4.4 Genetic engineering and
biotechnology
OBJECTIVES
• Outline the use of polymerase chain reaction
(PCR) to copy and amplify minute quantities of
DNA.
• State that, in gel electrophoresis, fragments of
DNA move in an electric field and are separated
according to their size.
• State that gel electrophoresis of DNA is used in
DNA profiling.
• Describe the application of DNA profiling to
determine paternity and also in forensic
investigations.
• Analyze DNA profiles to draw conclusions about
paternity or forensic investigations.
OBJECTIVES
• Outline three outcomes of the sequencing of the
complete human genome.
• State that, when genes are transferred between
species, the amino acid sequence of polypeptides
translated from them is unchanged because the
genetic code is universal.
• Outline a basic technique used for gene transfer
involving plasmids, a host cell (bacterium, yeast or
other cell), restriction enzymes (endonucleases) and
DNA ligase.
• State two examples of the current uses of genetically
modified crops or animals.
DNA
•
•
•
•
Double helix genetic material
Can be extracted by using chemicals in labs
Can be copied outside of cell
Can be transferred from one organism to
another organism (gene transfer)
• Can be used to analyze genetic properties of
owner of the DNA
DNA extraction
Breaking open the cells
Release of DNA by breaking cell wall
Filter cell debris
Breakdown proteins by protease
Precipitation of DNA by ethanol
Cutting up of DNA
• DNA can be by cut by restriction enzymes
• They form two types of DNA fragments (sticky
ends or blunt ends)
Exploring DNA
• Copying DNA in the lab (polymerasechain reaction) PCR
• Using DNA to reveal its owner’s identity
• Mapping DNA by finding where every A’T’C’G is
(Human genome project)
• Cutting and pasting genes to make a new
organism- gene transfer
• Cloning cells and animals
Figure 12.2_s4
1
DNA
Restriction enzyme
recognition sequence
A restriction
enzyme cuts
the DNA into
fragments.
2
Sticky
end
A DNA fragment
from another
source is added.
Restriction
enzyme
Sticky
end
Gene of
interest
3
Two (or more)
fragments stick
together by
base pairing.
4
DNA ligase
pastes the
strands together.
5
DNA ligase
Recombinant
DNA molecule
PCR
• PCR is a laboratory technique which takes a
very small quantity of DNA and copies all the
nucleic acids in it to make millions of copies of
DNA
• sample could be from the crime or from a
cheek smear ……
 Polymerase chain reaction (PCR) is a method of
amplifying a specific segment of a DNA molecule.
 PCR relies upon a pair of primers that are
• short,
• chemically synthesized, single-stranded DNA molecules,
and
• complementary to sequences at each end of the target
sequence.
 PCR
• is a three-step cycle that
• doubles the amount of DNA in each turn of the cycle.
Figure 12.12
Cycle 1
yields two molecules
Genomic
DNA
3
5
Cycle 2
yields four molecules
5
3
5
3
separates
DNA
strands.
5
5
3
Target
sequence
5
2 Primers bond
1 Heat
3
3
polymerase
adds
nucleotides.
with ends
of target
sequences.
3
5
Primer
3
3 DNA
3
5
5
5
5
5
3
New DNA
Cycle 3
yields eight molecules
Figure 12.12_1
Cycle 1
yields two molecules
Genomic
DNA
3
5
1 Heat
3
5
5
3
separates
DNA
strands.
3
5
5
2 Primers bond
with ends
of target
sequences.
Target
sequence
3
5
3
3 DNA
polymerase
adds
nucleotides.
3
5
5
3
5
3
Primer
5
5
5
New DNA
3
Figure 12.12_2
Cycle 2
yields four molecules
Cycle 3
yields eight molecules
 The advantages of PCR include
- the ability to amplify DNA from a small sample,
- obtaining results rapidly, and
- a reaction that is highly sensitive, copying only the target
sequence.
Gel electrophoresis sorts DNA
molecules by size
 Gel electrophoresis can be used to separate DNA
molecules based on size as follows:
1. A DNA sample is placed at one end of a porous gel.
2. Current is applied and DNA molecules move from the
negative electrode toward the positive electrode.
3. Shorter DNA fragments move through the gel matrix
more quickly and travel farther through the gel.
4. DNA fragments appear as bands, visualized through
staining or detecting radioactivity or fluorescence.
5. Each band is a collection of DNA molecules of the same
length.
DNA profiling
 DNA profiling is the analysis of DNA fragments to
determine whether they come from the same
individual (matching unknown DNA with known
DNA).
 DNA profiling
• compares genetic markers from noncoding regions that
show variation between individuals and
• involves amplifying (copying by PCR) of markers for
analysis.
DNA profiling has provided evidence
in many forensic investigations
 DNA profiling is used to
• determine guilt or innocence in a crime,
• settle questions of paternity,
• identify victims of accidents, and
• probe the origin of nonhuman materials.
Figure 12.11
Crime scene Suspect 1
1
DNA is
isolated.
2 The DNA of
selected
markers is
amplified.
3
The amplified
DNA is
compared.
Suspect 2
The goals of the Human Genome Project
(HGP) included
- determining the nucleotide sequence of
all DNA in the human genome and
- identifying the location and sequence of
every human gene.
HGP
• Human genome represents 30 000 – 45 000
genes
• A rice plant has 55 000 genes
• Complexity of the organism is not depend on
number of genes, it depends on how we
use/control those genes.
HGP
• Scientists know the location of gene that
cause genetic disease.
• We use gene therapy
e.g: familial hypercholesterolaemia. In this
disease cell membrane receptors for LDH are
defect and LDH accumulates in the blood.
- Search evolutionary relationship of two
organisms
Genes can be cloned in recombinant
plasmids
1. Recombinant DNA molecules are produced when DNA
ligase joins plasmid and target segments together.
2. The recombinant plasmid containing the target gene is
taken up by a bacterial cell.
3. The bacterial cell reproduces to form a clone, a group of
genetically identical cells descended from a single
ancestral cell.
Figure 12.1B
E. coli bacterium
Plasmid
Bacterial
chromosome
1
A cell with DNA
containing the gene
of interest
2
A plasmid
is isolated.
The cell’s DNA
is isolated.
Gene of
interest
3
DNA
The plasmid is cut
with an enzyme.
Examples of gene use
4
The cell’s DNA is cut
with the same enzyme.
Gene
of interest
5
6
The targeted fragment
and plasmid DNA
are combined.
DNA ligase is added,
which joins the two
DNA molecules.
Examples of protein use
Recombinant
DNA
plasmid
Gene
of interest
7
The recombinant plasmid
is taken up by a bacterium
through transformation.
Recombinant
bacterium
8
Clone
of cells
Genes may be inserted
into other organisms.
The bacterium
reproduces.
9
Harvested
proteins
may be
used
directly.
Recombinant cells and organisms can
mass-produce gene products
 Pharmaceutical researchers are currently exploring
the mass production of gene products by
• whole animals or
• plants.
 Recombinant animals
• are difficult and costly to produce and
• must be cloned to produce more animals with the same
traits
Recombinant goat produces human
protein
Cloning (producing identical things)
• Genes can be cloned
• Organisms can be cloned
• Asexual reproduction is a natural cloning
process in plants.
• Animals can be cloned by nuclear transfer
technique.
Human Cloning?
• Human cloning takes place naturally by
forming identical twins.
• It is forbidden to clone a human in the lab.
Instead scientist keep embryonic stem cell to
cure disease in the future.
Argument for and against human cloning
• Cloning could be used to produce an organ
not as a whole organism
• It could be used to produce “superior” race
• Cloned newborns may die in early age
• It might diminished sense of individuality
• Some aspects of human life should exist above
the values of standards of labs
• Some people believe that it is against God
Parents at high risk of producing offspring with
genetic disease can have healthy children
Organs can be produced and transplanted
Individuality is not affected because of
environmental factors