pairs it has by comparing to the standard curve.

Molecular Cloning: Copies of recombinant DNA are produced in a genetically engineered organism by ____.

DNA polymerase

Corn Genetics: You are analyzing the results of a dihybrid cross, resulting in four different phenotypes using a chi-square test. The calculated chi-square value is 5.72. Using a table below, determine the probability associated with this chi-square value and decide whether your results fit the expected genetic ratio.

Degree of freedom for this particular problem would be "3". A chi-square value of 5.72 fits between a probability of .5 and .1 . This would mean between 50% and 10% probability. The actual chi-square value would be closer to around 10% probability. Both of these percentages are above the cutoff of 5%. Anything above the 5% cutoff is deemed a good fit that chance alone is responsible for the deviation between the observed and expected genetic ratio.

SDS Electrophoresis: Read the question above about protein X and two gels. You were asked to select THE MOST PROBABLE positions of the band on gel A and gel B. In which situation(s) would you expect a different result? Please explain your reasoning.

If the protein X samples in both gels had both been exposed to B-mercaptoethanol, they would most likely be at the same positions in each gel. However, because only gel A was exposed to the reducing agent, it was able to interact with the SDS which allows the electrophoresis to take place and you should get your expected molecular weight (or very similar to the expected). Gel B protein X sample was not exposed to the reducing agent meaning that its interactions with SDS were very limited. This would mean that it remains denatured and would not be able to participate very well in the electrophoresis. I predict that it would remain near the negative electrode at the top which would place it at above the expected molecular weight. That is how I came up with my answers in the previous question. Vagin: However, most probably, the protein will run faster if some disulfide bonds are not disrupted, because it won't be fully linearized and hence will be shorter. If the protein doesn't have disulfide bonds, than the result will be the same in the absence and in the presence of a reducing agent.

SDS electrophoresis: What is a reducing agent? What is its role in SDS gel electrophoresis?

In this specific case, the reducing agent is B-mercaptoethanol. It is used in this procedure to break the disulfide bonds in the protein samples. Once this is accomplished, the proteins are now able to be denatured by through interactions with the detergent. In chemistry, a reducing agent causes a compound to be reduced, meaning that compound would now gains electrons.

Molecular Cloning: Why is it important to have an antibiotic resistance gene in plasmid cloning vectors?

It allows researchers to distinguish bacteria that contain the plasmid from those that do not.

mRNA Extraction: While analyzing the RNA fraction that you isolated from mouse cells, you got the following results for absorbance at different wavelenghts: 230 nm: 1.0 260 nm: 2.0 280 nm: 1.5 Select all correct statements about your results.

Low 260/280 ratio suggests the presence of protein contaminants

PCR: Which of the components for PCR is responsible for length of product?

Primer

PCR: Which of the components for PCR is responsible for specificity of product?

Primer

Mendelian Genetics: The ability of an individual heterozygous for two different genes to produce the four possible gamete types in equal numbers reflects Mendel's Law(s) of ____.

Segregation and Independent Assortment

Gene Expression: The cystic fibrosis protein, CFRTR, is a transmembrane protein that acts as a chloride channel. The deletion of the phenylalanine 508 (ΔF508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The ΔF508-CFTR mutation prevents normal folding of the protein in the ER. The patients homozygous for this mutation suffer from the impaired chloride transport across the plasma membranes of epithelial cells, which leads to various symptoms, including coughing, difficulty in breathing, and poor growth. Please explain why patients homozygous for ΔF508-CFTR mutation have abnormal chloride transport (2 points) and which stage of CFTR expression is impaired in these patients (2 points). Be detailed in your answers.

The patients have abnormal chloride transport, because the transmembrane proteins inserted in the plasma membrane wall are malfunctioning. If irregular folding occurs while the the protein is being trafficked or during quality control, it will change the protein's function and it may not be able to perform its normal function at all once the it is inserted into the plasma membrane. In this case, the transmembrane proteins were supposed to function as chloride channels. It seems that the deltaF508-CFTR mutation has led to irregular folding in the ER lumen and the remaining chaperones were not able to help these proteins correctly fold. The stage at which the CFTR expression has been impaired in these patients is the post-translational stage. Or more specifically, the protein trafficking/quality control stage. Chaperones are there in the ER lumen to make sure that proteins are not irregularly folded, because this can cause malfunctions when the transmembrane protein (or secreted protein) is trafficked to the plasma membrane. An irregular folding before the protein can reach the plasma membrane will change that protein's function and can be devastating.

Corn Genetics: You performed a chi-square test to analyze the results of Cross C during your Corn Genetics lab. You calculated the chi-square value and found that the probability associated with with this value is 0.567. What does this probability represent?

The probability for chi-square test results indicate whether chance alone is responsible for the deviation between expected and observed genetic ratio. Anything over 5% is usually deemed as a good probability that chance alone is responsible for the deviation. A probability value of 0.567 means that the percentage is 56.7%. This means that there is a very high probability that chance alone is responsible for the deviation between observed and expected genetic ratio.

mRNA Extraction: Why do we focus on ribosomal RNA when analyze the integrity of isolated RNA?

We focus on ribosomal RNA when we analyze the integrity of isolated DNA because ribosomal RNA is the most abundant form of RNA. It also contains large and small subunits. These large subunits can be viewed when doing electrophoresis so we use ribosomal RNA when trying to compare RNA to a DNA ladder. Vagin: rRNA constitutes for the majority of RNA molecules in total RNA isolated from cells.

Gene Expression: How is lac operon transcription regulated by the presence or absence of lactose?

When lactose is available, the repressor will bind to allolactose instead of blocking RNA polymerase.

Molecular Cloning: Describe similarities and differences between amplification of the gene of interest by PCR and by cloning in bacteria.

With PCR and cloning in bacteria, you can get a huge amount of copies of a gene of interest. However, there are some differences. With PCR, you will face some limits because of the reagents. However, with cloning in bacteria, there are not these same limits because we are amplifying genes by using the natural replication mechanism used by bacteria. Also, with PCR, we have to keep doing a huge number of rounds (around 30) to get a million copies. With cloning in bacteria, bacteria will keep replicating in a large colony. This means that with cloning in bacteria, we do not have to such a large number of rounds of replication like we do with PCR.

Mendelian Genetics: If purple flower color is dominant in pea plants, a cross between true breeding P generation purple and white plants will result in ____.

all purple flowers in the F1 generation

Corn Genetics: Yellow color of corn kernel endosperm is dominant over white color. A cross between true breeding plants with yellow endosperm and true breeding plants with white endosperm will result in a corn plant with ____ kernels having ____ endosperm.

all; yellow

Gene Expression: You hypothesize that Protein X is a transcription factor required for transcription of the gene coding for Protein Y. You have cells in culture that express both Protein X and Protein Y. To test your hypothesis, you should use _________ to prevent expression of Protein _______, and then use ________ to determine whether your procedure decreased the amount of mRNA that codes for Protein _____.

anti-X siRNA; X; RT-PCR; Y

SDS electrophoresis: You loaded the same protein sample, which contains purified protein X, on two SDS gels, gel A and gel B. You are going to run gel A in the presence of β-mercaptoethanol, and gel B - in the absence of β-mercaptoethanol. Based on the number of amoni acids in protein X, you calculated an expected molecular weight, 77 kDa. You will use the following molecular weight standards (25, 50, 75, 100 and 150 kDa) on both gels. Predict the most probable position of the band that corresponds to protein X relative to the molecular weight standards. Protein X band on gel A will be ____________. Protein X band on gel B will be _____________.

at 75 kDa; below 75 kDa

Restriction enzymes and DNA gel electrophoresis: Bacteria protect restriction sites in their own DNA from being cut by restriction enzymes by ____.

chemically modifying bases within the restriction sites

Mendelian Genetics: From his experiments, Mendel concluded that each individual carries two factors, or ____, that govern the inheritance of each trait. Different versions of each factor are known as ____.

genes; alleles

Mendelian Genetics: When an individual has two different alleles for a given gene, they are ____ for that gene.

heterozygous

Restriction enzymes and DNA gel electrophoresis: Agarose gel electrophoresis separates DNA molecules according to their ____.

length

SDS Electrophoresis: Proteins are separated by SDS-PAGE based on their ______.

length

mRNA Extraction: You want to determine which genes are activated by a new drug under development. You will incubate cultured cells in the presence or in the absence of the drug for 48 hours, then lyse the cells and isolate _____ from experimental and control cells for further analysis.

mRNA

Corn Genetics: Aleurone color in corn kernels is coded by ____ gene(s), and endosperm color is coded by ____ gene(s).

multiple; a single

Restriction enzymes and DNA gel electrophoresis: When the sequence of nucleotides read in the 5'-->3' direction on one strand is same as the sequence read in the 5'-->3' direction on the complementary strand, the sequence is called a(n) ____.

palindrome

Restriction enzymes and DNA gel electrophoresis: Restriction endonucleases break ____ bonds.

phosphodiester

SDS electrophoresis: In SDS gel electrophoresis, proteins move towards the _______ electrode due to _______.

positive; their interaction with SDS

mRNA Extraction: Oligo-dT is a single-stranded sequence of deoxythymine (​dT). Which property of mRNA explains why we use beads with attached oligo-dT to purify mRNA from total RNA?

the presence of a polyA tail

Gene Expression: The genes in an operon are ____.

transcribed into a single mRNA transcript

Gene Expression: Gene regulation in both prokaryotes and eukaryotes involves ____.

transcriptional control