Practice Exam Questions for Biotechnology Lecture 1. Eco RI is an example of which type of enzyme? a. b. c. d. e.

reverse transcriptase RNA polymerase restriction endonuclease [CORRECT] DNA polymerase DNA ligase

2. The diagram is a restriction map of a circular plasmid DNA showing the recognition sites for the restriction endonucleases xyz I and xyz II. Which of the 5 lanes (A, B, C, D, or E) in the agarose gel show the DNA the fragments expected following a restriction digest of the plasmid using enzyme xyz II ONLY? [D is CORRECT]

3. If you discovered a bacterial cell that contained no restriction endonuclease, which of the following would you expect to happen? a. b. c. d. e. The cell would be unable to replicate its DNA. The cell would destroy all plasmids. The cell would be easily infected and lysed by bacterial viruses. [CORRECT] The cell would become an obligate parasite. Both a and d would occur.

4. Which of the following is a technique that is used to separate DNA molecules from each other by differential movement due to size and electrical charge? a. b. c. d. e. restriction enzymes gene cloning agarose gel electrophoresis [CORRECT] ampicillin DNA ligase

5. Which of the following procedures would be involved in demonstrating a Restriction Fragment Length Polymorphism (RFLP) in human DNA? a. incubating a mixture of single strand DNA from two closely related species with the enzyme reverse transcriptase b. incubating DNA nucleotides with DNA polymerase c. Southern Blotting [CORRECT] d. incubating RNA with DNA nucleotides and reverse transcriptase e. incubating DNA fragments with "sticky ends" with ligase

Short Answer 1. 2. 3. 4. 5. Consider the DNA sequence: 5' ATCTA 3' 3' TAGAT 5' Is this sequence a "palindrome"? Yes, you could say so. Is this sequence an inverted repeat? NO In the restriction enzyme name "NotI", what does the "Not" mean? Refers to the latin binomial name of the bacterial species from which the enzyme was isolated. [Nocardia otitidis-caviarum, in case you are interested.] How do restriction enzymes differ from other (ordinary) nucleases? The most important difference is that they cut DNA molecules reproducably only at specific recognition sequences. How do these special properties make them useful in molecular biology? Allows a DNA sample to be repeatedly cut up into families of identical fragments with predictable sizes. What is the natural role of restriction modification systems in bacteria? Probably protection against infection by bacterial viruses. What 2 types of enzymes are involved in R-M systems? Endonuclease and methylase

6.

Complete the following example of a R-M sequence. The arrow represents a site of strand cutting and the "*" indicates a methylated base.

*
7. Compute the average frequency of the restriction site above in a DNA with 40% GC base pairs. (0.3) (0.2) (0.2) (0.2) (0.2) (0.3) = 0.00014 What would be the average restriction fragment size in kilobases? 1/0.00014 = 6944 bp

8.

The following diagram is a restriction map of a small linear DNA molecule containing one gene.
BamHI EcoRI BamHI
1.0 kb 0.5 kb

0.1 kb

0.3 kb GENE

0.9 kb

EcoRI

Samples of this DNA are digested in 4 separate tubes with different combinations of restriction enzymes as follows: Tube#: 1 2 3 4 Enzyme/s: Control EcoRI BamHI EcoRI + BamHI (no enzyme) Unfortunately, the 4 DNA digests were not labeled properly, and were mixed up while loading them onto an agarose gel. The stained gel looked like this:

Can you un-mix the samples? i.e. Can you determine which lane of the gel (A-D) corresponds to each tube (1- 4)? Circle all the bands on the gel that containing a DNA fragment with the complete sequence of the gene. Give the sizes of these fragments.

1
A

3
B

2
C

4
D

2.8 kb

1.2 kb

9. The following diagram is a restriction map of a small circular plasmid DNA. Two genes are shown, Amp-R and lacZ. The arrows indicate the direction of transcription for these genes.
EcoRI 0.4 kb BamHI 0.2 kb Amp-R EcoRI 0.7 kb SmaI 1.0 kb

1.1 kb lacZ

SmaI 0.3 kb EcoRI SmaI

What is the total size of the plasmid DNA in kilobases? 4.8 kb Determine the # and the sizes of the DNA fragments expected when the plasmid is fully digested by each enzyme combination below. List the fragments in order of decreasing size in each column of the table. BamHI EcoRI SmaI SmaI + BamHI SmaI + EcoRI + BamHI In the SmaI + EcoRI + BamHI column circle the fragment that would contain the promoter sequence for lacZ.
EcoRI

0.5 kb

0.6 kb

 BamHI 4.8 EcoRI 1.6 (X2) 1.0 0.6 SmaI 2.7 1.4 0.7 SmaI + BamHI 2.5 1.4 0.7 0.2 SmaI + EcoRI + BamHI 1.1 1.0 0.7 0.6 0.5 0.4 0.3 0.2

10. This problem is about the inheritance of color blindness caused by expression of an X-linked recessive allele. The recessive allele for color blindness is relatively common in the human population. A color-blind man (I-1) marries a woman with normal color vision (I-2). This couple's daughter (II-1) has normal vision and she marries a man with normal color vision (II-2). I. As a genetic counselor, what could you tell this family about the probability that their offspring (III-1) will be color-blind?

I-1

I-2

II-1

II-2

There is a 1/4 probability of a color-blind son.

III-1

You suggest that the family in question undergoes DNA testing. A pair of PCR primers is used to amplify a specific region from the human genome containing the gene for Xlinked color blindness. The normal allele of the gene has a variable restriction site for the enzyme Hind III that is missing in the color-blind allele. After amplifying DNA from the genomic DNA of each individual, the PCR product is digested with Hind III and the digests are then separated on an agarose gel.
I-1 I-2 II-1 II-2 III-1

800 kb 400 kb 200 kb

II. What would you tell the family now (based on the results of the DNA test)?

Now you can tell them they will have a daughter with normal vision but who is heterozygous for the recessive allele.