Directions: Complete the concept map by supplying the needed

terms.
Recombinant DNA Technology
 ACTIVITY: “Label it!”
Directions: Label the diagram on the steps of Recombinant DNA
by using the choices indicated below.
 ANALYSIS :“Your Say?”
Directions: According to Claude Vorilhon, “Cloning will enable
mankind to reach eternal life”. What is your thought about this
line? Write your answer in the space provided.
 Recombinant DNA
Recombinant DNA technology
 Allows DNA to be combined from different sources
 Also called genetic engineering or transgenics
 Recombinant DNA
Vector – DNA source which can replicate and is used to
carry foreign genes or DNA fragments.

Recombinant DNA – A vector that has taken up a

foreign piece of DNA.
 Restriction Enzymes
 Restriction enzyme – an enzyme which binds to DNA at a specific base sequence and then cuts the DNA
 Restriction enzymes are named after the bacteria from which they were isolated.

• Bacteria use restriction enzymes to chop up foreign viral DNA

 Restriction Enzymes
 Recognition site – specific base sequence on DNA where
a restriction enzyme binds.
• All recognition sites are palindromes, which means they read
the same way forward and backward.
example: RACECAR or GAATTC
CTTAAG
• Each restriction enzyme has its own unique recognition site.
 Restriction Enzymes
 After analyzing your results, you draw a
restriction map of the cut sites.
• A restriction map is a diagram of DNA showing the
cut sites of a series of restriction enzymes.
Restriction Enzymes
Restriction Enzymes
 Restriction Enzymes

 Most restriction enzymes cut within the

recognition site.
 When restriction enzymes cut in a zig zag
pattern, sticky ends are generated.
 Restriction Enzymes
 Overhanging sticky ends will
complementarily base pair,
creating a recombinant DNA
molecule.

 DNA ligase will seal the nick

in the phosphodiester
backbone.
 Transformation

 Transformation – the process by which

organisms take up and express foreign DNA

Griffith’s experiment
 Transformation
 Bacterial Transformation
 Bacteria, such as E.coli, can take up and express
foreign DNA, usually in the form of a plasmid.
 Transformation

 Gene cloning – using bacterial transformation to

make lots of copies of a desired gene.

Gene Cloning Animation

 Transformation
 Steps of Bacterial Transformation
1. Choose a bacterial host
a. E. coli is a model organism
i. Well studied
ii. No nuclear membranes
iii. Has enzymes necessary for replication
iv. Grows rapidly (20 min. generation time)
v. Inexpensive
vi. Normally not pathogenic
vii. Easy to work with and transform
 Transformation
 Steps of Bacterial Transformation
2. Choose a plasmid to transform
a. Characteristics of a useful plasmid
i. Single recognition site
• Plasmid only cuts in one place, so this ensures that the plasmid is
reformed in the correct order.
ii. Origin of replication
• Allows plasmid to replicate and make copies for new cells.
iii. Marker genes
• Identifies cells that have been transformed.
 gene for antibiotic resistance – bacteria is plated on media
with an antibiotic, and only bacteria that have taken up a
plasmid will grow
 gene that expresses color – bacteria that have taken up a
recombinant plasmid are a different color than bacteria
that have taken up a NONrecombinant vector.
 Transformation
 Steps of Bacterial Transformation
3. Prepare bacterial cells for transformation
a. Treat with calcium chloride – softens the phospholipid
bilayer of the cell membrane, which allows the plasmid to
pass through
b. Electroporation – brief electric pulse
c. Directly inject plasmid into bacterial host
 Transformation
 Steps of Bacterial Transformation
4. Plate transformation on
appropriate media
a. Contains nutrients for bacteria and
antibiotic to distinguish transformed
bacteria from NONtransformed
bacteria
5. Incubate plates overnight
a. E.coli grows at body temp. (37 °C)
6. Analyze plates

Gene Cloning Animation

 Sequencing

 Sequencing – determining the

order and arrangement of G’s,
A’s, T’s and C’s in a segment of
DNA.
 Sequencing

 Let’s review replication…..

 Sequencing

 The Sanger sequencing method uses dideoxy-

nucleotides to generate all possible fragments of
the DNA molecule to be sequenced.

deoxynucleotide dideoxynucleotide
 Sequencing

 Set up four different reactions:

 Sequencing

 Load the four reactions in different wells of a

polyacrylamide gel to separate the fragments
Sequencing

Sequencing Animation
 PCR

 Polymerase chain reaction (PCR)

 A lab technique used to amplify segments of DNA

"PCR has transformed molecular biology

through vastly extending the capacity to
identify, manipulate and reproduce DNA. It
makes abundant what was once scarce -- the
genetic material required for
experimentations."
 PCR
 Reaction requirements
 Template DNA – total genomic DNA
isolated from an organism that
contains a target region to be
amplified
 DNA primers - Short pieces of single
stranded DNA that flank the target
 Taq DNA polymerase - Attaches
nucleotides on the growing strand of
DNA
 Nucleotides (GATC) – Polymerase
adds complementary nucleotides to
the template
 PCR

 Reactions are placed in a machine called a

thermal cycler. The machine cycles through
three temperatures.
 PCR

1. Heat samples to 94°C for a minute or so to

denature the double stranded template DNA.
 PCR

2. Drop temperature to around 50 or 60°C to allow

primers to anneal.
 PCR

3. Maintain temperature at 72°C for a minute or two to

allow the polymerase to elongate the new DNA strands.
 PCR

 The thermal cycler repeats the denaturing,

annealing, and elongating temperatures
approximately 30 times.

PCR Animation
 PCR

 PCR amplification is logarithmic, meaning the

number of copies of the target is doubled every
cycle. (2n)
 PCR
 Cloning by PCR
• Design primer specific for gene of interest (must
know some of the sequence)
• Can use a T-vector because Taq polymerase adds an
A to the 3’ end of sequence
Applications of Recombinant DNA Technology
 I. Manipulating Genes
DNA Technology: technology involved
in genetic engineering that can be used
to cure diseases, to treat genetic
disorders, and to improve food crops.
 I. Manipulating Genes
A. Restriction Enzymes: bacterial enzymes that cut
long strands of nucleotides into smaller segments.
1. Recognize specific sequences of nucleotides (bases)
and cut the DNA at a specific site within the sequence.
2. In one chain, the sequence runs left to right and on
the complementary chain the sequence runs
right to left.
 I. Manipulating Genes
3. Single chain “tails” of DNA called
sticky ends are created on each DNA
segment by the action of the restriction
enzymes.

4. Sticky ends readily bind to

complementary chains of DNA.
 I. Manipulating Genes
5. Pieces of DNA that have been cut
with the same restriction enzyme can
bind together to form a new
sequence of nucleotides.
6. Restriction enzymes can be used to
isolate (cut out) a specific gene.
 I. Manipulating Genes
B. Cloning Vectors: A DNA carrier used to clone a
gene and transfer it from one organism to another.
1. Once a gene has been isolated, that small piece
of DNA can be placed into a cloning vector and can
be introduced into an organism.
2. Many bacteria contain a
cloning vector called a plasmid.
 I. Manipulating Genes
Plasmid: a ring of DNA found in bacteria in
addition to it’s main chromosome. It can be used
to transport genes from one organism to another.
 I. Manipulating Genes
Process of using a cloning vector:
a) A plasmid is removed from a bacterial cell
b) Using a restriction enzyme,
the plasmid is cut and a donor
gene is spliced in.
-Donor gene: a specific gene
that is isolated from another
organism and introduced
into a cloning vector.
 I. Manipulating Genes
c) the plasmid is returned
to the bacterial cell, where
it replicates as the bacterial
cell divides. This clones
the donor gene.
d) the bacteria containing
clones of the donor gene
can then be used to infect
other organisms and
transfer the
gene to them.
 II. Transplanting Genes
A. In some cases, plasmids are used to clone a
specific gene so that the bacteria will produce a
specific protein.
1. For example: insulin is a protein that controls
sugar metabolism.
II. Transplanting Genes
a) People who are diabetic do not make
the hormone insulin. Insulin allows sugars
to travel from the blood into the cells
where they are transformed into energy.
b) A large volume of insulin can be
produced by cutting out the human gene
and cloning it using a plasmid.
 II. Transplanting Genes
B. Process:
1. Isolating a gene:
a. DNA is removed from a human cell to isolate
the gene that produces insulin.
b. A restriction enzyme is used to cut the
human DNA into separate genes, this includes
the gene for insulin.
 II. Transplanting Genes
2. Producing Recombinant DNA:
a) Recombinant DNA: combination of
DNA from two or more sources.
b) Inserting a donor gene (human gene
for insulin) into a cloning vector
(bacterial plasmid) results in a
recombinant DNA molecule.
 II. Transplanting Genes
3. Cloning DNA:
a) the plasmid containing recombinant DNA is
inserted into a host bacterium.
b) the transgenic bacteria is placed into a nutrient
medium where it can grow and reproduce.
c) within each bacterium, the plasmid is copied
many times, and the donor gene for insulin is
cloned.
 II. Transplanting Genes
d) thousands of bacteria are produced very
quickly through mitosis.
e) the transgenic bacteria can be used to
produce large amounts of insulin.
C. Expression of Cloned Genes
1. Once a donor gene is transferred to a host
cell, it is transcribed and translated as though it
were in its own cell.
a) not all of the genes in a cell’s genome are
expressed.
b) genes are often turned off until
the proteins they code for are
needed
 III. DNA Fingerprints
Uses:
1. The banding patterns of DNA
fragments from two different
individuals may be compared to
see if they are related (ex.
Paternity tests)
2. DNA fingerprints of members
of two different species can be
compared to determine how
closely species are related.
3. Using DNA fingerprint to
compare samples of blood, hair,
or tissue found at a crime scene
with a suspect’s blood sample
may help in solving crimes.
C. Making a DNA Fingerprint
1. RFLP (restriction fragments
length polymorphism)
Analysis: method for
preparing a DNA fingerprint.
a) involves extracting
DNA from
blood, hair,
or tissue and cutting it
into
fragments using
restriction enzymes.
b) fragments of DNA are then
separated using a technique
called gel electrophoresis.
 C. Making a DNA Fingerprint
2. Gel electrophoresis:
separates nucleic acids
or proteins (amino
acids) according to their
size and charge.
a) Samples of DNA being
compared are placed in
wells made on the gel
b) An electric current is
then run through the
gel
c) DNA fragments (“-” charge)
migrate towards the “+“ end of the
gel
d) The smaller DNA fragments
migrate faster and farther down the
gel than the longer fragments.
e) The DNA fragments that have
been separated on the gel are
stained in order to be seen.
3. Accuracy of a DNA
fingerprint:
The complete nucleotide
sequence of each individual
is unique for each person
(except identical twins).
Therefore, everyone’s DNA
fingerprint will be different.
DNA fingerprints are
99.9999% accurate.
IV. Polymerase Chain Reaction
Can be used to make copies of
selected segments of DNA for a
DNA fingerprint.
Requires:
a) A primer: artificially
made
single-stranded
sequence of DNA.
b) When these ingredients (DNA
and Primer) are combined and
incubated, the selected regions
of DNA quickly double.
c) Every five minutes the sample
of DNA doubles again, resulting
in many copies of a sample in a
short amount of time.
d) the new copies of the DNA
sample can then be used to make
a DNA fingerprint.
e) Used in
Paternity tests
Diagnosis of genetic
disorders
Study of ancient fragments
of DNA
 V. Human Genome Project
A. Goals:
1. To Determine the nucleotide sequence of the Human
genome and to map the location of every gene on
each chromosome.
2. To compare the human genome with genomes of
other organisms in an effort to provide insight into
fundamental questions about how genomes are:
a. Organized
b. How cellular differentiation and growth are
under genetic control
c. How gene expression is controlled d. And how
evolution occurs.
B. It is hoped that the knowledge gained
from the Human Genome Project will improve
diagnosis, treatments, and even provide
cures for genetic diseases.
1. Identifying these genes and defective
proteins for which they code may make it
possible to design therapies aimed at
correcting the gene defects responsible for
the disorder.
 Human Genome Project
Initiated in 1990 with plan to
identify all human genes
• Analyze genetic variation among
humans
• Map and sequence genomes of
model organisms
• Develop new lab technology
• Disseminate genome information
• Consider ethical, legal, and
social issues that accompany
genetic research
 Human Genome Project
Francis Collins Craig Venter
 Human Genome Project
Consider ethical, legal and social issues
• Who owns your DNA?
 Human Genome Project
Develop new lab technology
• Automated Sequencing
 Human Genome Project
Disseminate genome information
• GenBank database
 Human Genome Project
Analyze genetic variation among humans
• The genome is approximately 99.9% identical
between individuals of all nationalities and
backgrounds.
 Human Genome Project
Map and sequence genomes of model organisms
• E.coli

• Arabidopsis thaliana

• Saccharomyces cerevisiae

• Drosophila melangaster

• Caenorhabditis elegans

• mus musculus
VI. Gene Therapy and Cloning
Treating a genetic disorder by introducing a
normal gene into a cell or by correcting a
gene defect in a cell’s genome.
A. Is well suited for treating genetic disorders
that result from a deficiency of a single
enzyme or protein.
Example: cystic fibrosis, lung cancer, AIDS,
ovarian cancer, brain cancer
A. Many medicine are proteins that can be mass-
produced in a less expensive way through DNA
technology.
Examples:
1. Insulin: controls sugar metabolism; used to treat
diabetes.
2. Colony-Stimulating Factors: used to treat immune
deficiency by stimulating the production of white
blood cells
3. Erythropoietin: used to treat anemia by stimulating
the production of red blood cells.
4. Human Growth Hormone: used as a
treatment for dwarfism; causes bones to
elongate.
5. Interferon: used to treat viral infections and
cancer by preventing the replication of
viruses
6. Interleukins: used to treat HIV and cancer by
activating and stimulating different kinds of
white blood cells.
- Genetically Engineered Vaccines
- Many diseases are combated by
prevention, using vaccines made by
genetic engineering.
 Increasing Agricultural Yields
1.DNA technology has been used to
develop new strains of plants,
which in turn can be used to
improve food and crop yields.
A. Examples: Tomatoes and
hornworms:
-by transferring genes for enzymes
that are harmful to hornworms
into tomato plants, scientists can
make tomato plants toxic to
hornworms and effectively protect
the plants from these pests.
B. Wheat, cotton, and soybeans:
-have been created to be resistant to
weed controlling chemicals
1) herbicide: weed controlling chemicals.
2) such herbicide resistant crops can be
protected from weeds more easily and
less expensively than crops that are
susceptible to the herbicides
-The examples given above are also
classified as therapeutic cloning
-Therapeutic cloning uses genetic
engineering to help treat and cure
diseases.
Safety and Environmental Issues
Many people are concerned about the
safety of genetically engineered foods.
the concern is that the food produced
by genetic engineering could contain
toxic proteins or substances that can
cause allergies in people who consume
them.
Foods produced by transgenic crops can
be sold without special permits or labels
if the product is identical to products
produced by nontransgenic crops.
Example: corn, tomatoes
Concerns:
a) genetically engineered crops could spread
into the wild and wipe out native plant
species.
b) transgenic crops could transmit their new
genes to other species in neighboring areas.
Example: superweeds produced by
rice and lawn grasses exchanging
pollen with native species
Reproductive Cloning
-Involves the making of a whole new
organism from a still living organism
-There is no sexual reproduction or
fertilization involved
-Sheep, donkeys, and a number of plants
have been cloned
-Human cloning is still far off in the future
Steps in Reproductive Cloning