THE APPLICATIONS
OF
RECOMBINANT DNA
Prepared by: Sir Cee Jae
LESSON OBJECTIVES:
give examples of products from recombinant DNA
technology;
illustrate the use of databases to search genes for desired
traits;
describe steps in PCR to amplify and detect a gene of interest;
identify the parts of an expression vector;
explain how genes may be cloned and expressed
REVIEW ON CENTRAL DOGMA OF
MOLECULAR BIOLOGY
DNA (gene) => RNA (transcript) => Protein (trait)
REVIEW ON CENTRAL DOGMA OF
MOLECULAR BIOLOGY
Different organisms have different traits based on
their genes (DNA sequences).
For example, frogs have antimicrobial peptides on
their skin. Some jellyfish have proteins that allow
them to glow in the dark.
Mutations in hemoglobin genes lead to anemia.
REVIEW ON CENTRAL DOGMA OF
MOLECULAR BIOLOGY
Based on the central dogma, if transcription and
translation of genes lead to some traits, then the
insertion of certain genes in a given organism may
provide it with new traits.
This is the basis for the development of genetically
modified organisms (GMOs).
THOUGHT EXPERIMENTATION
DESIGNER GENE GROUP WORK
CONSTRUCTING A GENETICALLY MODIFIED ORGANISM/TRAIT IN A FRUIT.
I. Arrange the learners into groups of 4 or 5
II. Have them identify a special trait (e.g. large fruit size)
III. Have them identify a source organism (e.g. jackfruit / langka)
IV. Have them identify a target organism (e.g. aratilis)
V. Have them identify the modified / added trait (e.g. langka-sized aratilis).
VI. Have the learners present their work to the rest of the class, and let the class
decide on the best proposal.
DNA RECOMBINATION
Modified Trait
Insulin Production
Gene
Modification
Insertion of Human
Insulin Gene
Recipient
Organism
Bacteria
Application
(Field)
(Medicine)
Production of
Human
Insulin in Bacteria
DNA RECOMBINATION
Modified Trait
Pest Resistance
Gene
Modification
Insertion of Bttoxin gene
Recipient
Organism
Corn / Maize
Application
(Field)
(Agriculture)
Production of corn
plants with
increased
resistance to corn
boxer
DNA RECOMBINATION
Modified Trait
Delayed Ripening
Gene
Modification
Disruption of a
gene for a ripening
enzyme (e.g.
polygalacturonase)
Recipient
Organism
Tomato plant
Application
(Field)
Agriculture)
Production of plants
with fruits that have
delayed ripening
fruits. These fruits will
survive longer
transport time,
allowing their delivery
to further locations
(i.e. export deliveries)
DNA RECOMBINATION
Modified Trait
Chymosin
Production
Gene
Modification
Insertion of a gene
for chymosin
Recipient
Organism
Bacteria
Application
(Field)
(Industry) Enhance large
scale production of
chymosin. This enzyme
serves as a substitute for
rennet in the coagulation of
milk. Rennet has to be
harvested from calves. The
large scale production of
this enzyme in bacteria
provides an abundant
supply of this important
component for the cheese
production industry.
DNA RECOMBINATION
For example, one would want to find out if any work has been
done on spider silks. The databases (e.g. Genbank:Nucleotide
database) may be searched for entries that contain information
on “Spiders, and Silk” (Result: 93615 entries). The results may be
screened for more specific studies (e.g. Malaysia, Spiders, and
Silk- Result two entries).
PCR
AMPLIFICATION
Once
a
desired
trait
is
chosen,
information must be acquired for either
its detection or expression in a
given organism.
DETECTION
SOME RESEARCHERS MAY BE INTERESTED IN DETERMINING IF A GIVEN GENE/TRAIT IS AVAILABLE IN A
PARTICULAR ORGANISM. IF NO PREVIOUS RESEARCH PROVIDES THIS INFORMATION, RESEARCHERS MAY
TEST THE DNA OF DIFFERENT ORGANISMS FOR THE PRESENCE OF THESE SPECIFIC GENES. A TECHNIQUE
THAT ALLOWS THE DETECTION OF SPECIFIC GENES IN TARGET ORGANISMS IS CALLED PCR.
DETECTION
PCR amplification is an in-vitro method that simulates DNA replication in
vivo. It utilizes a thermostable (heat-resistant) DNA polymerase that builds
single stranded DNA strands unto unwound DNA templates.
PCR uses repeated cycles of incubation at different temperatures to
promote the unwinding of the DNA template (~95°C); the annealing of a
primer (a ~20bp oligonucleotide sequence (recall RNA primers in DNA
replication) onto the ssDNA template strand (~54 - 60°C); and the
extension of the generated ssDNA strand through the binding of
complementary bases to the template strand (~72° C).
DETECTION
The thermostability of the polymerase allows it to survive the repeated
cycles of denaturation, annealing and extension with little loss of enzyme
function.
Each cycle of PCR doubles the amount of the target sequence. A typical
PCR experiment uses about 35 cycles of amplification. This increases the
original amount of the target sequence by 235 (i.e. ~34 billion) times.
DETECTION
Gene detection by PCR involves the design of primers that would
only bind to sequences that are specific to a target.
For example, researchers would want to find out if gene X (e.g. the
gene for insulin) is available in a target organism (e.g. a mouse, Mus
musculus).
Primers may be designed by looking at the available sequences for
gene X in the databases (e.g. all the genes for insulin in different
organisms; humans, pigs, cows, etc.).
DETECTION
The different gene X sequences must be aligned/ compared to
match areas of sequence similarity (conserved sequences) and
areas of sequence dissimilarity (non-conserved sequences).
Primers designed to have the same sequence as the conserved
areas will be specific for binding gene X sequences in all the target
organisms.
Primers designed to have the same sequence as the non-conserved
areas will only be specific for the organisms which match its
sequence.
DETECTION
Primers may be classified as forward or reverse primers. Forward
primers are complementary and bind to the reverse
complementary (non-coding) sequence of the gene.
Reverse primers arecomplementary and bind to the coding
sequence of the gene.
STEPS IN PCR AMPLIFICATION
Step 0: Undenatured Template ; Temp ~ 54 °"C;
Template: double stranded (ds) DNA strand. Complementary sequences are held
together by H-bonds
5’ A T GCGATGAGGATATGACCCGATAGATAGAGGTATCTAGAGAT 3’ (Coding strand)
3’ T A CGCTACTCCTATACTGGGCTATCTATCTCCATAGATCTCTA 5’ (Non-coding strand)
Step 1: Template denaturation ; Temp ~ 95 °"C;
Template: single stranded (ss) DNA strands; DNA strands are separated; H-bonds
between
complementary sequences are broken
5’ A T GCGATGAGGATATGACCCGATAGATAGAGGTATCTAGAGAT 3’ (Coding strand)
3’ T A CGCTACTCCTATACTGGGCTATCTATCTCCATAGATCTCTA 5’ (Non-coding strand)
STEPS IN PCR AMPLIFICATION
Step 2: Primer Annealing ; Temp ~ 54 °"C (dependent on primer melting
temperature); Template: ssDNA strands. H-bonds are formed between
complementary sequences on the primers and the target sequences.
5’ A T GCGATGAGGATATGACCCGATAGATAGAGGTATCTAGAGAT 3’ (Coding strand)
Direction of elongation <<<<<<< CCATAGATC (Reverse Primer)
5’ GCGATGAGG 3’ >>>>>> Direction of elongation (Forward Primer)
3’ T A CGCTACTCCTATACTGGGCTATCTATCTCCATAGATCTCTA 5’ (Non-coding strand)
STEPS IN PCR AMPLIFICATION
Step 3: New DNA strand elongation ; Temp ~ 72 °"C;
The two new dsDNA strands are formed by the elongation of the generated ssDNA and
the H-bonds between the complementary sequences on these new strands and their
templates. Each of the new dsDNA strands is made up of one old strand from the
original template, and one new strand that was generated as a reverse complement of
the template. This is called semiconservative replication of the sequence.
New Strand 1:
5’ A T GCGATGAGGATATGACCCGATAGATAGAGGTATCTAGAGAT 3’ (Coding strand) (old)
3’ CGCTACTCCTATACTGGGCTATCTATCTCCATAGATC-5’ (Reverse Primer) (new)
New Strand 2:
5’ GCGATGAGGATATGACCCGATAGATAGAGGTATCTAG-3’ (Forward Primer) (new)
3’ T A CGCTACTCCTATACTGGGCTATCTATCTCCATAGATCTCTA 5’ (Non-coding strand)
(old)
STEPS IN PCR AMPLIFICATION
Step 4: Repeat step 1 to 3 for N number of cycles (N is usually 35)
PCR Results
The expected product of PCR amplification will depend on the sequences / position
at which the primer sequences bind. If the forward primer starts binding at
nucleotide 3 (coming from the 5’ end) of a 43bp long gene, and the reverse primer
binds at a position complementary to nucleotide 39 of the coding strand, then a
37bp product is expected per cycle of PCR.
STEPS IN PCR AMPLIFICATION
Step 4: Repeat step 1 to 3 for N number of cycles (N is usually 35)
STEPS IN PCR AMPLIFICATION
Step 4: Repeat step 1 to 3 for N number of cycles (N is usually 35)
PCR APPLICATION
PCR may be used to detect the presence of a desired gene in an
organism.
Depending on the primer design, the expected product may
represent only a specific region of the gene or the entire gene itself.
The first case is useful for detection of the gene, or the detection of
organisms with that specific gene within a sample.
The second case is useful for the amplification of the entire gene for
eventual expression in other organisms.
The direct amplification/copying of a full gene is part of the process
for “cloning” that gene.
CLONING AND EXPRESSION
SOME GENES PROVIDE ECONOMICALLY, AND INDUSTRIALLY IMPORTANT PRODUCTS (E.G. INSULIN-CODING
GENES; GENES FOR COLLAGEN DEGRADATION). IN SOME CASES, SCIENTISTS WOULD WANT TO PUT THESE
GENES INTO ORGANISMS FOR THE EXPRESSION OF THEIR PRODUCTS. ONE EXAMPLE WOULD BE THE
INSERTION OF AN INSULINCODING GENE FROM THE HUMAN GENOME INTO BACTERIA. THIS ALLOWS THE
“TRANSFORMED” BACTERIA TO NOW PRODUCE HUMAN INSULIN AS A PRODUCT.
CLONING AND EXPRESSION
Certain types of bacteria are capable of this process since they are able to
take genes within their cell membranes for eventual expression.
The genes are normally in the form of small, circular DNA structures
called plasmids.
The genes found in the inserted plasmid DNA sequence will be expressed
as proteins that provide specific traits to the transformed bacteria.
The basic components of an expression plasmid are listed in the following
table. The purpose of each of these is also provided.
CLONING AND EXPRESSION
Component
Promoter
Purpose
Allows the controlled expression of the desired
gene in the presence of an inducing agent (e.g.
beta- galactosidase; heat treatment (~65°"C)
CLONING AND EXPRESSION
Component
Multiple Cloning Site
Purpose
DNA sequence or portion for the insertion of
the desired gene.
This section may contain sequences that will
be cut by specific restriction endonucleases (
cuts within the molecule) If both the amplified
gene and the plasmid are cut with the same
restriction enzyme, then complementary
sequences will be generated for each, allowing
them to bind together or anneal.
The desired gene is inserted into the multiple
cloning site through this process.
CLONING AND EXPRESSION
Component
Multiple Cloning Site
Purpose
CLONING AND EXPRESSION
Component
Purpose
PCR Primers:
5’ GCGATGAGG 3’ (Forward Primer)
3’ CCATAGATC 5’ (Reverse Primer)
Multiple Cloning Site
Forward Primer + EcoRI target sequence:
5’ GAATTCGCGATGAGG 3’
Reverse Primer + EcoRI target sequence:
3’ CCATAGATCCTTAAG 5’
CLONING AND EXPRESSION
Component
Inserted Gene Sequence
Purpose
Successful insertion of a gene allows the
expression of its protein product.
This usually provides a specific trait to the
“transformed” bacteria.
For example, if the gene for Green Fluorescent
Protein is placed within the expression
plasmid, bacteria transformed with this
plasmid will produce protein (GFP) that will
allow the bacterial cells / colonies to glow
green in the dark.
CLONING AND EXPRESSION
Component
Antibiotic Resistance
Gene
Purpose
Provides a way to screen a population of
bacteria for those that took up the plasmid.
For example, if an ampicillin resistance gene is
encoded in the plasmid, then only bacteria
which took up the plasmid will be able to grow
on media with ampicillin.
However, if the ampicillin resistance gene is cut
and the gene is inserted here for cloning, then
the cell will no longer be resistant to ampicillin.
CLONING AND EXPRESSION
Component
Antibiotic Resistance
Gene
Purpose
This is a way to select which among the colony
of cells actually contain the inserted gene
sequence.
Bacterial cells whose ampicillin resistance gene
have been cut will die in the presence (agar
plate) of ampicillin.
THINK FOR A MOMENT:
GIVE OTHER HYPOTHETICALLY MODIFIED OR GENETICALLY ENGINEERED
PLANTS AND ANIMALS WHICH CAN BE USED FOR HEALTH, INDUSTRY,
AGRICULTURE AND FOR THE PROTECTION OF THE ENVIRONMENT.
NOTE:
CAUTION!!!
Certain ethical principles should be followed and adhered to in the production of
genetically modified organisms. Animal welfare should be taken cared of and human
cloning must never be conducted.
THINK FOR A MOMENT:
Discuss how PCR may be used for the detection of disease
causing pathogens in a population.
Discuss how the cloning and expression of certain genes
allows for massive production of the desired product.