RESTRICTION ENZYME LAB
EcoRI
GAATTC
HindIII
CTTAAG
BamHI
GGATCC
TTCGAA
AluI
CCTAGG
SmaI
CCCGGG
AAGCTT
AGCT
TCGA
HbaI
GGGCCC
GCGC
CGCG
Restriction enzymes are proteins produced by bacteria to prevent or restrict
invasion by foreign DNA. They act as DNA scissors, cutting the foreign DNA into
pieces so that it cannot function.
Restriction enzymes recognize and cut at specific places along the DNA molecule
called restriction sites. Each different restriction enzyme has its own type of
site. In general it is a 4-6 base pair sequence that is a palindrome (read the same
on the top strand as backward on the bottom).
Restriction enzymes can produce two types of cuts:
1. Sticky end cuts, which leave one strand with bases exposed.
These type of enzymes are used to cut DNA that will be glued to another piece of
DNA.
2. Blunt end cuts, which cut through both strands at the same location. No bases
are exposed.
These type of enzymes are used for cutting DNA into fragments to be separated
by a process called DNA electrophoresis.
Another enzyme called Ligase glues DNA fragments with exposed bases together.
PROCEDURE WRITE YOUR QUESTIONS ON A SEPARATE PIECE OF PAPER.
1. Cut your DNA strips out.
2. Scan along the DNA sequence of strip 1 until you find the EcoRI site(s). Use a
highlighter or colored pencil to mark the recognition site. Make cuts between the
bases where the enzyme will cut. Be careful not to cut through both strands.
Then cut between the strips where hydrogen bonds would be broken. Label the
back of the strands EcoRI.
Question 1. Are these sticky end cuts or blunt end cuts? Explain your answer.
3. Repeat step 2 for strip 2 this time using the restriction enzyme SmaI. Label
the back of the strands SmaI.
Question 2. Are these sticky end cuts or blunt end cuts? Explain your answer.
4. Repeat step 2 for strip 3 using HindIII. Label the back of the strands
HindIII.
Question 3. Are these sticky end cuts or blunt end cuts? Explain your answer.
5. Repeat step 2 for strip 4 using EcoRI again. Label the back of the strands
EcoRI. W
Question 4. Can the fragments from strip 1 and strip 4 that were cut with
the same restriction enzyme be bonded together? Explain your answer.
Question 5. Could fragments cut with HindIII be stuck to fragments cut with
EcoRI? Explain your answer.
Biotechnology
1. What is a restriction enzyme?
2. What is a recognition site?
3.What kind of restriction enzyme would be used when combining two pieces of
DNA (recombinant DNA)? Explain why.
4. What king of restriction enzyme is used for separating DNA by fragment size?
Explain why.
5. If DNA is cut with two different restriction enzymes, can those pieces be
stuck together into one piece? Why or why not?
6. What does DNA electrophoresis do?
7. What is recombinant DNA?
8. What is recombinant DNA used for?
9. What is the most common type of organism that contains recombinant DNA?