2011 May Applied DNA Technology

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Multiple Choice
Identify the choice that best completes the statement or answers the question.

Small circular molecules of DNA in bacteria are called

a.	plasmids.
b.	desmids.
c.	pili.
d.	F particles.
e.	transferrins.

Enzymes used to cut DNA molecules in recombinant DNA research are

a.	ligases.
b.	restriction enzymes.
c.	transcriptases.
d.	DNA polymerases.
e.	replicases.

Plasmids

a.	are self-reproducing circular molecules of DNA.
b.	are sites for inserting genes for amplification.
c.	may be transferred between different species of bacteria.
d.	may confer the ability to donate genetic material when bacteria conjugate.
e.	all of these

The "natural" use of restriction enzymes by bacteria is to

a.	integrate viral DNA.
b.	destroy viral DNA.
c.	repair "sticky ends."
d.	copy the bacterial genes.
e.	clone DNA.

RNA can manufacture DNA via the action of

a.	DNA polymerase.
b.	RNA polymerase.
c.	reverse transcriptase.
d.	ligase.
e.	restriction endonuclease.

For polymerase chain reaction to occur,

a.	isolated DNA molecules must be primed.
b.	all DNA fragments must be identical.
c.	the DNA must be separated into single strands.
d.	a sticky end must be available for the ligase enzyme to function.
e.	isolated DNA molecules must be primed and the DNA must be separated into single strands.

The enzyme used in the polymerase chain reaction is

a.	a restriction enzyme.
b.	reverse transcriptase.
c.	DNA polymerase.
d.	RNA replicase
e.	all of these

Suppose the lacI repressor gene were permanently silenced by an alteration to the DNA sequence. What would be the impact on the function of the lac operon?

a.	The lac operon would be transcribed but at a low level.
b.	The lac operon would be transcribed at a high level.
c.	There would be no real impact on lac operon expression.
d.	The lac operon would only be expressed when lactose was present.
e.	The lac operon would not be expressed even when lactose was present.

Some restriction endonuclease cuts the DNA in such a way that short, single stranded regions are created. We call these regions

a.	hydrogen-bonding ends
b.	sticky ends
c.	tacky ends
d.	blunt ends
e.	jagged ends

10.

I need to determine if the gene for a particular protein is the same length( same number of base pairs) in frogs, humans, and trees. Which method or methods will be most helpful to me?

a.	PCR and agarose gel electrophoresis
b.	DNA cloning and DNA hybridization
c.	restriction digest of each organisms’ genomic DNA and then agarose gel electrophoresis
d.	isolate each version of the gene using the edited mRNA and reverse transcriptase the sequnce for restriction digest
e.	agarose gel electrophoresis alone

The following image shows different plamids after a restriction digest. Each lane contains a different restriction digest with a different enzyme.

Use the figure above for the following question(s).

11.

In the figure above, which DNA fragment is the smallest?

a.	A
b.	B
c.	C
d.	D
e.	E

12.

In the figure above, which lane shows a very large DNA plasmid that was cut by twice with an enzyme?

a.	1
b.	2
c.	3
d.	4
e.	5

The following plasmid map shows the three genes on the plasmid pBIO. The GFP gene and ampicillin resistance gene (AMP^R) are regulated by a promoter that is always “on”. Therefore, the genes are always expressed once in a cell. The kanamycin resistance gene (KAN^R) is regulated by the Lac operon. Kanamycin is another antibiotic that prevents the grow of bacteria.

Plates labeled with “Amp” and “Kan” contain the corresponding antibiotics. The plate labeled with “lactose” contains the sugar lactose. A bacterial culture is transformed with pBIO and successfully transformed cells are plated on all four plates shown below.

Answer the questions based on your understanding of transformation, regulation by the Lac operon, and gene expression.

13.

You transform bacteria (E.coli) with the plasmid pBIO. The successfully transformed cells are plated onto the four plates above. On which plate will you not see any growth?

a.	LB
b.	LB/Amp
c.	LB/Amp/Kan
d.	LB/Amp/Kan/lactose
e.	All plates will have some growth

14.

Which of the following is not an observation you will make?

a.	The LB plate will show lawn growth because all plated bacteria, including those that did not take up a plasmid, will be able to grow.
b.	The LB/Amp plate will show colony growth. Each colony represents a single bacterium that took up the plasmid pBIO and grew into millions of cells during incubation.
c.	All colonies on the LB/Amp/Kan/Lactose plate will GLOW because GFP is expressed.
d.	The colonies on the LB/Amp plate will GLOW.
e.	When untransformed E.coli is plated onto LB/Amp/Kan/lactose, you will see lawn growth because lactose will help make bacteria resistant to the antibiotics.

15.

Which of the following is false about the plasmid pBIO?

a.	It is a circular piece of DNA.
b.	It codes for enzymes that make bacteria resistant to antibiotics.
c.	It codes for enzymes that make the antibiotic kanamycin.
d.	Contains three genes.
e.	It codes for a gene that makes a protein that will fluoresce.

Below is the plasmid map of pAMP. The plasmid has a single ampicillin resistance gene and an origin of replication. Restriction sites and corresponding nucleotide numbers are shown. The entire size of pAMP is 4539 base pairs (bp).

The plasmid was cut with the enzymes BamHI and EcoRI. This reaction produced two fragments with sizes 1120bp and 3419bp.

You accidentally mix up the digestion reaction with another that you were using for a separate experiment (this is why you should always label tubes!). Unable to figure out which tube contained your original digestion reaction, you run samples of both tubes in a gel along with a ladder.

The ladder standards are DNA fragments with the following sizes: 5000, 4000, 3000, 2000, and 1000.

The diagram to the right shows the resulting gel.

16.

Which of the following statements are true?

a.	Lane 1 contains pAMP digested with BamHI and EcoRI.
b.	Lane 2 contains pAMP digested with BamHI and EcoRI.
c.	This gel does not provide sufficient information to identify your pAMP digestion.
d.	Without knowing how much DNA was loaded into each well in the gel, it is not possible to determine which lane contains your pAMP digestion.
e.	Both lane 1 and 2 seems to contain the same sample.

17.

You prepare another restriction digestion and digest pAMP with the enzyme BglI. When you run this DNA sample in a gel, how many bands should you see?

a.	5
b.	4
c.	3
d.	2
e.	1

18.

You wish to clone a human gene (e.g. insulin) into the plasmid pAMP so that you can use it to produce the human protein in bacteria. You want to choose a restriction enzyme or enzymes to “open” the plasmid so that you can insert the human gene. You will then use ligase to “close” the plasmid before transforming the plasmid into bacteria. Which of the following approaches would be most likely to work?

a.	The human gene you want to clone has a HindIII site on one end and a BamHI site on the other. You will use the restriction sites for HindIII and BamHI to “open” pAMP and create your recombinant DNA.
b.	You will use BglI to “open” the plasmid to insert the human gene. You find BglI sites on both sides of the human gene so that they can be ligated into the plasmid. Although there are multiple BgI sites, the enzyme will cut just two of these locations so that the human gene can be inserted.
c.	The human gene has EcoRI sites on both ends and one in the middle of the gene. You hope that the enzyme will only cut on the two sites at the ends of the gene so that it can be inserted into pAMP that has also been “opened” with EcoRI.
d.	All of the methods above will work equally well.
e.	None of the above methods are likely to work.

19.

Imagine you are trying to cut open plasmid A to ligate an insert from duckweed. Which of the following choices are appropriate protocols for this outcome? The labeled sites on the plasmid are restriction sites recognized by each enzyme denoted. Assume the insert will have complementary ends to whatever answer you choose. NOTE: The antibiotic resistance gene is found between RI and Pst1

a.	Digest with RI and Hind III then ligate in the insert
b.	Digest with RI and PstI and then ligate in the insert
c.	Digest with RI only and ligate in the insert
d.	Digest with PstI and HindIII and ligate in the insert
e.	both c and d are correct

20.

The above image shows a plasmid digest with Ava I enzyme on three different plasmids.
Which of the following explanation best describes the results observed from the gel.

a.	Lane 1 shows a plasmid with possibly one cut site
b.	Lane 1 shows a plasmid with two possible cut sites
c.	Lane 2 shows a digest with two cut sites
d.	Lane 3 shows a plasmid with three cut sites
e.	All of the above are correct

The following diagram describes how the human insulin gene can be cloned into a plasmid and transformed into bacteria to produce human insulin for therapeutic use. Steps 1 through 3 describe essential steps necessary to create a copy of the gene that can be expressed in bacteria. Each numbered step is a enzyme-facilitated step.

Answer the following questions based you your understanding of the diagram.

21.

Why are steps 1 through 3 necessary to create a version of the insulin gene that can be expressed in bacteria? In other words, why can’t the original gene simply be cut out from human DNA and inserted into a plasmid?

a.	Bacteria do not have mechanisms to splice introns.
b.	The gene must be transcribed into RNA, which can then be inserted into the plasmid.
c.	The original gene lacks the 5’ cap and 3’ poly-A tail necessary for gene expression in bacteria.
d.	Both (a) and (b) above.
e.	All of the above.

22.

Step 2 represents:

a.	Transcription.
b.	Removal of introns.
c.	Translation.
d.	Replication.
e.	Digestion by restriction enzymes.

23.

The enzyme used in step 3:

a.	is reverse transcriptase.
b.	is RNA polymerase.
c.	uses RNA as a template to create double stranded DNA.
d.	Both (a) and (c).
e.	Both (b) and (c).

24.

The enzymes used in step 4 is (are):

a.	DNA polymerase and ligase.
b.	EcoRI and HindIII.
c.	Ampicillin and ORI.
d.	RNA polymerase.
e.	Reverse transcriptase.

25.

After bacteria is transformed with the recombinant plasmid (step 6), the culture was plated on the two plates shown in the diagram (LB/Amp and LB/Kan). Which of the following statements is true?

a.	There will be colony growth on both plates.
b.	The bacteria growing on the LB/Kan plate contains the recombinant plasmid and is capable of producing human insulin.
c.	Only the LB/Amp plate will have colony growth.
d.	Only the LB/Kan plate will have colony growth.
e.	There will be lawn growth on the LB/Amp plate.

Short Answer

26.

Draw the possible plasmid maps for each plasmid shown in the gel image. Plasmid maps require the labeling of the restriction sites.