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Gene Technology
Gene Technology Jargon
 Gene technology: a general term covering a
variety of technics involving genetic material
including genetic engineering, creation of
gene libraries & DNA frequencing.
 Genetic engineering: the transfer of a gene
from one organism (the donor) to another
organism (the recipient) e.g. the gene of
insulin
Gene Technology Jargon
 Restriction enzyme/ restriction
endonuclease: an enzyme which cut the
DNA at a particular site determined by the
base sequence.
 Marker: an easily recognised gene which is
transferred along with the target gene
during genetic engineering e.g. fluorescent
pigments
Gene Technology Jargon
 Promotor: a section of DNA directly before
the gene where RNA polymerase attaches to
the DNA allowing transcription to begin.
 Vector: a carrier DNA molecule which
allows the gene which is to be transferred to
be inserted into the recipient organism e.g.
viruses or bacterial plasmids
Gene Technology Jargon
 Genetic fingerprinting: an analytical process
which is used to determine the relativeness of
two DNA sample.
 DNA sequencing: the process which
determines the precise sequence of
nucleotides in a sample of DNA
Synthesise human insulin
 Why produce human insulin
– It is identical to human insulin therefore liver
cells react more quickly, the hormone is more
easily broken down, and does not elicit an
immune response.
– Some religions groups e.g. Muslins & Jews might
object to use pig insulin
– It avoids contamination e.g. prion proteins 
Bovine spongiform encephalopathy
Synthesise human insulin
 Using internet find out the stage of synthesis
human insulin. – 15 mins
 Using the information which you find, write
down the stages. – 15 mins
 Present you work – 30 mins
 Remarks: detail stages, including which RE it
use, which vector it use. etc.
Stage 1:
 Identification and isolation of the gene.
 Pancreas cells were used because they contain
large amounts of mRNA. mRNA for the insulin
gene was isolated and purified. This mRNA has
the advantage that the junk DNA sequences
(introns) have already been removed.
 The mRNA was incubated with DNA nucleotides
and a reverse transcriptase to make DNA i.e. the
insulin gene
 Then the single strands of DNA are incubated
with DNA polymerase to produce double strands.
Stage 2:
 Short sequences of single strand DNA are
added to the ends of the gene. These sticky
ends correspond to other sticky ends
produced when DNA is cut by a particular
restriction enzyme. e.g. ecoR1
Sticky
end
 The restriction enzyme which is chosen
must not cut the gene i.e. the sequence must
not appear in the DNA sequence of the gene.
Stage 3:
 DNA plasmids from bacteria are cut using
the same restriction enzyme. Then the genes
are incubated along with the plasmids and
hopefully the gene DNA will become
inserted into the plasmid DNA.
 NB: a plasmid is the natural method by
which bacteria transfer genes between one
another. As such, it allows us to transfer
genes into the bacterial genome, because the
bacteria have a natural tendency to acquire
plasmids.
Stage 4:
 Plasmids containing the human genes are
mixed with bacteria e.g. Escherichia coli
(E.coli) and incubated. Some of the bacteria
will take up the plasmid and incorporate it
into there own genome.
Stage 5:
 The bacteria are spread over agar plates
and allowed to grow. Then bacterial colonies
which have incorporated the insulin gene
are identified. Originally, this was done by
transferring a sample from each colony onto
another agar plate which contains
antibiotics for which the resistance gene was
transferred along with the insulin gene as a
marker. Only the bacteria with the insulin
gene will grow. These can be separated and
cultured in large quantities.
Issues associated with genetic
engineering
1. Promoters: are necessary to regulate the
transcription of genes. The original genetic
engineering involving insulin used the lac
operon. The lac operon act as follows.
–
Lac operon – a system of promoter & gene for
breaking down lactose – promoter & gene for
β–galactosidase enzyme & gene for lactose
permease.
Issues associated with genetic
engineering
 There is a protein know as the lac repressor
which attaches to the DNA next to the
promoter sequence preventing the
attachment of RNA polymerase. However in
the presence of lactose, lactose molecules
attach to the repressor changing its
configuration so that it no longer attaches to
the DNA. This allows RNA polymerase to
attach & express the gene. So in the original
experiment, the bacteria would produce
insulin protein in the presence of lactose.
Issues associated with genetic
engineering
2. Markers: originally antibiotic-resistant
marker were used. However, there are 2
problems using these two markers.
 The antibiotic-resistant genes could spread to
pathogenic species. e.g. cholera
 Antibiotic resistance occurs naturally in bacteria
therefore it is possible for resistance strains to
not contain the insulin gene.
So nowadays, genes which are harmless and not
naturally occurring in bacteria are used. e.g.
fluorescent protein from jellyfish – glows under
UV light or a gene for substances which are
easily stained.
Issues associated with genetic
engineering
3. Eukaryotic organisms: insulin is a simple
protein which could be successfully
produced by bacteria. However, large
proteins with more complicated
configurations can only be produced by
eukaryotic organisms. So for these
proteins, we would use single-celled
eukaryotic organisms e.g. yeast
DNA fingerprinting
 This process allows us to analyse and identify
individuals from samples of DNA. DNA finger
printing involves cutting the DNA into
fragments using restriction enzymes. Different
people have different DNA especially highly
repetitive nonsense sequences or VNTR
(variable number tordem repeat). These
sequences are different but also inherited e.g.
someone may have 50 or 150. The variable
number means the size of the fragments in
different individuals is variable.
Stage 1
 DNA is extracted from a sample of tissue.
This DNA is treated with specific restriction
enzymes. Chosen because around the
repetitive sequences.
Stage 2
 Electrophoresis
 DNA molecules
are negatively
charged and
therefore move
towards the
anode. They
become
separated into
bands because
the different size
molecules move
at different
speeds.
Stage 3
 When electrophoresis is complete, a layer of
nylon is laid on top of the gel. The absorbent
material e.g. tissue is pressed onto the nylon.
The tissues absorbs water from the gel by
capillary action in doing so the DNA
fragments become attached to the nylon
membrane.
Stage 4
 The DNA is heated cause denaturation i.e.
the two strands separate. Then a DNA
probe is added. This is a short single strand
of DNA containing radioactive phosphorus.
It attaches to the exposed DNA fragments in
locations where the DNA sequence matches.
Stage 5
 A piece of X-ray film is placed over the
nylon sheet and as the radioactive isotope
decays, the radioactivity released, creates a
pattern of lines on the film.
DNA sequencing
 This process involves determining the
sequence of bases found in a molecule of
DNA.
DNA sequencing
 The DNA molecule is multiplied, then broken
down are base at a time. The remaining
fragments vary in lengths from one base to the
whole molecule can be separated by
electrophoresis. A second process of
electrophosesis can determine which base is on
the end of each fragments and therefore the
entire sequence can be deduced.
 Most recently, DNA sequencing has been used in
a large scale in the human genome project (HGP).
This involve sequencing of the entire human
genome so as to identify genes and mutations.
Gene therapy – e.g. cystic fibrosis
 Gene therapy is a process which aims to
cure or prevent disease by changing the
genotype.
 There are 2 potential approaches:
1. Germ cell therapy i.e. sperms, eggs, zygotes.
This has the advantage that all the cells in the
body derived from the germ cells will contain
the same alteration. However, it is not carried
out for ethical reasons since the potential
beneficiary is unable to consent to the
procedure.
Gene therapy – e.g. cystic fibrosis
2. Somatic cell therapy. This involves
changing the genotype of body cells by
adding/removing a particular gene. In the
case of cystic fibrosis, this is made easier
because it is recessive, and therefore only
requires a functioning copy of the gene
into the nucleus of the cell to allow the
production of the ion channels. Somatic
therapy has 3 main drawbacks:
Gene therapy – e.g. cystic fibrosis
 3 main drawbacks
– The gene must be inserted into all somatic cells
individually.
– The difficulty of delivering the gene.
– The limited lifespan of the affected cells.
 For cystic fibrosis it is estimated that less
than 10% of the epithelial cells needs to be
functioning to reduce the symptoms
dramatically.
Gene therapy – e.g. cystic fibrosis
3. Methods of delivery have been attempted:
 Virus – e.g. Adenovirus. Viruses are
specialized to deliver DNA into cells. If the
harmful genes of the virus are replaced by the
chloride transport protein genes, the genes
can be incorporated into the cells’ genome.
The virus can be delivered through an aerosol.
 Liposome – A sphere of lipid surrounding the
DNA which when introduced through aerosol
will fuse with cell membrane introducing the
gene into the cells.
Gene therapy – e.g. cystic fibrosis
Microinjection – Physical injection of DNA into
the cell. This is only practical in laboratory
situation.
Real trials of gene therapy were carried out in
Jam, 1985 using a liposome delivery system.
Viruses were not used because they stimulate
an immune response. The study produced a
20% improvement in potential difference a
cross the cells of the nose lining. However, the
effect lasted for about a week only.
Genetic screening
 This is the process by which samples of
DNA by individuals are tested for the
presence or absence of individual alleles and
therefore the risk of passing on an inherited
condition. Screening can take place at 3
stages:
Genetic screening – 3 stages
1.
2.
Before pregnancy – obviously at this stage we will
screen the parents looking for recessive alleles e.g. cystic
fibrosis. A DNA probe labelled with radioactive markers
can be added to the DNA and will attach to the mutant
gene if it is present.
During pregnancy – looking at the embryo or the fetus's
DNA. There are 2 techniques for gathering cells from
embryo


3.
Amniocentesis – a hypodermic syringe is inserted into the
amnion carefully avoiding the fetus. A sample of amniotic fluid
is withdrawn which contains cells from the fetus which can be
cultured and tested.
Chorionic villus sampling – a catheter is inserted through the
virgina to the uterus gathers cells from the placenta.
After pregnancy – e.g. PKU(phenylketonuria) are tested
from a blood test on the new-born infant.
Genetic counseling
 The process of genetic screening can produce
worrying and upsetting results therefore genetic
counseling is used to try to help people deal with
the results of genetic screening. The kinds of
questions involved are:
– Is it right to have the test
– What are the risks associated with becoming
pregnancy.
– What action can be taken e.g. selection of embryo
from I.V.F or abortion.
– The effects of the genotype of the offspring.
Discuss the possible benefits &
dangers of gene technology
 Practical
 Ethical
 Social