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DNA & BIOTECHNOLOGY
DNA & BIOTECHNOLOGY
Keywords
Keywords
Biotechnology
 Recombinant DNA
 Gene splicing
 Vector
 Plasmid vector
 Phage vector
 Transgenesis
 Transgenes
 Cloning
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
Polymerase chain
reaction
 Primer
 Short tandem repeats
 Gel electrophoresis
 DNA blot / Southern
blot
 Capillary
electrophoresis
 DNA micro-array
WHAT IS BIOTECHNOLOGY?
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Biotechnology is using
living things to create
products or to do tasks
for human beings.
It is the practice of using
plants, animals and
micro-organisms and
their biological processes
to some benefit eg. in
medicine, agriculture and
industry
Researchers use DNA,
genes, yeast, bacteria
and cells
WHY USE BIOTECHNOLOGY?
For ourselves
For the environment
Biotechnological
research has been used
to assist human health in
many areas:
Biotechnology is a tool
used:
antibiotics
 vaccines
 IFV
 genetic disorders
 dna profiling &
forensics
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to help control pests
 for conservation of
plant & animal species
 leach metals from the
soil for cleaner mining
 clean up heavy metal
contamination
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WHY USE BIOTECHNOLOGY
For food & agriculture
Selective or conventional
breeding of plants and animals
changes the genetic make-up of
organisms. Gene technology
makes these changes more
specific.
Biotechnology is used to create:
 Frost, salt and drought
tolerant plants
 Salad vegetables that do not
‘brown’
 Fruits and vegetables with
extra vitamins
 Slow-ripening tomatoes &
pineapples
 Produce blowfly-resistant
sheep
 Increase wool production
For horticulture
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Long-lasting flowers
Blue roses
For research & medicine
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Transgenic mice
Production of insulin
Production of antibiotics
BIOTECHNOLOGY TERMS
Term
Meaning
Gel
electrophoresis
The use of an electric charge applied to a gel in order to
separate fragments of DNA according to their size
Gene splicing
Inserting foreign DNA into the DNA of a cell
Gene vector
Mechanical or biological, used to transfer DNA into a host
cell.
Phage vector (viral) or plasmid vector (bacterial)
Ligase
An enzyme which can be used to join DNA strands or the
‘sticky’ ends of DNA strands.
Polymerase Chain
Reaction
A means of producing large quantities of a small sample of
DNA by heating and cooling it many times in the presence of
a heat tolerant enzyme called polymerase. The polymerase
enables free nucleotides to combine with the heat separated
threads of DNA as they are cooled, therefore doubling the
amount of DNA each cycle.
Recombinant gene A gene which has been inserted on a foreign organism’s
DNA
RECOMBINANT DNA & GENE SPLICING
 Recombinant
DNA is
a method of cutting
and pasting a foreign
piece of DNA into the
DNA of a cell.
 It brings together
genetic material from
multiple sources,
creating new
sequences of DNA.
 Enables the genome
to be manipulated
very precisely
Transgenesis
 Transgenesis is when
specific genes from one
organism are placed in
the DNA of another
organism of a different
species
 The recombinant genes
are called transgenes
Example:
Genetically modified mice
for the purposes of
scientific studies
TRANSGENESIS
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Another example of
transgenesis is for the
production of specific
proteins
The first chemical
produced by
transgenesis was human
insulin in the late 1980’s
The human gene is
placed into a bacterium
which can then use the
genetic information to
produce the human
hormone.
The hormone is refined
from the culture of
bacteria.
RECOMBINANT DNA & GENE SPLICING
Steps to gene splicing:
1. Restriction enzymes
 Cut the DNA at a specific location
 Leaves the DNA strand with ‘sticky
ends’
2. Sticky ends
 Unattached (unpaired) nucleotides
 Match up with the DNA to be
inserted
3. Ligation
 Ligase enzymes help form the
hydrogen bonds between
nucleotides
 DNA-ase helps form the bonds
between the side strands
(backbone)
Fluorescent green protein
transgenic mouse
RECOMBINANT DNA
& GENE SPLICING
RECOMBINANT DNA & GENE SPLICING
Sticky ends
BamHI:
HindIII
EcoRI
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Vectors are the carriers of
the recombinant DNA
They can be phage (viral
DNA) or plasmid (bacterial
DNA) vectors
Plasmids are small
sections of DNA separate
to the chromosomal DNA
Most often, plasmids are
genetically engineered
Many bacteria however,
also contain plasmids
naturally
VECTORS AND TRANSGENESIS
RECOMBINANT DNA & GENE SPLICING
Sticky ends
The vector used
in this diagram is
a plasmid vector
Some strains of the bacterium E. coli have resistance to the
antibiotic kanamycin, others have resistance to tetracycline.
Plasmid* carrying gene for Plasmid* carrying gene for
kanamycin resistance
tetracycline resistance
K
E. coli with kanamycin
resistance
T
E. coli with tetracycline
resistance
* Plasmids are small circular pieces of DNA that occur in bacteria
and protozoa. They are not in a chromosome but can replicate
independently in a host cell.
K
Plasmids can be cut at
specific points using
restriction enzymes
T
The cut plasmids are
mixed with DNA ligase to
form recombinant DNA
K
T
Plasmid reintroduced
into E. coli
E. coli with tetracycline
and kanamycin resistance
POLYMERASE CHAIN REACTION
The polymerase chain
reaction (PCR) is a process
that reproduces large
quantities of small sections
of DNA.
Background information
 The process involves
heating and cooling cycles.
 It has been made possible
with the discovery of the
Taq polymerase enzyme
which is heat tolerant.
 This enzyme was originally
discovered in a bacteria
(Thermus aquaticus) that
lives in hot springs
 It can replicate a 1000
base pair strand of DNA in
less than 10 seconds at
72°C.
Young man with cystic fibrosis taking medication using a nebuliser.
Wellcome Library
The gene responsible for cystic fibrosis has been
identified and it is hoped that, using recombinant
DNA technology, it will be possible to transfer a
normal copy of the gene into affected cells.
GENE THERAPY
It has long been anticipated that cystic fibrosis will
be one of the first diseases to be treated by gene
therapy. However, since the first clinical trials in the
early 1990s numerous problems have been
encountered.
 It is expected that a clinically effective treatment will
be available in the next 10 years.
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POLYMERASE CHAIN REACTION
Steps
1. The DNA to be copied is
heated to 90ºC, which causes
the strands to separate
(denature).
2. Large amounts of primers are
added to the single strands of
DNA.
3. These primers bind to the
matching DNA sequences in
front of the gene that is to be
copied. This tells the
polymerase enzyme where to
start copying.
4. The primers are both forward
and reverse sections of DNA
(5’ to 3’ and 3’ to 5’).
5.
6.
7.
8.
9.
The mixture is then cooled so that
the primers can anneal (bind) to
the single stranded templates.
The polymerase enzyme is added
and the mixture is heated again to
about 70ºC.
DNA polymerase begins adding
nucleotides onto the ends of the
annealed primers.
At the end of the cycle, which lasts
about 5 minutes, the temperature is
raised and the process begins
again.
The number of copies doubles after
each cycle. Usually 25 to 30 cycles
produce a sufficient amount of
DNA.
PCR MACHINE
POLYMERASE CHAIN REACTION
POLYMERASE CHAIN REACTION
DNA SEQUENCING / PROFILING
DNA sequencing is
used to work out the
exact order of the base
pairs in a section of
DNA. Knowing the base
sequence can be helpful
in locating and identifying
specific genes.
 Gene probes can then
be made and used to
locate these genes
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DNA profiling is used to
identify species or
individuals
 Gel electrophoresis is
used in DNA profiling.
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SHORT TANDEM REPEATS
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DNA profiling uses the "repeat" sequences of non-coding DNA
that are found between the genes that code for proteins.
These sequences can vary a great deal between individuals
(polymorphic).
They are called short tandem repeats (STRs).
The number of repeats is inherited. Therefore, unrelated
individuals are extremely unlikely to have the same number.
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Gel electrophoresis
separates these fragments of
DNA (STRs) using an electric
current.
DNA has a slight negative
charge. It will migrate
towards the positive end of
the gel.
Smaller fragments move
faster through the gel than
larger fragments.
The gel can be upright like
this one or it an be a flat bed
gel – both work in the same
way.
At the end of the ‘run’ a
pattern of bands will be
produced. Each band
represents a fragment size of
DNA from the sample.
Different samples will have
different patterns.
GEL ELECTROPHORESIS
FRAGMENT LENGTHS AND GEL ELECTROPHORESIS
SHORT TANDEM REPEATS
Blotting is a method of ‘photographing’ the resulting sequence of DNA
fragments once they have gone through the process of gel
electrophoresis.
DNA BLOT ANALYSIS
In the example below, what is the genotype of the father? Rule out all the
mother’s alleles. The ones left are from the father.
DNA BLOT ANALYSIS
Who was adopted?
DNA PROFILING - SUMMARY
Steps in DNA profiling
1. Collect sample of material
containing cells
2. Extract DNA from sample
3. Use PCR to increase the
size of the sample
4. Add restriction enzymes to
DNA sample
5. Place solution of DNA and
restriction enzymes into gel
electrophoresis
6. Run gel
7. Process gel to see location
of DNA bands
8. Photograph the gel (DNA or
Southern blot)
CAPILLARY ELECTROPHORESIS
• STR profiles are more easily stored and compared in
the form of numbers and letters rather than pictures of
lines.
• Capillary electrophoresis is a way of collecting
numerical data that is plotted on a line graph.
• The peaks on the graph represent the different STRs
Sample
Amelogenin D3S1358
vWA
FGA
D8S1179
D21S11
D18S51
Victim
XY
14, 15
18, 20
24
13, 16
28, 30.2
14, 15
Suspect
XY
14, 15
15, 18
21, 22
13, 14
30
14, 15
Blood Stain
from Crime
Scene
XY
14, 15
15, 18
21, 22
13,14
30
14, 15
Capillary electrophoresis graph
USES OF DNA PROFILING
Identification
 Criminals
 Victims – crimes, disasters
 Family members
 Species – quarantine, smuggling
 Genetic differences between populations
Information is kept in data banks
GENE PROBES
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The search for a particular
gene uses a single-stranded
piece of DNA called a gene
probe.
To test a DNA sample, it is
first treated so that the
double-stranded molecule
unzips into single strands.
The probe is then added to
the solution
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Probes are constructed with
a radioactive or a
fluorescent section (tag) to
that they can be detected
after attaching to the DNA.
The probe gives infomration
about which chromosome
the gene is on, and where
the gen is on the
chromosome.
We know the base
sequences in a number of
disease-causing genes.
Gene probes can detect if
these genes are present in
individuals being tested.
DNA MICRO-ARRAYS
o A DNA mico-array allows scientists
to perform an experiment on
thousands of genes at the same
time.
o Each spot on a micro-array contains
multiple identical strands of DNA.
o The DNA sequence on each spot is
unique.
o Each spot represents one gene.
Thousands of spots are arrayed in
orderly rows and columns on a solid
surface (usually glass).
o The precise location and sequence
of each spot is recorded in a
computer database.
o Microarrays can be the size of a
microscope slide, or even smaller
http://learn.genetics.utah.edu/content/labs/microarray/