Transcript Recombinant DNA - Richmond School District

Recombinant DNA
rDNA
“rDNA”



contains DNA
from 2 or more
different
sources
the DNA has
been “spliced”
together!
See page 501
(Mader)
Making rDNA:
animation of "sticky ends"
Need 2 Enzymes:
1. Restriction Enzymes
(“molecular scissors”)
-a specific
restriction enzyme
will cut DNA at a
specific cleavage
site (a sequence of
bases)
animation of section of DNA inserted into a plasmid

Restriction
enzymes
recognize a
specific short
nucleotide
sequence
This is known as a
Restriction Site
2. DNA ligase
- seals any breaks in
the DNA molecule.
Need a “VECTOR”
(something to carry the
DNA into a host cell)
-we often use bacterial
PLASMIDS (small,
circular pieces of bact.
DNA)
The
Procedure:

1. A bacterial plasmid
is isolated.

2. The desired human gene is “cut” out of the
human DNA using restriction enzymes (example of
a restriction enzyme is “EcoR1”.)

3. A bacterial plasmid is also “cut” with the SAME
restriction enzyme. (this leaves the human DNA and the
plasmid DNA with the same “sticky ends”)

4. The two kinds of DNA are mixed together
with some DNA ligase.

5. Bacteria are
mixed with the
newly
engineered
plasmids. (they
readily take up
plasmids especially if you
treat them with
CaCl2, which
makes them
more porous)

6. Bacteria are
allowed to replicate.

7. Bacteria are tested for the presence of the
human gene.
NB: The gene that is
inserted into the plasmid
will only work if it
DOESN’T have any
introns. One way to do
this is to synthesize the
gene in a machine.
Another method is to
isolate the mRNA for the
gene and use “REVERSE
TRANSCRIPTASE” to
make a DNA copy of it.
 (= complementary DNA
(cDNA))
v. detailed animation of the whole process

Uses for rDNA:
Mass production of
hormones ,proteins,
and chemicals
ex. Insulin
1.
2. Producing safer
vaccines.
 Eg. Hepatitis B
vaccine
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3. Producing
Transgenic plants and
animals
: pest -resistant crop
species
: larger cows (they
produce more growth
-hormone)
Farms in the Future?
4. Gene Therapy (See page
508, Mader)
- Ex vivo (outside the body)

Cells from the patient are
given normal genes to
replace defective ones,
then the cells are
returned to the patient to
replicate and grow.
In vivo (inside the body)
 eg. A virus carrying a
normal gene is inhaled by
the patient. The virus is
able to provide the patient
with the normal gene
product that the patient
was missing due to a
genetic disorder.