DESIGN

D ESIGN A
ANNE
EXPERIMENT
XPE R I M E NT 4
4B
B
Inquiry Skills
Questioning Observing Determining the Right Tool for the Job
Hypothesizing Concluding
Restriction enzymes are an invaluable tool for scientists today. Scientists have found hundreds
Predicting Inferring
of these specialized DNA scissors among many species of bacteria. Restriction enzymes recog-
Conducting Classifying nize and cut at specific places along the DNA molecule called restriction sites. Each restriction
Recording Identifying site contains a DNA base palindromic sequence of four to six base pairs. A DNA palindrome is
Analyzing Measuring a sequence in which the “top” strand read from 5’ to 3’ is the same as the “bottom” strand read
Evaluating Reporting from 5’ to 3’. For example
Synthesizing Creating Models
5’ GAATTC 3’
3’ CTTAAG 5’
Communicating
is a DNA palindrome. These enzymes will function under optimum pH and temperature to cut
a piece of DNA into a series of fragments. The number and sizes of the fragments depend on the
Materials number and location of restriction sites or palindromic sequence sites for that enzyme. Another
feature of some of these enzymes is that often a single-stranded tail is produced when the
◾ paper template of human cell DNA enzyme cuts at the restriction site. For example EcoRI makes one cut between the weak hydrogen
supplied by your teacher bonds of G and A in each of the DNA strands seen below:
◾ template of bacterial plasmid sup-

→
plied by your teacher
5’ GAATTC 3’ Eco RI 5’ G AATTC 3’
◾ envelope of restriction enzymes 3’ CTTAAG 5’ Enzyme 3’ CTTAA G 5’
◾ scissors

←
◾ tape Notice the single-stranded tail produced in this example. “Sticky ends” allow for the attachment
of another piece of DNA with a complementary sticky end. Using the same restriction enzyme
to cut a bacterial plasmid and a desired gene, the desired gene can be inserted into the bacterial
plasmid. The plasmid can be put into bacteria and copies of the gene or the gene product can be
harvested when the bacteria multiply.
In this investigation, you will be acting as a biochemist who works for a pharmaceutical
company that produces the protein erythropoietin, which is used to prevent anemia in patients
undergoing kidney dialysis or cancer therapy. Your task is to choose from eight restriction
enzymes in the company’s laboratory and find a restriction enzyme that will cut open your plas-
mid at ONE site only. This same enzyme must be able to cut your human cell DNA containing the
erythropoietin gene at TWO sites. This cut must be made close to the desired gene.

Pre-Lab Questions
1. Why is the same restriction enzyme used to cut both the plasmid and desired gene?
2. An enzyme is needed to join the two DNA pieces together. From your knowledge of DNA
replication what enzyme would serve this purpose?
3. Would this procedure work on DNA from any organism? Why or why not?
4. Why must the human cell DNA be cut at two sites?

Procedure
1. Construct the Plasmid
Cut the plasmid strips along the solid lines. Discard any two of the strips. Tape the remain-
ing four strips in any order to make a circular plasmid.

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 Bacterial Plasmid Plan and Conduct
GC AT TA CG TA TA 1. Your biochemistry team is to design a strategy to determine
CG GC AT GC GC AT which restriction enzyme is the best suited to produce
CG AT GC AT GC AT erythropoietin in your bacterial plasmid. Devise and write down
CG AT GC GC TA GC the procedure you will use to determine the best restriction
AT AT CG TA GC CG enzyme for this task.
GC AT CG TA GC CG 2. Design and construct a chart to record your results.
AT TA CG AT GC GC
GC GC CG AT GC TA 3. Use your selected restriction enzyme to construct your
TA TA CG CG GC AT recombinant DNA plasmid.
TA GC TA CG CG GC
TA TA TA TA AT GC
CG GC TA AT AT TA
Restriction Enzymes
TA TA TA GC GC TA
TA CG TA GC GC CG
AT CG AT AT TA GC CG T A CG
AT AT GC GC TA AT CG T A CG
GC GC GC GC AT AT T A Ava II CG Hind III T A Bam HI
GC TA GC GC TA CG GC GC A T
TA AT AT CG AT TA
GC A T GC
CG GC CG CG CG CG
TA GC TA CG TA CG A T GC

T A CG
CG GC T A
2. Construct the Cell DNA
T A Bgl II GC Hpa II T A Eco RI
Cut the human cell DNA into strips. Using tape, assemble the
DNA in the order at the bottom of each strip. A T CG A T
GC CG A T
A T GC

erythropoietin gene =
CG GC
Example of Human Cell DNA Strips T A GC
TA GC TA TA GC TA CG Sac I GC Xma I
GC AT TA TA TA TA GC CG
GC GC CG CG AT CG A T CG
GC AT GC GC AT GC GC CG
CG TA AT TA TA AT
CG TA AT CG AT AT
TA CG GC AT TA CG
AT TA GC TA TA GC Analyze and Interpret
GC TA TA GC CG GC 4. Analyze your results and determine the best enzyme to use.
GC AT AT TA CG GC
CG AT CG GC TA GC
AT GC AT CG CG CG Conclude and Communicate
CG TA TA CG CG CG 5. Provide several reasons why certain enzymes were eliminated
AT CG AT TA TA CG
and justify why your enzyme choice was the best.
GC AT AT TA TA TA
GC AT CG TA AT AT 6. What did the tape represent in this activity?
GC GC GC TA AT GC 7. Discuss your results with the rest of the class. Did all the
CG CG TA AT GC GC biochemistry teams use the same restriction enzyme? Why or
CG AT CG AT AT AT why not?
CG GC TA AT AT CG
8. Restriction enzymes are used as tools in other biotechnological
GC GC CG TA TA CG
applications. When would a multiple cutting restriction enzyme
1 2 3 4 5 6
be used?

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