GENETIC ENGINEERING

GENETIC ENGINEERING
• Genetic engineering is the process of using recombinant DNA
(rDNA) technology to alter the genetic makeup of an organism.
• Traditionally, humans have manipulated genomes indirectly by
controlling breeding and selecting offspring with desired traits.
• Genetic engineering involves the direct manipulation of one or
more genes.

• Most often, a gene from another species is added to an

organism's genome to give it a desired phenotype.
DEFINITIONS

Recombinant DNA (rDNA) is a form of artificial DNA that is

created by combining two or more sequences that would not
normally occur together through the process of gene splicing.

Recombinant DNA technology is a technology which allows

DNA to be produced via artificial means. The procedure has
been used to change DNA in living organisms and may have
even more practical uses in the future.
 Enzymes Involved in Gene Cloning
1. Reverse Transcriptase: It is obtained from Avian myeloblastosis virus. It
catalyses the synthesis of copy DNA (cDNA).
2. DNA Ligase: It’s action is quite opposite to restriction endonuclease (RE). It is
known as molecular suture. It joins two different pieces of DNA. The source of
enzyme is T4 virus or E.coli. The two fragments are brought together by
formation of phosphodiester bond between free 5’ – PO4 group on one strand
and 3’ – OH group on the other strand

3. Restriction Endonuclease: Restriction endonucleases are enzymes that

recognize a specific DNA sequence, called a restriction site, and cleave the
DNA within or adjacent to that site
STEPS INVOLVED IN GENE CLONING PROCESS

Isolation of a desired Gene

Insertion into a vector to construct rDNA
Introduction of rDNA into host cells
Identification and isolation of the
transformed cells
Cloning i.e replication of rDNA to produce
multiple copies of itself
Expression of foreign gene to obtain the
desired gene product.
 Step-1: Gene of Interest
1. Genomic DNA
2. Complementary/Copy DNA (cDNA)
Isolate whole genomic DNA from organism

DNA extraction easily performed using:

• SDS (detergent) to break up cell membrane and organelles.

• Salt (NaCl) lyses cells and binds the DNA strands together.

• Proteinase K to digest proteins bound to DNA (essential to

remove eukaryotic chromatin).

• Ethanol (EtOH) to precipitate and wash DNA.

• Water to resuspend and store DNA.

Step 2-Cut DNA with restriction enzymes

Restriction enzymes recognize specific bases pair sequences in DNA called

restriction sites and cleave the DNA by hydrolyzing the phosphodiester
bond.

✔ Cut occurs between the 3’ carbon of the first nucleotide and the phosphate
of the next nucleotide.

✔ Restriction fragment ends have 5’ phosphates & 3’ hydroxyls.

restriction
enzyme
 Tools of Recombinant DNA Technology

RESTRICTION ENDONUCLEASES

o This enzyme is first discovered by Hamellton Smith in

Haemophilus influenzae bacteria.
o This enzyme is also known as ‘ Molecular scissors’.
o These are used to cut DNA within recognition site.
Actions of restriction enzymes-overview
Creating a cDNA library

• Anneal a short oligo dT (TTTTTT) primer to the poly-A tail.

• Primer is extended by reverse transcriptase 5’ to 3’ creating a mRNA-DNA

hybrid.

• mRNA is next degraded by Rnase H, but leaving small RNA fragments intact to
be used as primers.

• DNA polymerase I synthesizes new DNA 5’ to 3’ and removes the RNA primers.

• DNA ligase connects the DNA fragments.

• Result is a double-stranded cDNA copy of the mRNA.

 Step-2: Insertion into vector

Vectors
– Nucleic acid molecules that deliver a gene into a cell

– Useful properties

• Small enough to manipulate in a lab

• Survive inside cells
• Contain recognizable genetic marker
• Ensure genetic expression of gene
– Include viral genomes, transposons, and plasmids
Step 3-Splice (or ligate) DNA into some kind of cloning vector
to create a recombinant DNA molecule

Six different types of cloning vectors:

1. Plasmid cloning vector

2. Phage λ cloning vector

3. Cosmid cloning vector

4. Yeast artificial chromosome (YAC)

5. Bacterial artificial chromosome (BAC)

1. Plasmid Cloning Vectors:

• Bacterial plasmids, naturally occurring small ‘satellite’ chromosome, circular

double-stranded extrachromosomal DNA elements capable of replicating
autonomously.

• Plasmid vectors engineered from bacterial plasmids for use in cloning.

• Features (e.g., E. coli plasmid vectors):

1. Origin sequence (ori) required for replication.

2. Selectable trait that enables E. coli that carry the plasmid to be

separated from E. coli that do not (e.g., antibiotic resistance, grow
cells on antibiotic; only those cells with the anti-biotic resistance grow
in colony).

3. Unique restriction site such that an enzyme cuts the plasmid DNA in
only one place. A fragment of DNA cut with the same enzyme can
then be inserted into the plasmid restriction site.

4. Simple marker that allows you to distinguish plasmids that contain

inserts from those that do not (e.g., lacZ+ gene)
Construction of rDNA
*Cut with same
restriction enzyme
*DNA ligase
Vectors and their maximum hold size for foreign DNA

Type of Vector Maximum insert size (Kbp)

Plasmid vector 15

Bacteriophage Vector 20

Cosmids 45

BAC 300

YAC 2000
Step-3: Recombinant DNA

Gene of interest is inserted in vitro into the vector to synthesize

rDNA
1. REs generating cohesive ends: If type II RE is used to generate
cohesive ends of desired gene and the same RE is used to cut the
vector. Then if vector and desired genes are brought together, in
vitro annealing occurs.

The ends of two DNAs can be joined by DNA ligase at temperature

of 4-110C requiring 12-24 hrs.
Recombinant DNA
2. RE generating blunt ends: Ligation of desired gee and vector
having blunt ends can be brought about by using a high
concentration of DNA ligase than that of ligating cohesive ends.
It is the T4 DNA ligase that employed rather than Escherichia coli
DNA ligase to join blunt ends
3. Homopolymer Tailing: By using RE, if blunt ends are generated
then the homo polymer tailing technique is useful for inserting the
desired gene into vector.
Recombinant DNA
4. Linkers and Adapters: It is another technique to convert blunt ends
to cohesive ends.
Linkers are synthetic oligonucleotides having predetermined
recognition and cleavage sites for particular RE that generates
cohesive ends on cutting.
The linkers are blunt ended on the both sides. First they are
phosphorylated using polynucleotide kinase and then ligated to the
blunt ended DNA fragment using T4 DNA ligase.

Now the desired DNA fragment is treated with the particular RE,
generating the sticky cohesive ends. Then it can be ligated.
 Step-4: Introduction of rDNA into a host cell

• Inserting DNA into Cells

• Goal of DNA technology is insertion of DNA into cell
• Natural methods
• Transformation

• Transduction

• Conjugation

• Artificial methods
• Electroporation

• Protoplast fusion
• Injection: gene gun and microinjection

23
 TRANSFORMATION

✔ Certain bacterial species of genera Streptococcus, Bacillus,

Haemophilus, Neisseria and Rhizobium are able to take up
the DNA fragments spontaneously under physiological
conditions.

✔In some species such as E.coli success rate of

transformation is low. Such cells are chemically treated to
enhance the ability to take up the foreign DNA.

✔Such treated cells are said to be competent.

TRANSFORMATION

✔Competent cells are prepared by treating with 50mM

calcium chloride ice cold solution and then heat shock
raising the temperature to 42°C to make the movement
of foreign DNA into the competent cell.
 The 4 steps in Transformation

1. A donor bacterium dies and is degraded 2. A fragment of DNA from the dead donor
bacterium binds to DNA binding proteins
on the cell wall of a competent, living
recipient bacterium

3. The Rec A protein promotes genetic

exchange between a fragment of the donor's
4. Exchange is complete
DNA and the recipient's DNA
Transduction
Genetic recombination in which a DNA fragment is transferred
from one bacterium to another by a bacteriophage

Structure of T4 bacteriophage Contraction of the tail sheath of T4

 Transduction (Contd...)
• There are two types of transduction:

Generalized transduction: A DNA fragment is transferred from one

bacterium to another by a lytic bacteriophage that is now carrying
donor bacterial DNA due to an error in maturation during the lytic
life cycle.
Specialized transduction: A DNA fragment is transferred from one
bacterium to another by a temperate bacteriophage that is now
carrying donor bacterial DNA due to an error in spontaneous
induction during the lysogenic life cycle
Seven steps in Generalised Transduction

1. A lytic bacteriophage adsorbs to a

susceptible bacterium.

2. The bacteriophage genome enters the

bacterium. The genome directs the
bacterium's metabolic machinery to
manufacture bacteriophage components and
enzymes
3. Occasionally, a bacteriophage head or
capsid assembles around a fragment of
donor bacterium's nucleoid or around a
plasmid instead of a phage genome by
mistake.
Seven steps in Generalised Transduction (cont..))

4. The bacteriophages are released.

5. The bacteriophage carrying the

donor bacterium's DNA adsorbs to a
recipient bacterium
Seven steps in Generalised Transduction (contd)

6. The bacteriophage inserts the

donor bacterium's DNA it is carrying
into the recipient bacterium .

7. The donor bacterium's DNA is

exchanged for some of the recipient's
DNA.
 Bacterial Conjugation
Bacterial Conjugation is genetic recombination in which there is
a transfer of DNA from a living donor bacterium to a recipient
bacterium. Often involves a sex pilus.
 Artificial methods of inserting DNA into cells:
Electroporation

Pores in wall and

Chromosome membrane
Cell
synthesizes
new wall
Electrical
field applied
Competent Recombinant
cell DNA from cell
Electroporati
on another
source
 Artificial methods of inserting DNA into cells: Protoplast fusion

Cell
Cell
synthesizes
walls new wall

Polyethyl
Enzymes ene
remove glycol
cell walls
Recombina New
nt cell wall
Protoplast Protopl Fused
fusion asts protoplasts
 Step-5: Identification & Isolation of Transformed
cells
The transformed cells are identified on the basis of some selective
property that has been acquired by the transformed cells.

Most frequently markers coding for specific antibiotic resistance

are used.

• Resistance against antibiotics, heavy metals

• Production of antibiotics, bacteriocins, enterotoxins, H2S

• Metabolism/degradation of aromatic compounds, sugars,

haemoglobin.
• Induction of plant tumour
 Bacterial
cell DNA
containing
gene of
interest

Bacterial Plasmi
chromoso d
me Isolate Gene of
plasmid. interest Enzymatically cleave
DNA into fragments.

Isolate fragment with the gene of

interest.

Insert gene into plasmid.

Insert plasmid and gene into

bacterium.

Culture bacteria.

Harvest copies of gene

Harvest proteins
to insert into plants or coded by gene
animals

Eliminate Create beneficial

Produce vaccines, antibiotics,
undesirable combination of
hormones or enzymes
phenotypic traits traits
ADVANTAGES:
• Provide substantial quantity.

• No need for natural or organic factors.

• Tailor made product that you can control.

• Unlimited utilisations.

• Cheap

• Resistant to natural inhibitors.

DISADVANTAGES:

• Commercialised and became big source of income for

business man.
• Effects natural immune system of the body.
• Can destroy natural ecosystem that relies on organic cycle.
• Prone to cause mutation that could have harmful effects.

• Major International concern: Manufacturing of

biological weapons such as botulism and anthrax target
humans with specific genotype.

• Concern of creating super human care.