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The use of living things, biological systems and
processes for the benefit of humans.
1. Manipulating DNA
EL: To learn how to cut and paste
DNA
Tools for manipulating DNA
1. CUTTING
DNA into fragments using restriction enzymes:
these molecules cut at specific DNA sequences
and are only found in prokaryotic organisms
2. PASTING
Pasting DNA fragments together using enzymes
called ligases. We can join fragments of DNA to
make what is called “recombinant DNA”. DNA
ligases found in many species including humans.
3. COPYING
Making many copies of DNA (amplification)
using DNA polymerases in the Polymerase Chain
Reaction (PCR) technique. All organisms contain
DNA polymerases as they all need to copy their
DNA.
4. TRANSFERRING
DNA into cells using vectors such as “plasmids”.
This technique is called a transformation in
prokaryotic cells.
1. Cutting DNA
• Restriction enzymes (molecular scissors) are
found in prokaryotic organisms
Specificity
Restriction enzymes are
specific:
• The DNA and the enzyme
need to be mixed together
and incubated at a
temperature that will result
in maximum activity of the
enzyme.
• Each restriction enzyme will
only cut the DNA at a
specific sequence of A, G, T
and Cs. We call this place a
recognition site.
Different
restriction
enzymes
recognise
specific
recognition sites.
Cutting Specificity
• When DNA is cut with a restriction enzyme the resulting
fragments are left with either a short overhang of single
stranded DNA called a sticky end or no overhanging DNA
which is called a blunt end (snake demo)
EcoRI – leaves sticky ends
HpaI – leaves blunt ends
GAATTC
GTT
AAC
GTTAAC
CTTAAG
CAA
CAATTG
TTG
- site where enzymes cuts through the sugar phosphate backbone of the DNA strand.
What’s in a name!
• Restriction enzymes are named after the organism
from which they were isolated.
– E.g. Escherichia coli
EcoRI
• The Roman number indicates the order of discovery
• If another letter is placed in front of the Roman number
it signifies a particular strain of the bacterium. R =
resistance
Restriction Enzyme
EcoRI
BamHI
HindIII
HpaI
Restriction Site Overhang Type
E = genus Escherichia
co = species coli
R = strain RY13
I = first endonuclease isolated
GAATTC
CTTAAG
STICKY
B = genus Bacillus
am = species amyloliquefaciens
H = strain H
I = first endonuclease isolated
GGATCC
CCTAGG
STICKY
H = genus Haemophilus
in = species influenzae
d = strain Rd
I = third endonuclease isolated
AAGCTT
TTCGAA
STICKY
GTTAAC
CAATTG
BLUNT
H = genus Haemophilus
pa = species parainfluenzae
I = first endonuclease isolated
Fragments are sorted by Gel Electrophoresis
• This technique is used to separate out fragments, obtained by a
restriction digest, of DNA according to their size (length in base
pairs).
• DNA fragments are separated into bands containing fragments
of the same length by electrical separation in a gel matrix.
• DNA molecules migrate to the positive electrode, when an
electric field is applied to the gel matrix, as they are negatively
charged.
• This technique is used to isolate DNA fragments containing
genes which are subsequently used to make recombinant DNA.
Fragments are sorted by Gel Electrophoresis
2. Pasting
• When two samples of
DNA are combined using
DNA ligases.
• Any 2 DNA strands can be
joined that have
complementary exposed
nucleotides (i.e. cut with
same restriction enzyme).
Activities
• Watch DNAi animations
• Complete activity 9.1 “Gel electrophoresis
analysis” & 9.2 “Detecting traits in families….”
Reflection
Summarise in your own words – restriction
enzymes, cutting, pasting and gel
electrophoresis
• What learning was new today?
• What learning was revision or built on what I already know?
• What did I find most challenging and what strategies will I put
in place to help me?
• What percentage of the class did I spend on task and how can
I improve this if needed
2. Manipulating DNA
EL: To learn how to copy and transfer
DNA.
3. Copying (Amplification) of DNA
using the Polymerase Chain Reaction
“I was working for Cetus, making
oligonucleotides (primers). They were
heady times. Biotechnology was in flower
and one spring night while the California
buckeyes were also in flower I came across
the polymerase chain reaction. It was the
first day of the rest of my life”.
Kary Mullis 1972
• The $300 million dollar man.
Why PCR?
• To amplify a small
amount of DNA into an
analysable quantity
– E.g. crime scene, fossils
etc
PCR Tools
• Taq DNA Polymerase – is an enzyme that
works well at 72°C.
PCR Tools
Primers:
• Synthetic short segments of DNA up to 25 nucleotides
long.
• Probe for a specific sequence or gene along a strand of
DNA.
• Hybridise with a sequence of bases on the template
DNA through complementary base pairing.
• Indicate to Taq DNA polymerase where to start building
the complementary strand by extending the primer.
Find the starting point for copying STR
regions
Select your primer
Start region
Sequence to be copied by extending the primer.
Step 1. Denaturation
Step 1: Denaturing the DNA – 2 minutes
T A C C G T A A
A T G C C A T T
Step 1:
92°C
Thermocycling
machine
• At this temperature the hydrogen bonds are broken resulting in two
single strands of DNA.
Step 2. Attachment of Primers
T A C C G T A A
A T G
T A A
Step 2:
55°C
A T G C C A T T
• Step 2: Primer annealing– 2 minutes
The temperature is lowered to allow the primers to bind
(anneal) to their complementary bases on each of the single
strands of DNA.
Step 3: Extension
T A C C G T A A
A T G GTaqC
A
Step 3:
72°C
T
T A C
G TaqG T A A
A T G C C A T T
• Step 3: DNA synthesis – 1 minute
Taq DNA polymerase extends the DNA strand from
the primers using the base pairing rule.
T
And you can repeat the three
step cycle over and over!
PCR song
• http://www.youtube.com/watch?v=dD3faDLE
vmY&feature=related
4. Transferring
Fluorescent
jellyfish
Jellyfish
• Because DNA is the
same in all organisms,
we should be able to
take a piece of DNA from
one organism and put it
into another organism.
• You can change the way
an organism looks or
behaves!
• This process of taking
DNA from one organism
and putting into another
is called transformation.
Plasmid
Fluorescent
Jellyfish and plasmid DNA is cut with the same
restriction enzyme.
Vectors
• Gene inserted into a vector that will carry the gene into
the desired organism.
• Common vectors are:
• Viral vectors (eg. Adenovirus and retorovirus) – must
have disease symptom genes removed first!
• Liposome vectors – small circular molecules
surrounded by phospholipid bilayer
• Plasmid vectors – small circular piece of bacterial
DNA. Plasmids are used as vectors in bacterial
transformations.
Plasmids are not naturally attracted to
bacteria!
Bacterium
Transformation of Bacteria with a
Recombinant DNA Plasmid
Making the bacteria more ‘attractive’ to plasmids
Plasmids are
now attracted
to the bacteria
Bacterium
The Transformation
• Now give the bacteria some food and the right
temperature to reproduce.
• Any bacteria with the plasmid inside will start making
the jelly protein, that results in fluorescence.
HEAT SHOCK
Activity
• In pairs, complete Activity 12.2 “finding a
gene”
• Quick check qu 1-3 (pg 426), 4-6 (pg 431)
• Ch 12 review qu 3, 4, 5, 6, 7, 8, 12
Reflection
Summarise in your own words – copying
(include PCR) and transferring
• What learning was new today?
• What learning was revision or built on what I already know?
• What did I find most challenging and what strategies will I put
in place to help me?
• What percentage of the class did I spend on task and how can
I improve this if needed
3. Applications of DNA
manipulation
EL: To explore the uses of DNA
manipulation.
Gene Sequencing
Gene sequencing is identifying the nucleotide order in a
segment of RNA or DNA.
A G G A C T C AT G G A G AA G AA C T T T . . .
Our genome has been sequenced. We have 3,100,000,000 base pairs, what a big book!
Gene Cloning
• Making identical copies of sequences of DNA that code
for proteins using plasmids
1. extract plasmid from bacteria
2. Cut plasmid DNA and DNA of the gene to be inserted
with same restriction enzyme
3. Paste 2 pieces of DNA using DNA ligase to create a
recombinant plasmid.
4. Add recombinant plasmid to bacterial culture, where
some are taken up and replicate (called transformation)
5. Isolate and analyse bacteria containing recombinant
plasmids.
PRACTICAL APPLICATION: Production of human growth hormone
DNA Profiling
• Compares base sequence of 2 or more individuals
• Short tandem repeats (STRs) and variable
nucleotide tandem repeats (VNTRs): non-coding
sections of DNA repeated many times between
genes
– E.g. GAGAGAGAGAGAGA
• There are more than 10,000 STR
loci in one set of human
chromosomes!
DNA Profiling
• The repeat is present in all
members of the
population, but the
number of repeats varies
among individuals and is
inherited.
• DNA profiling allows us to
view these patterns in our
DNA.
• Uses PCR and gel
electrophoresis – smaller
fragments will migrate
further on the gel.
DNA Profiling
DNA Profiling
•
•
Loci of STR regions found to vary from person to person with a high frequency
13 are used in America, but only 9 are used in Australia – why?
Activities
• Genetic Engineering: A model (Biol: The
Common Threads, pg 175)
• DNA fingerprinting (Biol: The Common
Threads, pg 179)
• Quick check qu 7-10 (pg 433), 11-14 (pg 437),
15-17 (pg 443), 18-21 (pg 448), 22-24 (pg
455), 25&26 (pg 457)
• Ch 12 ch review qu 9, 10, 11 (pg 461)
Reflection
Summarise in your own words gene
sequeencing, cloning and DNA profiling
• What learning was new today?
• What learning was revision or built on what I already know?
• What did I find most challenging and what strategies will I put
in place to help me?
• What percentage of the class did I spend on task and how can
I improve this if needed