Download Recombinant DNA Simulation

Investigation 6: Recombinant DNA Simulation
Introduction:
One of the most important processes developed by biotechnologists was the procedure where a gene is removed
from the DNA of one organism and inserted into the DNA of another organism. This technique is called
Recombinant DNA. The entire procedure is dependent upon using the correct restriction enzymes which cuts and
removes the desired gene and then inserting the gene into a different DNA strand. Once this recombinant vector
DNA has been made it then can be inserted into a cell that will then express the protein coded for in the new gene.
An example of this technique would be the insertion of the human insulin gene into bacteria for the purpose of
having the bacteria make human insulin for the treatment of persons with diabetes.
Objectives:
1. To learn the technique of making Recombinant DNA.
2. To understand what a restriction enzyme does.
3. To understand the importance of using the correct enzyme in this process.
Procedure:
1. Obtain supplies
2. Bacterial Plasmid DNA sheet Yellow Paper (One partner should work on this step while the other partner
works on step 3).
a. Notice that coded on the DNA base pair sequences are four genes. Be sure to save the gene key at
the bottom of the page!
b. Cut out the plasmid strips of DNA base pair sequences.
c. Find the strip that has the Plasmids Origin of Replication.
d. Select Any 3 of the 5 additional plasmid strips.
e. Discard the 2 remaining strips.
f. Tape/Glue together the 4 selected strips (the one with the Plasmid of origin of replication plus the
3 additionally selected plasmid strips) end to end. This will form a plasmid “Ring.”
3.
4.
5.
Human Cell DNA with insulin gene Blue paper (One partner should work on this step while the other
partner works on step 2)
a. Cut and save the key from the bottom of the page.(Do NOT cut off the strip numbers!)
b. Cut the Human Cell DNA stips out in order (1Æ6)
c. Tape the strips together in order indicated by the number at the bottom of each DNA strip (ie.
DNA strip 2 is taped to the bottom of strip 1 and then strip 3 is taped to the bottom of strip 2, etc.)
This will form a Linear piece of DNAÆ
After completing steps 2 and 3, use the Plasmid Map to map the relative locations of the genes for
antibiotic resistance.
Test each of the 8 restriction enzymes.
a. Use the restriction enzyme handout for this step.
b. One Partner’s job is to find which restriction enzyme(s) will cut open the Yellow Plasmid
DNA at ONE site and does NOT cut the Origin of Replication and does NOT cut any of the
antibiotic resistance genes 1.
c. The other Partner’s job is to find which of the Possible restriction enzyme(s) (See
Column 2) will cut the Blue Human cell DNA on either side of the insulin gene without cutting
the insulin gene.
d. Mark all possible restriction enzymes on the plasmid or the cell DNA.
e. Choose a restriction enzyme to use. This restriction enzyme must meet the following
criteria:
1. Cuts the plasmid at one site only and does NOT cut any of the antibiotic
resistance genes or the DNA replication site .
2. Cuts the human cell DNA twice – once above and once below the insulin gene
but does not cut the insulin gene.
3. Cuts closest to the insulin gene.
f.
Fill in the information on the data table as to which restriction enzyme you used
and the reasons why you did or did not use a restriction enzyme. You should only
have one restriction enzyme marked used.
6.
7.
Use a pair of scissors to CUT the Yellow plasmid DNA and the Human Cell DNA in the ZIG-ZAG pattern
made by the chosen restriction enzyme.
The Restriction enzyme cuts will produce “sticky ends,” which will stick to each other by complementary
base pairing.
a. Use tape to splice (stick) together the human insulin gene into the Yellow Plasmid.
b. Mark on your plasmid map where you have inserted the insulin gene.
c.
8.
Congratulations you have engineered a Recombinant DNA Plasmid.
Answer the Conclusion Questions
a. What do restriction enzymes do?
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b.
c.
d.
e.
f.
What is the importance of “sticky ends”?
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What is recombinant DNA?
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What Restriction Enzyme did you end up using to make the recombinant DNA? ____________
Explain why you chose that restriction enzyme ?
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Based on your experience from this lab, explain why you can’t use just any restriction enzyme
on any Bacterial Plasmid?
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