Download MCDB 1041: Using DNA To manipulate DNA in the laboratory, one

MCDB 1041: Using DNA
To manipulate DNA in the laboratory, one needs to be able to cut it, make copies of it, and visualize it.
Cutting DNA:
Restriction enzymes are proteins that have the ability to cleave DNA. They are found naturally in bacteria
where they cut viruses that infect bacteria. Different Restriction Enzymes (REs) cut DNA at different, specific
sites. Scientists have isolated many different kinds of these restriction enzymes from bacteria, and use them to
cut any kind of DNA.
RE sites are always “palindromes”: In English, a palindrome is when a sentence reads the same whether it is read from the right or left Examples: live not on evil & madam Im adam In DNA: Palindromes read the same 5' to 3' on each DNA strand, and the DNA can be cut with a particular RE (in the example above, the restriction enzyme Eco RI recognizes the sequence GAATC and cuts between the G and the A, on both strands. You have separate pieces of paper at your tables that look like the diagram below, representing a piece of double stranded DNA with restriction enzyme sites as marked: E
P
B
H
B The letters E, P, B, and H along the DNA signify specific RE sites: nucleotide sequences where restriction enzymes cut the DNA. For example “E” is where a restriction enzyme called Eco RI cuts. EcoRI cuts between the G and the A in the following sequence: GAATTC (and also in that location on the complimentary strand). The other letters represent different restriction enzymes. 1. Each line on the DNA strand represents 1 KB (kilo bases or 1,000 base pairs) of DNA. What is the length of the DNA above in KB? 2. When DNA is cut into pieces and run through gel electrophoresis, which will move further in a gel, short fragments or long fragments? Why? Visualizing DNA At the top of the gel below are shown Lanes 1-­‐6. The labels indicate the enzymes that have been used to cut the DNA (ie, the “E” column contains DNA that is cut with the Eco RI restriction enzyme. The “H + E” column contains DNA is cut with the H restriction enzyme and the Eco RI restriction enzyme, and so on). 3. Take your piece of paper and CUT IT as if your scissors were a Restriction Enzyme. Begin by cutting the paper at the Eco RI site (cut the paper at E). What lengths of DNA do you get? In the “E” column, draw horizontal lines to represent the DNA band(s) you would expect to see if you ran this DNA on the gel. Now take a new strand of DNA, and cut with restriction enzyme Hind III. Repeat for each column, and fill in the rest of the columns on the gel. Write the size of the DNA next to each band you draw on the gel. Cut DNA is
“Ladder”(DNA of
loaded into
known sizes run on
lanes 1-6 the gel for reference)
Lane 1 2 3 4 5 6 7 DNA cut with
DNA cut with
DNA cut with
DNA cut with DNA cut with
DNA cut with
Enzyme E
Enzyme P
Enzyme B
Enzyme H
Enzymes H+E Enzymes B+P
- 12 KB
11 KB
10 KB
9 KB
6 KB
3 KB
1 KB
+ 4. If you add the sizes of the DNA bands together in each column, what number would you expect to get? Try it out: add up the sizes of the bands in each column. a. How do your results compare to your prediction? If your results were different, what accounts for the difference? b. If you didn’t already do this, go back to your gel and make the bands that contain more DNA thicker than the bands that contain less DNA. 5. The gene for sickle cell anemia is 23 KB in length. You have isolated this fragment from human cells with
PCR. You also know that this gene has two sites for the same restriction enzyme, located at 10 KB and at 18
KB in the sequence.
a. How many pieces of DNA will be generated by cutting this piece of DNA with this restriction
enzyme? List the sizes of the DNA that will be produced.
b. Draw them on the “gel” below
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