Recombinant DNA Technology
double stranded DNA that can self-
- Sometimes called Genetic
Engineering.
- Is the use of laboratory techniques
to isolate and manipulate
fragments of DNA.
Molecular Cloning
- Isolation and amplification of a
specific DNA fr5agment.
- Set of experimental methods in
molecular biology that are used to
assemble rDNA molecules and to
direct their replication within host
organism.
Restriction enzymes/Restriction endonuclease
 It is an enzyme used to cleave or cut
segments of DNA.
Vector
 It is a DNA molecule used as a vehicle to
carry the gene of interest into another
cell.
Steps in Molecular Cloning
1. Isolation of DNA fragments (Gene of
Interest) to be cloned.
In this step, the gene of interest or the
target DNA is being isolated using a
restriction enzyme.
2. Selection of an appropriate vector in
which would propagate the DNA.
In this step, in choosing a vector we
need to consider the size of the DNA
fragment or gene of interest. There are
two types of vector: plasmid vector and
bacteriophage (lambda phage).
Plasmid- it is a relatively small, circular
replicate.
 pUC18 is the most commonly
used plasmid vector because it
has a polylinker. When the gene
of interest is inserted into the
tetracycline part, it will
inactivate the resistance to
tetracycline.
 It means if we put it in a
solution with a tetracycline, it
will not survive.
3. Insertion of gene of interest into a
suitable vector
In this step, using the DNA ligase the
gene of interest is inserted into the
vector to form recombinant DNA.
4. Introduction of RDNA into suitable host
organism.
The rDNA is now being introduced into
host organism using different techniques
depending on the cell type. Electroporation for
mammalian cells, Biolistics for plant cells,
Plasmid insertion by Heat Shock Treatment and
Transformation for bacterial cells.
5. Selection process to screen which cells
actually contain the gene of interest.
There are some methods to screen the
recombinant cells. One example is the “blue-
white” screening process. When a cell turns to
blue it does not contain the rDNA while the cells
that turns to white it contain the rDNA. Another
example is using the antibiotic resistance like
the ampicillin and tetracycline.
Polymerase Chain Reaction Expression Vector

 It was invented by Kary Mullis in 1983.  It is also known as expression construct.

 It is a laboratory procedure to amplify  Usually a plasmid or vires designed for
and copy segment of DNA. gene expression in cells.
 Expression vector is to express a gene
There are four basic ingredients in PCR:
or specific trait in host cells while
1. Taq Polymerase cloning vector is to amplify or replicate
2. Nucleotides the rDNA/host cell.
3. PCR Primers There are components of an expression
4. DNA template vector:
1. Promoter
 allows the controlled
expression of desired gene
Taq polymerase
in the presence of an
 It is isolated from a heat-loving inducing agent (beta-
bacterium called thermos aquaticus. galactosidase).
 It initiates the transcription
PCR Primers process.
2. Multiple Cloning Site
 Short strand/segment of DNA. It serves
 DNA sequence or portion
as starting point.
for the insertion of the
Steps in PCR desired gene. This section
may contain sequences that
1. Denaturation will be cut by specific
 The temperature required in this restriction enzyme or
stage is 94˚-95˚C. restriction endonucleases.
 High temperature causes the  If the gene of interest and
hydrogen bonds to break and the plasmid are cut by
double stranded DNA separates. restriction enzyme, then
2. Annealing complementary sequences
 The temperature is lower down into will be generated for each,
54˚-60˚C. allowing them to anneal.
 It causes the primers to bind at the  The desired trait is inserted
side of target DNA. into the MCS through this
3. Elongation process.
 The temperature rises to 72˚C.
 It causes the Taq polymerase to EcoRI Target Sequence:
extend the primers.
5’ GAATTC 3’
 Freely floating nucleotides bind
with the single stranded DNA. 3’ CTTAAG 5’
PCR Primers:

5’ GCGATGAGG 3’ (Forward Primers)

3’ CGCTACTCC 5’ (Reverse Primer)

Forward Primer + EcoRI target sequence:

5’ GAATTCGCGATAGG 3’

Reverse Primers + EcoRI target sequence

3’ CCATAGATCCTTAAG 5’

3.Inserted Gene Sequence

 Successful insertion of a gene allows the

expression of its protein product. This
usually provides a specific trait to the “
transformed” bacteria.

4.Antibiotic Resistance Gene

 Provides a way to screen a population

of bacteria for those that took up the
plasmid.
 Since plasmid has an ampicillin
antibiotic resistance, if we put the cells
into a solution with an ampicillin those
that took up the plasmid will survive.