F215 control, genomes and environment
Module 2 – Biotechnology and gene
technologies
Learning Outcomes

Outline the steps involved in
sequencing the genome of an
organism.
Genomes

1950’s
 Learnt that DNA is the genetic material

Gene technology
 Use of DNA to produce something that we want
Developing rapidly
Becoming more and more able to alter
genes within organisms
 Point to think about


 Just because we can do something does that
mean that we should do it?
Manipulating DNA

Advances in DNA technology
 DNA profiling (genetic fingerprinting)
 Genomic sequencing
 Comparative genome mapping
 Genetic engineering
 Gene therapy
Genome


All the genes possessed by an
individual organism, or a population of
organisms.
The whole sequence of bases in all of
the DNA in an organism.
Human Genome Project

1988
 International project started to discover
the sequence of bases in each of the 23
different types of chromosomes found in
human cells

2000
 A working draft sequence was produced
Facts about human genome
99.9% of the base sequence in our DNA
seems to be identical in all humans
 Variation is caused by the variable 0.1%

 This 0.1% is very variable
 Variations can be used for DNA profiling

2% of human genome codes for the
manufacture of proteins
 Giving around 20 000 genes in the human
genome (even mice have more!!)

The rest of the “junk” genome, may be
involved in gene expression
Sequencing a genome
The genome is broken up and sequenced in
sections
 Sequencing is carried out on overlapping
regions
 Stages

 Genome mapping
 Mechanically break into smaller sections
 Carry out sequencing on overlapping sections
 Analyse and put back together to form the
complete code
Sequencing a genome

Look at the worksheet
 “sequencing a plant gene”


Make multiple copies of the genome
using PCR
DNA randomly broken up into lengths
2000bp – 10000bp long
 These lengths can then be broken up
further
Sequencing a genome

Make multiple labelled copies of each
small length of DNA
 Lengths of DNA mixed with
▪ DNA Polymerase
▪ Primer
▪ “normal” DNA nucleotides
▪ “labelled” DNA nucleotides
▪ dideoxy nucleotides
▪ Four colours of dye used for bases A, T,G and C
▪ If incorporated in nucleotide chain – chain stops
growing
Sequencing a genome

Result
 Many different chains of different lengths
 Each length ends with a labelled nucleotide

Mixture of lengths of DNA separated using
electrophoresis
 The shorter the length of DNA the faster it travels
Computer records the colours as they pass
the end of the tube, if there are enough
fragments then every base in the complete
chain will be represented.
 Computer works out the sequence of the
length of DNA

Sequencing a genome

Process is largely automated
 Put your DNA sample into a sequencing
machine
 Get a print out from the bottom

Preparation of DNA and analysis is still
time consuming
Learning Outcomes

Outline how gene sequencing allows
for genome-wide comparisons
between individuals and between
species.
Comparing Genomes

Comparative gene mapping has a wide
range of applications
 Identification of genes for proteins gives clues to
relative importance of these genes to life
 Modelling the effects of changes to DNA can be
carried out
 Compare pathogenic and non-pathogenic
organisms
▪ Identify targets for drug treatments and vaccines
 Analysis of individuals DNA
▪ Presence of alleles associated with disease
 Determine evolutionary relationships
 Classification of organisms
Learning Outcomes
Define the term recombinant.
Explain that genetic engineering involves
the extraction of genes from one organism,
or the manufacture of genes, in order to
place them in another organism (often of a
different species) such that the receiving
organism expresses the gene product.
 Describe how sections of DNA containing a
desired gene can be extracted from a
donor organism using restriction enzymes.


Genetic Engineering

Genetic engineering
 the use of technology to change the genetic
material of an organism.
 Involves taking genes from an organism or one
species and placing them in another

Recombinant DNA
 DNA that contains lengths of DNA from different
species

Recombinant organism
 Organism to which the new gene has been
added
 AKA transgenic organism or transformed
organism
Gene transfer

Identify gene that is required
 Cut out of chromosomes
 Made by “reverse transcription” of mRNA
Multiple copies make using PCR
(polymerase chain reaction)
 Gene inserted into a vector

 Vector is an organism or structure that can
deliver the gene into required cells e.g. Plasmid,
bacteriophage, liposomes


Vector inserts gene into cells
Transformed cells identified and cloned
Extracting the gene


A length of DNA known to contain
HGH gene is treated with restriction
enzymes
Restriction enzymes
 Cut DNA at specific base sequences
 BamH1 always cuts DNA where there is a
GGATCC sequence on one DNA strand
 Cut the two DNA strands at different
positions, leaving sticky ends
▪ Short lengths of unpaired bases on both pieces.
Cutting DNA with a restriction
enzyme
Extracting the gene

After cutting with restriction enzymes
 Get a mixture of lengths of DNA
 Required length of DNA can be identified
using
▪ DNA probes
▪ Electrophoresis
 Multiple copies of the DNA made using
PCR.
Learning Outcomes


Explain how isolated DNA fragments
can be placed in plasmids, with
reference to the role of ligase.
State other vectors into which
fragments of DNA may be
incorporated.
Inserting gene into vector
Plasmids are used if the gene is to be
inserted into a bacteria
 Plasmids often contain genes that confer
resistance to antibiotics

Inserting the HGH gene into plasmid

Plasmid cut using the same restriction
enzyme
 Leaves sticky ends that are complementary to
those on the HGH gene

Plasmids and HGH genes are mixed
together
 Sticky ends of plasmid match up with sticky ends
of HGH gene
 DNA ligase used to link the deoxyribosephosphate backbones

Produce a closed circle of double stranded
DNA containing HGH gene
 not all plasmids will take up HGH gene.
Inserting the HGH gene into plasmid
Getting plasmids into bacteria



Plasmids are mixed with a culture of
bacteria
Calcium ions are added to affect the
cell walls and plasma membranes
1% of bacteria take up the plasmids
containing the HGH gene
Sorting out the transformed
bacteria

Plasmid pBR322 contains
two antibiotic resistance
genes
 Tetracycline
 Ampicillin


The restriction enzyme
BamH1 cuts right through
the tetracyclineresistance gene.
So when HGH gene is
inserted it inactivates the
tetracycline resistance
gene.
Replica plating

The bacteria are grown on
agar jelly containing
ampicillin
 Any that survive have taken
up the plasmid

Samples of each colony
are grown on a plate
containing tetracycline
 Colonies that are unable to
grow must have taken up the
HGH gene
 These colonies are selected
from the first plate
HGH production



The genetically modified bacteria are
cultured on a large scale in fermenters
They secrete HGH
HGH is extracted, purified and sold
Vectors

The method for getting the vector into
the cell depends on the type of cell
 Electroporation
▪ High voltage pulse used to disrupt membrane
 Microinjection
 Viral transfer
 Ti plasmids
 Liposomes
Learning Outcomes

Outline how the polymerase chain
reaction (PCR) can be used to make
multiple copies of DNA fragments.
Polymerase Chain reaction
Stage 1



The reactants are
mixed together in a
PCR vial.
The mixture contains
the DNA which is to
be amplified, the
enzyme DNA
polymerase, small
primer sequences of
DNA and a good
supply of the four
nucleotide bases
A,T,C and G.
The vial is placed in a
PCR machine.
Polymerase Chain reaction
Stage 2
The reaction mixture
is heated to 90-95oC
for about thirty
seconds.
 At this temperature
the DNA strands
separate as the
hydrogen bonds
holding them
together break
down.

Polymerase Chain reaction
Stage 3
The mixture is cooled
down to 55-60oC. At this
temperature the primers
bind (or anneal) to the
single DNA strands.
 The primers are short
sequences of nucleotide
bases which must join to
the beginning of the
separated DNA strands
for the full copying
process to start.

Polymerase Chain reaction
Stage 4
In the final step the
mixture is heated up
again to 75oC for at least
a minute.
 This is the optimum
temperature for the DNA
polymerase enzyme.
 The enzyme adds bases
to the primers segments
to build up
complementary strands
of DNA identical to the
original molecule.

PCR


These last three steps can be
repeated around thirty times to give
around 1 billion copies of the original
DNA.
The whole process takes only about 3
hours – and much of that is the time
taken heating and cooling the
reaction mixture in the PCR machine
Summary of PCR

Denaturing of double-stranded DNA
molecules to make single stranded
 High temperature 95oC

Annealing primers to the ends of the
single-stranded DNA molecules
 55-60oC

Building complete new DNA strands
using DNA polymerase
 72oC
Learning Outcomes


Outline how DNA fragments can be
separated by size using
electrophoresis.
Describe how DNA probes can be
used to identify fragments containing
specific sequences.
Electrophoresis

Electrophoresis separates different
fragments of DNA according to their sizes.
 Tank set up containing agarose gel
 Direct current is passed continuously through the
gel
 DNA fragments carry a small negative electric
charge
 DNA fragments are pulled through the gel
towards the anode
 The smaller the fragments the faster they move
through the agarose matrix.

When the current is turned off
 DNA fragments will have ended up in
different places
 These can be transferred onto absorbent
paper or by a technique called southern
blotting
Using electrophoresis

A radioactive
probe is added to
bind to the invisible
bands of DNA, so
they can blacken
an X-ray film
electrophoresis
After
electrophoresis and
labelling of DNA
samples, you can
compare the DNA
from different
individuals.
 This is DNA profiling

Gene Probes
A gene probe is a length of single stranded
DNA that has a complementary base
sequence to the gene you want to extract
 The probe is “labelled”

 E.g. with nucleotides containing an isotope of
phosphorous, 32P, which emits beta radiation

When the probe is mixed with DNA
fragments it forms hydrogen bonds with
stretches of DNA complementary to its own
base sequence (annealing)
Using probes

Probes can be used to locate specific
sequences
 Identify the same gene on a variety of
different genomes
 Locate a specific desired gene
 Identify the presence or absence of an
allele for a genetic disease
Revision of DNA sequencing
Automated DNA Sequencing


Previous methods of DNA sequencing
were slow and time consuming.
The current cutting edge approach
uses an automated process involving
interrupted PCR with modified
nucleotide bases.





The PCR sequence starts as before, with the primer
annealing to the DNA fragment, allowing the DNA
polymerase to attach.
The DNA polymerase starts to add complementary
nucleotides.
Eventually, a modified nucleotide will be added,
which prevents addition of any further nucleotides
to the DNA strand.
This generates many fragments of DNA that all end
in a modified nucleotide, located in different
positions on the unknown strand.
These fragments are read by the automated
sequencer, and the unknown sequence is
revealed.

The PCR mixture contains:
 Primers
 DNA polymerase
 Surplus nucleotide bases
 Multiple copies of the single stranded DNA
fragment to be sequenced

Modified nucleotides with different
coloured fluorescent markers
An unknown sequence has a
known initial fragment
(CATGATA)
Primer binds and free &
tagged nucleotide bases are
added with a polymerase
enzyme.
Terminator bases produce fragments of varying
length.
Electrophoresis allows
fragments to be sorted
by size, slowly revealing
the complementary
sequence to the
unknown section.
The sequence of
fluorescent colours is
then read by a laser,
providing the complete
sequence of bases.
Learning Outcomes
Explain how plasmids may be taken up by
bacterial cells in order to produce a
transgenic micro organism that can express
a desired gene product.
 Describe the advantage to microorganisms
of the capacity to take up plasmid DNA
from the environment.
 Outline how genetic markers in plasmids
can be used to identify the bacteria that
have taken up a recombinant plasmid.

Genetic markers

To identify transformed bacteria the
following genetic markers can be
used
 Antibiotic resistance genes
 Gene that causes fluorescence
▪ fluoresces bright green in UV light
Learning Outcomes

Outline the process involved in the
genetic engineering of bacteria to
produce human insulin.
Human Insulin production

Stages to isolate the gene
 Remove mRNA from β-cells in islets of
langerhans
 Incubate mRNA with reverse transcriptase
▪ Produces complementary single stranded DNA
▪ This is converted to double stranded DNA –
insulin gene
Human Insulin production

Preparing the gene and vector
 Add lengths of single stranded DNA made
from guanine nucleotides to create “sticky
ends”
 Lengths of cytosine nucleotides were
added to the cut ends of the plasmids
Learning Outcomes

Outline the process involved in the
genetic engineering of Golden
RiceTM.
Golden Rice TM

Vitamin A
 Required for the formation of rhodopsin
 Involved in the synthesis of glycoproteins
 Needed for the maintainance and
differentiation of epithelial tissues and
helps to reduce infection
 Essential for bone growth
Sources of Vitamin A in the diet



Meat products, esp. Liver
β-carotene (precursor) in carrots – can
be used to make retinol
In countries where vitamin A
deficiency is significant they rely on
rice as there staple food.
Golden RiceTM

Two genes were inserted into the rice
genome
 Gene coding for phytoene synthase
(daffodils)
 Gene coding for carotene desaturase
(bacterium Erwinia uredovora

The first rice produced did not
produce significant quantities of βcarotene
Golden RiceTM

Versions were made of Golden RiceTM
using genes from the maize plant and
the rice itself.
Are GMOs safe?



Do they pose risks to health?
Do they damage the environment?
Are they hugely beneficial to humans and
the environment?

Two issues
 Could genetically modified crops cause harm to
other organisms in the environment?
 Is it safe to eat food from genetically modified
plants?
GM crops


The majority of GM crops have been
developed to benefit the grower and
the retailer.
Would GM crops be more acceptable
if the benefits to health were clearly
demonstrated?
Learning Outcomes

Outline how animals can be
genetically engineered for
xenotransplantation.
xenotransplantation

Transplanting tissues or organs
between animals of different species.

Human organ transplantation
 Shortage of organs
 Rejection of transplanted tissue
▪ Compatability checked
▪ Immunosupressor drugs
xenotransplantation

Using organs from a pig
 Similar size and structure to human organs
 Risk of human immune response
▪ Human antibodies attach to glycoproteins on
pig plasma membranes
▪ One of these glycoproteins is made by an enzyme
GGTA1 (1, 3-galactosyltransferase)
▪ If the sugar is not present, then antibodies don’t attach
and immune attack is weakened.
▪ Genetically engineered pigs do not contain the
gene that codes for the GGTA1 enzyme.
Physiological problems




Slight differences in organ size
Is knocking out one gene enough to
reduce the immune response
sufficiently
Body temperature of pigs is 39oC
Pigs have much shorter lifespans than
humans
Ethical and medical problems


Is it right to genetically modify pigs for
our benefit?
Is it acceptable to place an organ
from another animal into a human
body?
 Religious beliefs

Disease transfer from pigs to humans
Learning Outcomes


Explain the term gene therapy.
Explain the differences between
somatic cell gene therapy and germ
line cell gene therapy.
Gene Therapy


Gene therapy is the treatment of a
disease by manipulating the genes in
a person’s cells.
Two examples of gene therapy
 SCID
 Cystic fibrosis
SCID

SCID
 severe combined immunodeficiency
disease
 Caused by a faulty allele coding for the
enzyme adenosine deaminase (ADA)
 This enzyme is essential for the healthy
working of the immune system
Gene therapy for SCID

Gene therapy
 Removal of patient’s T cells and insertion
of the correct allele into them using a
vector (retrovirus)
 Cells that have taken the allele up
successfully are cloned and replaced into
the patient’s body

Alternative treatment
 Daily injections of adenosine deaminase
Problems with SCID gene
therapy

Some patients who appeared to have
been successfully treated, went on to
develop leukaemia

Is the risk of cancer acceptable when
the patients would have died anyway
from this rare and fatal disease?
Cystic Fibrosis
Abnormally thick mucus is produced in the
lungs and other parts of the body.
 Caused by a recessive allele of the gene
that codes for the CFTR protein.
 CFTR gene

 Sits on chromosome 9
 Commonest defective allele is a result of the
deletion of three bases
 Machinery of cell recognises that the protein is
not right and does not insert it in the cell
membrane
The CFTR protein forms channels for
chloride ions in the plasma membrane
Gene therapy for cystic fibrosis

Normal allele inserted into liposomes
 Sprayed as an aerosol into the nose
 Liposomes are lipid soluble and able to move
through the lipid layers of the plasma membrane
of the cells lining the respiratory passages
 Effect only lasted a week
▪ Cells have a short lifespan and are continually replaced

Introducing the gene using adenovirus
 Unpleasant side effects – trials stopped
Gene Therapy

Somatic therapy
 Body cells
genetically modified
 Modified genes will
not be passed on to
any offspring

Germline therapy
 Changing genes in
cells that would go
on to form gametes
 All of the cells in the
new organism would
carry the genetic
modification
 Modified genes in
gametes could be
passed on to
offspring
Germline cells

Each cell of an early embryo is a stem
cell
 It can divide and specialise to become
any cell type within the body
 It has the potential to become a new
being
 These are germline cells
Learning Outcomes

Discuss the ethical concerns raised by
the genetic manipulation of animals
(including humans), plants and
microorganisms.
Benefits and risks of genetic
engineering
organism
benefit
Risk
Micro-organism
GM bacteria can be
used to produce
useful products
Antibiotic resistance
genes are used as
genetic markers
Plants
Animals
Humans
Use your textbooks to
complete this table.