Recombinant DNA Technology Flashcards
Also Genetic Engineering
Recombinant DNA Technology
Refers to various techniques and procedures used in gene
manipulation
Recombinant DNA Technology
Involves modifying and recombining DNAs to produce desired
products (e.g. proteins, or animals and plants with desirable
traits)
Recombinant DNA Technology
▪involves the use of molecular techniques to
modify the traits of target plant(s). The
resulting plants are often referred to as
transgenic plants or genetically modified
organisms (GMOs)
Genetic engineering
▪Achieved by adding a specific gene or genes
to a plant, or by knocking down a gene RNA,
to produce a desirable phenotype.
Genetic engineering
▪This gene can be from the same species or
different species organism
Genetic engineering
▪Sometimes genetic modification can produce
a plant with the desirable trait or traits faster
than classical breeding because the majority of
the plant’s genome is altered.
Genetic engineering
▪Practices involve focus on the mating of
organisms with desirable qualities. This relies
heavily on the naturally occurring plant life
cycle and homologous recombination to
generate genetic diversity and to eliminate
undesirable traits
Classical engineering
▪Involve the same species of plants.
Classical engineering
▪Interbreeding (crossing) can be only carried
out with closely or distant related plants.
Classical engineering
▪It is time-consuming as breeding needs
frequent crossing and self-fertilization.
Classical engineering
Tools used in Recombinant DNA
Technology
- Target DNA
- Restriction enzyme
- DNA cloning vectors
- Host cell
- Modifying enzymes
Also known as gene of interest
Target DNA or gene
A particular gene or DNA being studied
and manipulated in the experiment
Target DNA or gene
Identify a specific base sequence (restriction site) and cut
the DNA at specific point between two nucleotides in the
site
Restriction Enzymes
Different enzymes cleave at different base sequences.
Restriction Enzymes
Read 5’ to 3’ direction
Restriction Enzymes
Cut in staggered way resulting in DNA fragments with
unpaired bases at the end
Restriction Enzymes
are DNA molecules that carry foreign DNAs into
a host cell.
Cloning vectors
small, circular DNA molecules in bacteria - < 10kb
fragments
* Found in bacterial cell and in some eukaryotes
* Gene carried in plasmids provide bacteria with genetic advantages
(antibiotic resistant)
Plasmid
carries a foreign gene
Recombinant plasmid
linear double helix molecule (bacteria-infecting
viruses) - >10kb
Bacteriophage
are viruses that
infect and replicate within bacteria.
Bacteriophages
are highly specific
to the type of bacteria they infect,
making them potential tools for
treating bacterial infections.
Bacteriophages
Characteristics of Cloning Vectors
- able to accept foreign DNA in multiple cloning sites.
- able to replicate freely and rapidly.
- contain genes which are useful for bacteria
(amp – code for antibiotic resistance)
A cell which is able to accept
foreign DNA (cloning vectors) and
allow them to multiply.
Host Cell
Prokaryotic cell (E.coli) or
eukaryotic cell (yeast or animal
cell)
Host Cell
Characteristics of a Host Cell
- Able to accept rDNA plasmids through transformation
- Able to maintain the structure of rDNA from one
generation to another - Able to amplify(multiply) the gene product of rDNA
join fragments of
DNA (target gene + plasmid =
recombinant DNA)
DNA ligase
– replace DNA in
PCR (polymerase chain reaction), a
method for amplifying short
segments of DNA
Taq polymerase
gene of interest
Target DNA
used to cut enzymes into fragments
Restriction enzyme
to carry target gene into a host cell
(plasmid or bacteriophage)
DNA cloning vectors
bacterial cell that allows the cloning vectors to
replicate with it
Host cell
DNA ligase and Taq polymerase
Modifying enzymes
Methods in Gene Cloning
- Isolation of the target gene
- Insertion of the target gene into a vector
- Introduction of the vector into a host
- Amplification of the target gene by the host cell
(cloning) and screening
The desired gene is identified by:
- by cutting the gene from a complete chromosome using a restriction enzyme
and - by producing complementary
fragments of various lengths
DNA fragments
reverse transcription
DNA (cDNA)
separate various fragments according to sizes
Gel electrophoresis
to locate the target gene
DNA probe
Insertion of the target gene into a vector
- Target gene is inserted into vectors (plasmid or
bacteriophage) resulting in recombinant DNA,
combinations of DNAs from two sources. - The plasmids (opened rings) with sticky ends are mixed
with the target gene and then joined by DNA ligase. - Results: plasmids that carry the target gene and/ or
otherwise ( non-recombinant plasmid)
Introduction of the vector into a host
- The plasmids carrying the target gene (recombinant
plasmids) must be introduced into a host cell.
Results: - Bacteria that do not take up any plasmid
- Bacteria that take up non-recombinant plasmid
- Bacteria that take up recombinant plasmid
Amplification of the target gene by the host
cell (cloning) and screening
- The bacteria is cultured in a medium containing
ampicillin and the sugar, X-gal. - E. coli will be able to grow and form colonies (resistant to
ampicillin) - The bacteria will divide and produce clones containing
recombinant plasmids.
A method of amplifying segments of DNA in vitro.
Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR) Tools:
Tools: thermocycler, a test tube with the DNA sample, Taq
polymerase, primers and free DNA nucleotides
Steps in PCR
- Denaturation
- Annealing
- Elongation (Extension)
The DNA is heated to break the hydrogen
bond and separate the strands.
Denaturation
Each fragment is then cooled.
- Add Taq polymerase and primers so they
can bind to the ends of the DNA
Annealing
Add free DNA nucleotide and
raise the temperature.
Elongation (Extension)`
Applied to the bacteria
E. coli (Escherichia coli)
to produce synthetic
human insulin
commercially
Production of Insulin
Transgenic
animals – animals which genetically-
modified
Agriculture
Transgenic plants
– plants which
genetically-
modified
