Recombinant DNA Technology

What is genetic engineering

Genetic engineering is the process of manually
adding new DNA to an organism.

The goal is to add one or more new traits that are not
already found in that organism.

Examples of genetically engineered (transgenic)

organisms currently on the market include plants with
resistance to some insects, plants that can tolerate
herbicides, and crops with modified oil and nutrient
content.
What is genetic engineering
Genetic engineering, also known as recombinant
DNA technology, means altering the genes in a
living organism to produce a Genetically Modified
Organism (GMO) with a new genotype.

Various kinds of genetic modification are possible:

inserting a foreign gene from one species into another,
forming a transgenic organism;
altering an existing gene so that its product is changed;
changing gene expression so that it is translated more
often or not at all.
Basic steps in genetic engineering
1. Isolate the gene of interest
2. Insert it in a host using a vector
3. Produce as many copies of the host as possible
4. Separate and purify the product of the gene
The process: Once a goal is in
mind
1) First, find an organism 2) The DNA is extracted
that naturally contains from that organism. This
the desired trait. is like taking out the
entire cookbook.
The process: Once a goal is in
mind
3) The one desired gene 4) The gene may be
(recipe) must be located modified slightly to work
and copied from in a more desirable way
thousands of genes that once inside the recipient
were extracted. This is organism.
called gene cloning.
The process: Once a goal is in
mind
5) The new gene(s) -transgene is delivered into cells of
the recipient organism. This is called transformation.
The transgene is inserted into the bacteria, which then
delivers it into cells of the organism being engineered.
Or the gene gun method, shoots microscopic gold
particles coated with copies of the transgene into cells
of the recipient organism.
With either technique, genetic engineers have no
control over where or if the transgene inserts into the
genome. As a result, it takes hundreds of attempts to
achieve just a few transgenic organisms.
The process: Once a goal is in
mind
6) Once a transgenic organism has been created,
traditional breeding is used to improve the
characteristics of the final product.
So genetic engineering does not eliminate the need for
traditional breeding. It is simply a way to add new
traits to the pool.
Gold rice
produces more
beta-carotene
Genetic Engineering
Genetic Engineers can alter
the DNA code of living Recombinant DNA
organisms.
It involves the following:
PCR

Gel Electrophoresis

Transgenic Organisms

Selective Breeding
Step 1: Alternative method (using reverse
transcriptase)
Reverse transcriptase (enzyme)
mRNA converted into cDNA

Complementary DNA strand produced using DNA

polymerase

Advantage more mRNA in cell than DNA

What do we already know
DNA Double Helix Structure
Each spiral strand is composed of a sugar
phosphate backbone and attached bases
4 Bases: Adenine (A), Guanine(G),
Cytosine (C), and Thymine (T).
Form Base Pairs; A with T and C with G in the
complementary strand via
hydrogen bonding (non- covalent)

The strands can be cut by

restriction enzymes, e.g. ECOR1
Step 1: Isolating the gene using
restriction enzymes
Step 1: this requires restriction enzymes
Restriction Endonucleases
(Restriction Enzymes)
 Restriction Endonucleases
(Restriction Enzymes)
Enzymes that cut DNA EcoR1 is from E. coli
at specific sites or base strain R.
sequence. HindIII is from
Haemophillis influenza
Restriction enzymes HpaI is from
are named after the Haemophilus
bacteria species they parainfluenzae
came from
 Restriction Endonucleases
(Restriction Enzymes)
restriction enzymes usually recognize palindromic sequences
 Restriction Endonucleases (EcoRI)
They may make a
staggered cut, forming

sticky ends because they
have short stretches of EcoRI digestion
single-stranded DNA produces "sticky" ends,

These sticky ends will

stick (or anneal) to
another piece of DNA by
complementary base
pairing, but only if they
have both been cut with
the same restriction
enzyme.
Many restriction nucleases produce staggered cuts in
DNA which leave short single stranded "tails" at the
ends of DNA fragments
Single stranded tails produced in the result of DNA cleavage (cutting)
by restriction nucleases are known as cohesive or "sticky" ends. They
can form complementary base pairs with the DNA ends produced by
the same restriction enzyme.
Restriction Endonucleases (SmaI)
Some RE cut straight whereas SmaIl (SmaI)
across both chains restriction enzyme cleavage
Forming blunt ends produces "blunt" ends
Some restriction enzymes, for example, Sma I, cut both
DNA strands in the middle of the recognition sequence
and produce "blunt-end" DNA fragments
 Bacterial Structure
Bacteria are often used in biotechnology as they have
plasmids
A plasmid a circular piece of DNA that exists apart
from the chromosome and replicates independently of
it.
steps involved in
Recombinant DNA
The ability to combine
the DNA of one
organism with the DNA
of another organism.

Recombinant DNA
technology was first used
in the 1970s with
bacteria.
Recombinant Bacteria
 Recombinant Bacteria
1. Remove bacterial DNA (plasmid).

2. Remove DNA(containing gene of interest) from cell.

3. Cut the Bacterial DNA with restriction enzymes.

4. Cut the DNA from another organism with the same restriction
enzyme.

5. Combine the cut pieces of DNA together with another enzyme

DNA ligase. Inserting them into bacteria which is now a
vector, and then insert the plasmid vector into bacteria.

6. Reproduce the recombinant bacteria.

7. The foreign genes will be expressed in the bacteria.

 Plasmids
A typical plasmid contains 3-5 genes. These include:

Cloning site where foreign DNA can be inserted

Drug resistance gene which destroys antibiotics to

allow selective growth of the host cell

Replication origin allows plasmid to replicate in host

cell

Plasmids are copied separately from the main bacterial

DNA and so they are passed on to daughter cells
 Cloning site

Drug resistance gene

 R-Plasmid (pBR322)

One of the most common plasmid is the R-plasmid

(pBR322). It contains:
A replication origin

Several recognition sequences for different restriction

enzymes such as PstI, EcoRI

Two drug resistance or marker genes, which

resistance to the antibiotics amplicilin and tretracycline

Step 2: Sealing with DNA ligase
This enzyme DNA ligase repairs cut DNA by joining
two nucleotides in a DNA strand. (anneal = join)

Sticky ends allow two restriction fragments to

anneal, but only by weak hydrogen bonds.

DNA ligase completes this process by forming

covalent bonds between nucleotides.
 Step 1&2:
removing plasmid
& foreign DNA
Reverse transcriptase
(enzyme)
mRNA converted into
cDNA
Complementary strand
produced using DNA
polymerase
Advantage more mRNA
in cell than DNA
This can be done using:
Vector method
Gene gun
 Gene Transfer
Once the vectors receive the required gene it is transferred
into a cell (host) at point the cell is said to be
transformed.

In order for transfer to take place the membrane of the host

cell must be made permeable. There are several ways of
doing this:
Heat shock

Electroporation

Viruses
 Vectors
In genetics a vector is a length of DNA that carries the
gene we want into a host cell.

A vector is needed because the length of DNA

containing the gene you want will not survive on its
own in a host cell as its not apart of the cells normal
genome.
 Vectors
A vector bypasses these problems by :

Being big enough to hold the required gene plus a few

more

Its a closed loop which makes it less likely to be broken

down

It contains controlled sequences such as replication

origin, transcription promoter, antibiotic resistance

 Step 3: inserting vector into host

Possible treatments that can help the hosts to take up the

vectors include; shocking, temperature shock, calcium
ions all help the cells to uptake the plasmids

A common marker, used in plasmids, is a gene for resistance

to an antibiotic such as tetracycline.

Bacterial cells taking up this plasmid are resistant to this

antibiotic.
Step 3: inserting vector into host
So if the cells are grown on a medium containing
tetracycline
all the normal untransformed cells (99%) will die.

Only the 1% transformed cells will survive, and these can

then be grown and cloned on another plate.

Gene Transfer-Inserting vector into host
 There are different types of vectors, namely:

Cloning vector contains sequences that cause the gene

to be copied inside a cell, but not expressed.

Expression vector - contains sequences causing the gene

to be expressed inside a cell.

Below are names of some vectors:

 Multiplication of the host cells by cloning
The required genes can be cloned by cloning the
bacterial cells that contain them
Before this can be done the amount of DNA present
must be amplified by PCR polymerase chain reaction
PCR is DNA replication where a length of DNA is
mixed with the four nucleotides and the enzyme DNA
polymerase in a test tube causing the DNA to be
repeated many times
 Purification of DNA - Gel
Electrophoresis
This is a form of chromatography used to separate
different pieces of DNA on the basis of their length.
It might typically be used to separate restriction
fragments.
Gel Electrophoresis

This technology allows

scientists to identify
someones DNA!
Steps Involved in Gel Electrophoresis
1. Cut DNA sample with
restriction enzymes.

2. Run the DNA fragments

through a gel.

3. Bands will form in the gel.

4. Everyones DNA bands are

unique and can be used to
identify a person.

5. DNA bands are like genetic

fingerprints.
Polymerase Chain Reaction
PCR
PCR allows scientists to
make many copies of a
piece of DNA.

1. Heat the DNA so it

unzips.

2. Add the complementary

nitrogenous bases.

3. Allow DNA to cool so the

complementary strands
can zip together.
Selective Breeding
Breed only those plants
or animals with desirable
traits

People have been using

selective breeding for
1000s of years with farm
crops and domesticated
animals.
genetically modified fruits & vegetables
Benefits of Recombinant Bacteria
1. Bacteria can make human insulin or human growth
hormone.

1. Bacteria can be engineered to eat oil spills.

The DNA of plants and animals
can also be altered.

PLANTS

1. disease-resistant and
insect-resistant crops

2. Hardier fruit

3. 70-75% of food in
supermarket is
genetically modified.
How to Create a Genetically
Modified Plant
1.Create recombinant
bacteria with desired
gene.

2. Allow the bacteria to

infect" the plant cells.

3. Desired gene is inserted

into plant chromosomes.
What do you think about eating genetically
modified foods?
Genetically modified organisms are called
transgenic organisms.

TRANSGENIC ANIMALS

1. Mice used to study human

immune system

2. Chickens more resistant to

infections

3. Cows increase milk supply

and leaner meat

4. Goats, sheep and pigs

produce human proteins in
their milk
 Transgenic Goat

.
This goat contains a human
gene that codes for a blood
clotting agent. The blood
Human DNA in
clotting agent can be harvested
a Goat Cell
in the goats milk.
How to Create a Transgenic Animal

Desired DNA
is
added to an
egg cell.
Ha Ha Ha!