Bio Lab

Genetic Engineering
& PCR Technique
2nd semester
Objectives
• Genetic engineering
• Host genome manipulation.
• Recombinant technology.
• PCR Technique.
• Applications.
 Genetic engineering
• Process of inserting new genetic information into
existing cells in order to modify a specific organism for
the purpose of changing its characteristics (alters the
genetic structure of an organism by either removing or
introducing DNA).
• Or it refers to the direct manipulation of DNA to alter
an organism’s characteristics (phenotype) in a
particular way. This may mean changing one (A-T or
C-G), deleting a whole region of DNA, or introducing
an additional copy of a gene.
Also called genetic modification or genetic manipulation, is the
direct manipulation of an organism's genes using biotechnology.
It is a set of technologies used to change the genetic makeup of
cells, including the transfer of genes within and across species
boundaries to produce improved or novel organisms.
Genetic engineering is used by scientists to any organism,
from a virus to a sheep.
 New DNA is obtained by either isolating and
copying the genetic material of interest using recombinant DNA methods or by artificially
synthesizing the DNA.
1*The first recombinant DNA molecule was made by Paul Berg in 1972
by combining DNA from the monkey virus SV40 with the lambda virus.

2*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.

3*An organism that is generated through genetic engineering is

considered to be genetically modified(GM) and the resulting entity is a
genetically modified organism (GMO). The first GMO was a bacterium in
1973.
 Inserting DNA into the host genome
**There are a number of techniques used to insert genetic material
into the host genome. Some bacteria can naturally take up foreign
DNA.
**This ability can be induced in other bacteria via stress (e.g. thermal
or electric shock), which increases the cell membrane's
permeability to DNA; up-taken DNA can either integrate with the
genome or exist as extrachromosomal DNA. DNA is generally
inserted into animal cells using microinjection, where it can be
injected through the cell's nuclear envelope directly into the
nucleus, or through the use of viral vectors.
**In animals it is necessary to ensure that the inserted DNA is
present in the embryonic stem cells, Bacteria consist of a single
cell and reproduce clonally so regeneration is not necessary, But
in plants the DNA is often inserted using specific bacteria
mediated recombination Selectable markers are used to easily
differentiate transformed from untransformed cells.
These markers are usually present in the transgenic organism.
*Further testing using PCR, Southern hybridization, and DNA
sequencing is conducted to confirm that an organism contains
the new gene.
Recombinant Technology:
-Basically it is the potential genetic engineering field by
using PCR or microorganisms.
-Genes are Isolated ,Modified &Inserted into an organism
they are made up by recombinant technology through:
–Cutting DNA and recombine their pieces
–Amplify modified pieces.
Ex.
-Plasmid is small circle of bacterial DNA
-Foreign DNA can be inserted into plasmid
–Forms recombinant plasmids
–Plasmid is a cloning vector
–Can be used to deliver DNA into another cell
Genetic Transfer and Recombination in Bacteria

Genetic Transfer is a process whereby genetic material from one bacterium(donor cell)
is transferred to a nother bacterium (recipient cell ) .
Like sexual reproduction in eukaryotes, genetic transfer in bacteria is thought to
enhance the genetic diversity of bacterial species .
For example, a bacterial cell carrying a gene that provides antibiotic resistance may
transfer this gene to another bacterial cell, allowing that bacterial cell to survive
exposure to the antibiotic .
In bacteria there are three different mechanism for transfer of genetic material :
1- Transformation
2- Transduction
3- Conjugation
as in ( figures below)
1- Transformation :
The recipient cell takes up free DNA fragment released from donor cell .
Transformation known to occur naturally and also used in genetic engineering in
recombinant DNA study . and example of genes transferred by transformation the
genes for polysaccharide capsule .

Transformation Steps :
1- Donor DNA fragments binds to a cell surface receptor .
2- An extracellular endonuclease cuts the DNA into smaller fragments .
3- One of the DNA strands is degraded and the other which contains the gene enters
the bacterial cell .
4- The DNA strand is incorporated into the bacterial chromosome via homologous
recombination ( crossing over ) . Fig-1-
Figure 2
2- Transduction :

Occur when bacterial genes are carried from a donor cell to a recipient cell by a
bacterial virus ( bacteriophage ) . Facilitating subsequent recombination of the genetic
markers of the two cells .
Phage attaches to host cells receptors and injects DNA leaving the capsid outside .
Inside the cell, viral DNA can either :
a- Replicate to form phage and lyse the host to release the phage progeny ( process called
lytic cycle ) . Or
b- Integrate into the bacterial chromosome ( process called lysogenic cycle ) .
Prophage : the viral DNA that integrates into the bacterial chromosome ..
Genes transferred by transduction :
-Genes of toxins such as ( botulinum, diphtheria , cholera )
-Genes of enzymes for sugar fermentation and genes of drug resistance .

Fig-3-
 Some phage progeny released from the lytic may contain host DNA
(transducing phage ) which is transferred into a new host in the next
infection cycle . The foreign host DNA can integrate by homologous
recombination the process is called Generalized Transduction .
The prophage may be exits from the bacterial chromosomes carrying
small segment of host genes with it and enter the lytic cycle .
Transducing phages infect anew host cells where recombination
( crossing over ) occur , the process is called Specialized Transduction .
3- Conjugation :
Requires direct cell contact and involves the transfer of donor DNA
to recipient cells through a conjugation tube that forms between the two
cells . Fig-4-
Genes transferred by Conjugation :
Genes for drug resistance , resistance to metals , toxins
production, enzymes, adherence molecules , degredation of
toxic substances , uptake of iron .

Some bacteria may also possess Trasposons or Jumping

genes: that means genes or DNA segment that moves to
another chromosome .
Figure 5-
 What is PCR ?
• It is Polymerase Chain Reaction that results in the
selective amplification of a chosen region of a DNA
molecule.
• It is done in a single test tube simply by mixing DNA
with a set of reagents and placing the tube in a
thermal cycler (a device)that enables the mixture to be
incubated at a series of temperatures.
PCR device
PCR-Reaction Mixture
•Copies of DNA to be sequenced
•Primer of (piece of DNA)
•TaqDNA polymerase ( an enzyme)
•Standard nucleotides
•Modified nucleotides
PCR Steps:
A- Denaturation step :
It causes melting of DNA template by disrupting the hydrogen bonds
between complementary bases of the DNA strands, producing single strands
of DNA.
B– Primer Annealing step :
Annealing of the primers to the single stranded DNA template .
C–Extension or Elongation step :
DNA polymerase synthesizes a new DNA strand complementary to the DNA
template strand by adding deoxynucleotide triphosphate (dNTPs) that are
complementary to the template in 5 to 3 direction .
PCR cycle diagram
 Agarose gel
electrophoresis
*At the end of a PCR a sample of the
reaction mixture is usually analyzed by
agaros gel electrophoresis technique
,this provide useful information about
the DNA region(band) that has been
amplified, or the PCR product can be
examined by DNA sequencing method.
By repeating the cycle 30 times the double-stranded molecule that we
began with is converted into over 130 million new double-stranded
molecules.
 Genetic engineering Applications:
• *Genetic engineering has been applied in numerous fields
including: research(Micro organisms &proteins),
medicine(human insulin, growth hormone, enzymes,
drugs,vaccines & heart disease, diabetes, arthritis, ..),
industrial(Genetically modified food) biotechnology(GM
animal ,GM bacteria,..) and agriculture(GM crops).
In research GM organisms are used to study gene
function and expression through loss of function, gain of
function, tracking and expression experiments.
Applications:
• Engineered Proteins (Hormons-Insulin, and blood-clotting factors
&Vaccines by using bacteria)
• Forensic Investigation : identification of suspect based on crime scene
samples .
• 2 – Paternity test.
• Produce Drugs,foods,enzymes.
• Cleaning Up the Environment-Microorganisms can break down
organic wastes and cycle materials.
• Basic Researchs(genetic processes).

• Engineered Mammals(mice, Cattle& chicken)

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