Bio H – Molecular Genetics
Restriction Enzymes –
Introduction: Bacterial cells have circular pieces of DNA called Plasmids. In genetic engineering we can use
these plasmids to make protein for humans. For example, if you cut open the plasmid, you can stick in a
particular gene, like the insulin gene, and glue it back together using the enzyme ligase. Now when we put the
plasmid containing the insulin gene back into the bacteria, the bacteria can transcribe and translate the gene and
make insulin protein. We can then purify the insulin protein and use it to treat people with diabetes.
Within the sequence of these plasmids are small, but specific, sequences called restriction sites. When mixed
with the correct restriction enzymes these sites will be cut, creating breaks in the DNA.
For example: The enzyme EcoR1 recognizes the DNA sequence GAATTC. If you mix EcoR1 with DNA it
will make a cut whenever it sees the sequence GAATTC. If it sees multiple GAATTC’s it will make multiple
cuts.
Look at the picture of the Sample Plasmid below. All the little dashes represent restriction sites (the associated
restriction enzyme is listed next to where it cuts).
Figure 1
- Example: how many cuts does EcoRI make in this plasmid?
How many fragments of DNA are created by EcoR1’s cuts only?
- How many DIFFERENT enzymes can cut this plasmid?
- How many fragments could you get if all of the enzymes cut at all of their restriction sites?
The number in the middle
indicates how many bases
(nucleotides) make up the
WHOLE circle. The unit kB
means kilobases. 1 kilobase =
1000 bases.
The numbers between the
restriction sites indicate the
distance (in nucleotides) between
one site and the next.
Notice if you add up all the
pieces you get 5400 bases or 5.4
Kb
By cutting the plasmid with different combinations of enzymes we can get different length pieces of DNA.
DNA that is similar (from related organisms or individuals) will create similar fragments. Therefore we can use
these fragments to determine how closely related two samples are.
Once we chop up the DNA in a test tube we inject it into a jelly like block, called an agarose gel and run
electricity through the gel causing the DNA pieces to move through the pores in the gel. The smaller pieces
Bio H – Molecular Genetics
will work their way through the maze-like pores faster than the long pieces. For this reason, we can separate
out the DNA fragments based on size: long pieces on the top of the gel, small piece nearer to the bottom. This
is called gel electrophoresis. See diagram below
Figure 2
Make Gel Block with “wells” for the DNA
Inject Marker – a mix of DNA fragments of KNOWN sizes
Inject your DNA samples of unknown sizes
Apply an electric current to the top of the gel (DNA is inherently negative and will be repelled by the
negative side and attracted to the positive side)
5. Wait… it takes time for all the DNA fragments to move
6. Compare the Bands of DNA in your samples to the known fragments in the marker to determine their
size. Remember small bands travel farther than long bands!!!
1.
2.
3.
4.
Things to note about electrophoresis:
1. Each differently sized fragment will make a distinct band
2. If you have two bands the same length they will blend together and look like one. If you have MANY
fragments the same length, they will still show up as one band on the gel, but may look thicker or darker.
3. The left most lane always is filled with a marker or “ladder” rather than a sample. The fragments in the
ladder are of known length. We use this to compare the bands that come from our unknown samples.
Bio H – Molecular Genetics
Example: Let’s say we digest (cut) your sample plasmid (fig. 1) with EcoRI and BamHI.
- you will end up with 4 pieces.
o Piece one (from EcoRI  BamHI) 800 base pairs (bp)
o Piece two (from BamHI  EcoRI) 1400 bp
o Piece three (from EcoRI  EcoRI) 800bp
o Piece four (from EcoRI  BamH1) 800 bp
o Piece five (from BamH1 through HindIII to EcoRI) 1400+200 = 1600
Your Gel will look like this (L = Ladder; S = Sample)
L
S
3000
2000
1000
800
600
400
100
Notice there is 1-1600bp band, 1-1400bp band and only 1-800 band even though we had three 800bp fragments.
However, if there were 100 800bp fragments, the 800bp band might look thicker and darker.
Bio H – Molecular Genetics
Plasmid Analysis Questions: Use your assigned plasmids (not fig. 1) to answer the questions below in
complete sentences
PLASMID ID #: ___________________(number in parentheses in upper right corner)
1. What is the total length of your plasmid in kB? In bp?
2. How many DIFFERENT enzymes cut your plasmid? Name them.
3. If you cut your plasmid with EcoRI and BamHI how many pieces will you end up with and what will their
sizes be?
4. Use the ladder as a guide and draw in the fragments above as they would appear when you added your
sample to the gel. Add in the exact lengths to the right of each band.
L
3000
2000
1000
800
600
400
100
S
Bio H – Molecular Genetics
5. Let’s say that your lab partner set up the plasmid and the enzymes and forgot to write down which enzymes
he or she added. You check your sample on the gel and get the following band pattern. What enzymes were
used? Explain.
L
S
3000
2000
1000
800
600
400
100
6. Now you are adding in your insulin gene which is 1.5 kB long. However, you do all of this in a test tube.
So when you “seal” the gene in the plasmid, you need to test whether the gene got added into the
plasmid or if the plasmid sealed up by itself. Explain how you can use restriction enzymes and
electrophoresis to tell whether your gene got inserted.
7. In DNA fingerprinting, you can use restriction enzymes and electrophoresis to match blood samples or
determine paternity (whether a child belongs to a particular parent). Explain how this process would be able
to differentiate between people who are related and people who are not.