RECOMBINANT DNA
TECHNIQUE
Objective
Students will model the process of
using restriction enzymes and
plasmids to form recombinant
DNA.
Background
Information
major tools of recombinant DNA
technology are bacterial enzymes
called restriction enzymes.
Each enzyme recognizes a short,
specific nucleotide sequence in DNA Result: a set of double-stranded
molecules
DNA fragments with singlestranded ends, called “sticky
ends”.
Sticky ends are not really sticky
bases on the sticky ends
form base pairs with the
complementary bases on other DNA
molecules.
Enzyme cuts the backbones of the
molecules at that sequence.
sticky ends of DNA fragments can be
used to join DNA pieces originating
from different sources.
In order to be useful, the recombinant
DNA molecules have to be made to
replicate and function genetically
within a cell.
Small DNA fragments can be
inserted into the plasmids, which
are then introduced into bacterial
cells.
One method for doing this is to use
plasmid DNA from bacteria.
As the bacteria reproduce, so
do the recombinant plasmids.
The result is a bacterial colony in
which the foreign gene has
been cloned.
Key Ideas
 Plasmids - ideal vectors for genetic engineering
 replicate in the bacteria cell
 gene promoter
 antibiotic resistance as a selectable marker,
 can be transferred into bacteria by conjugation and transformation
 Restriction enzymes – key to the creation of a recombinant
plasmid.
 cut DNA at specific sequences
 sticky ends allow strands to join
TG B-10, C-10 Science background-Plasmids
 Bacteria cells produce restriction enzymes as a defense
against bacteriophages
 BamHI: Bacillus amyloliquefaciens
 HindIII: Haemophilus influenza
Where do we get restriction enzymes?
TG B-12, C-12 Science background
 An enzyme that cuts DNA at a specific site (sequence)
Blunt Ends
“Sticky” Ends
What is a restriction enzyme?
 Cut at Palindromes: the complementary DNA is a palindrome
to the DNA strand
 Palindromes: Same forwards/backward:
 A nut for a jar of tuna
 Eva, can I stab bats in a cave?
How do restriction enzymes cut?
 Sticky ends: want to form hydrogen bonds
 H-Bonds form easily
Why are sticky ends important for
ligation?
Scientists can build
designer plasmids that
contain specific restriction
sites
This allows scientist to cut
out and recombine
genes to allow for cloning
and gene expression.
Why use restriction enzymes in
Biotechnology?
Cloning vector plasmid
Engineered to replicate
in high numbers within
bacteria
Plasmids
Expression vector
plasmid
Carries the gene of
interest in a specific
location that allows the
bacteria to express the
gene
Cloning Vector plasmid
Contains:
1. Origin of
replication
2. Selectable
marker gene
3. Multiple
cloning site
Expression vector
plasmid
Contains
1. Origin of replication
2. Selectable marker
gene
3. An inducible
promoter
4. Transcription and
translation initiation
sequence
pARA-R plasmid
A sequence for initiating
DNA replication
Regulates expression
of arabinose genes
Promoter site
Ampicillin resistant
Restriction digest of pARA-R
Biotech Experience
Recombinant plasmid of interest
pARA-R
5,302 bp
PBAD-rfp
806 bp
Restriction analysis of pARA-R
Biotech Experience
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4,496 bp
Hind III
BamH I
806 bp

Clone That Gene activity
RM2 and RM3
1. Cut the plasmid and the human
DNA with the appropriate
restriction enzyme
2. Insert the insulin gene into the
plasmid DNA
3. Determine which antibiotic you
would use to identify bacteria
that have taken in the plasmid
Plasmid DNA
Human DNA
Materials For each group:
 Handout: Plasmid Base Sequence Strips
 Handout: DNA Base Sequence Strips
 Handout: Restriction Enzyme Sequence Cards
 Scissors
 Tape
 Pencil
 Paper
Instructions
 1.Cut out the plasmid strips along the dotted lines.
 2. Show me the strips and tape them together to
form a single long strip.
 Reminder: The letters should all be in the same
direction when the strips are taped. The two ends of
the strip should then be taped together with the
genetic code facing out to form a circular plasmid.
 3. Cut out the DNA base sequence strips, and tape
them together to form one long strip. The pieces
must be taped together in the order indicated at
the bottom of each strip.
 4. Cut out the restriction enzyme cards. The
enzyme cards illustrate a short DNA sequence that
shows the that shows the sequence that each
particular enzyme cuts.
Instructions
 5. Compare the sequence of base pairs on an
enzyme card with the sequences of the plasmid
base pairs.
 If you find the same sequence of pairs on both
the enzyme card and the plasmid strip, mark the
location on the plasmid with a pencil, and write
the enzyme number in the marked area. Do this
for each enzyme card.
 Some enzyme sequences may not have a
corresponding sequence on the plasmid, and
that some enzyme sequences may have more
than one corresponding sequence on the
plasmid.
Instructions
 6. Once you have identified all corresponding
enzyme sequences on the plasmid, identify those
enzymes which cut the plasmid once and only
once.
 Discard any enzymes that cut the plasmid in the
shaded plasmid replication sequence. Record
your findings on a separate piece of paper.
Instructions
 7. Next, compare the enzymes you listed against
the cell DNA strip. Look for any enzymes that will
make two cuts in the DNA, one above the
shaded insulin gene sequence and one below
the shaded insulin gene sequence.
 Mark the areas on the DNA strip that each
enzyme will cut.
Instructions
 8. After you have compared each enzyme with
the DNA strip, select one enzyme to use to make
the cuts.
 The goal is to cut the DNA strand as closely as
possible to the insulin gene sequence without
cutting into the gene sequence.
 Make cuts on both the plasmid and the DNA
strips. Make the cuts in the staggered fashion
indicated by the black line on the enzyme card.
 Tape the sticky ends (the staggered ends) of the
plasmid to the sticky ends of the insulin gene to
create your recombinant DNA.
Discussion Questions
 1. Why was it important to find an enzyme that
would cut the plasmid at only one site? What could
happen if the plasmid were cut at more than one
site?
 2. Why was it important to discard any enzymes that
cut the plasmid at the replication site?
 3. Why might it be important to cut the DNA strand
as closely to the desired gene as possible?
 4. In this activity, you incorporated an insulin gene
into the plasmid. How will the new plasmid DNA be
used to produce insulin?
Discussion Questions
 1. Why was it important to find an enzyme that
would cut the plasmid at only one site? What
could happen if the plasmid were cut at more
than one site?
Cutting at only one site is important for
controlling the variables that will be
reproduced. If the restriction enzyme cut
more than one site, then the plasmid
might recombine with different DNA
fragments.
Discussion Questions
 2. Why was it important to discard any enzymes
that cut the plasmid at the replication site?
If the plasmid were cut at the
replication site, it would not
be able to reproduce and
transfer genetic information
to its host bacterial cell.
Discussion Questions
 3. Why might it be important to cut the DNA
strand as closely to the desired gene as possible?
To make sure that the desired
information is transferred to the
plasmid without adding extra
unknown or undesirable sequences.
Discussion Questions
 4. In this activity, you incorporated an insulin
gene into the plasmid. How will the new plasmid
DNA be used to produce insulin?
The new plasmid DNA will be
introduced into bacterial cells,
where it will reproduce, creating
clones of the insulin gene.