Key area 2: Replication of
DNA
Unit 1: DNA and the Genome
How is DNA replicated?
Now you know the structure of DNA...
…how is it copied?
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DNA and
the
Genome
Why do cells need to copy their
DNA?
DNA is copied during cell division
(mitosis) to ensure that new cells have
the same number of chromosomes and
to ensure that all cells have the same
genes.
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DNA and
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Genome
Hypothesise…
Key
Original DNA
New Copied DNA
Take a 5 minutes to
discuss in groups
ways in which DNA
might copy itself.
Use colouring
pencils to put your
ideas on the idea
sheet.
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DNA and
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Genome
Three possible hypotheses
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DNA and
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Meselson and Stahl
Meselson and
Stahl were two
scientists who, in
1958, carried out
an ingenious
experiment to
solve this
question.
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DNA and
the
Genome
This is their experiment:
They grew E. coli
bacteria in “Heavy”
nitrogen.
This nitrogen was used
to make DNA.
They grew the cells
first in “heavy
nitrogen” then
switched them to
“light nitrogen” .
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DNA and
the
Genome
When you spin DNA
with “Heavy
nitrogen” in a
ultracentrifuge it
sinks to the
bottom. “Light
nitrogen” stays at
the top.
DNA with LIGHT
nitrogen only
DNA with both
heavy AND light
nitrogen
DNA with HEAVY
nitrogen only
In groups fill in a results prediction sheet. Each group will
be allocated either: Conservative, Semi-conservative or
Dispersive.
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DNA and
the
Genome
The results…
Generation Result
1
2
Lighter
Heavier
So which model was correct?
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DNA and
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Genome
Summary
In your jotter, create a summary of:
• The 3 models of DNA replication
• Medelson & Stahl’s experiment design
• Their results.
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DNA and
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Genome
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DNA and
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Genome
DNA is a unique molecule because it can
direct its own replication and reproduce
itself.
DNA replicates by semi-conservative
replication.
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DNA and
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AT
C G
T A
DNA parental strand
composed of two
complementary strands
GC
C G
AT
STEP 1:
Hydrogen
bonds
between the
bases break
– separating
the strands
A
T
C
G
T
A
G
C
C
G
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T
DNA and
the
Genome
STEP 2: Free
nucleotides
start to line up
with
complementary
nucleotides
A T
A
T
C G
C
G
T A
T
A
G C
G
C
C
G
A
T
T
C
A
G
C
T
A
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G
DNA and
the
Genome
A T
A T
C G
C G
A
T A
G C
G C
C G
C G
A T
A T
T
STEP 3: Sugar-phosphate bonds form.
Two DNA molecules identical to the parental
molecule have been formed.
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DNA and
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Enzyme control of DNA
replication
DNA replication is a complex process
involving many enzymes.
The enzyme DNA polymerase controls
the formation of the sugar-phosphate
bonds when making the new strand.
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DNA and
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The leading strand
DNA polymerase can only join nucleotides
onto the 3’ end of a growing DNA
strand.
Therefore…
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DNA and
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5’ end
of
DNA
strand
DNA
polymerase
enzyme
3’ end
of
DNA
strand
Primer
Leading
strand of
replicated
DNA
Start of
complementary
strand of replicated
DNA
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Replication of the
leading strand of
DNA
DNA and
the
Genome
1. After the hydrogen bonds break, the
DNA unzips.
2. A DNA primer (a short stretch of
complementary DNA) attaches to the
start of the piece of DNA being copied.
3. DNA polymerase the attaches free
nucleotides to the 3’ end of the primer.
4. This continuous process till leading
strand is copied.
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DNA and
the
Genome
Replication of the
lagging strand of
DNA
3’ end
of
DNA
strand
Primer
DNA
polymerase
Ligase
5’ end
of
DNA
strand
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DNA and
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DNA polymerase can only add onto the 3’
end of a primer. So for the other strand:
• Many primers attach along the strand.
• These are extended by the DNA
polymerase.
• The fragments are then joined by the
enzyme ligase
• This is a discontinuous process creating
the lagging strand.
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DNA and
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Replication bubbles and forks
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DNA and
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When copying a long chromosome many
replication forks operate simultaneously
to speed up the replication process.
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DNA and
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Requirements for DNA
replication
For DNA replication to occur, the nucleus
must contain:
• DNA (to act as the template)
• Primers
• A supply of the 4 types of nucleotide
• DNA polymerase and ligase enzymes
• A supply of ATP (energy)
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DNA and
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The polymerase chain
reaction (PCR)
Unit 1: DNA and the Genome
Prior knowledge
• The structure of DNA.
• DNA replication process.
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What is PCR?
PCR (Polymerase chain reaction) was
developed by Kary Mullis in the mid1980s.
For which he received the Nobel Prize.
It has revolutionized molecular biology.
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DNA and
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Genome
What is PCR?
PCR allows specific sections of DNA to be
amplified in vitro (replicated out with a
cell in a test tube (in vitro = in glass)).
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DNA and
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Millions of copies of a specific piece of
DNA can be created in a few hours in a
thermocycler.
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DNA and
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The
first
cycle
5’
3’
Single copy
of DNA
3’
5’
Step 1: The DNA is heated at approx. 95 oC for
a few seconds. This causes the DNA to
denature and the strands to separate.
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3’
5’
5’
3’
3’
PCR
primer
5’
5’
3’
Step 2: The DNA is cooled to approx. 50-65 oC
for a few seconds. This makes short primers to
bond to the separated DNA strands.
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3’
5’
3’
5’
5’
3’
5’
3’
Step 3: The DNA is heated again to approx. 72oC
for a few minutes. This allows a heat-tolerant
DNA polymerase to replicate the DNA.
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5’
3’
3’
5’
5’
3’
3’
5’
Step 4: Heat the DNA up to 95 oC again.
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DNA and
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5’
3’
5’
3’
5’
5’
3’
3’
3’
5’
3’
5’
3’
5’
3’
5’
Step 5: Cool to between 50 – 65 oC again. The
primers now bond to the original fragments and
the copies.
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DNA and
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Genome
5’
3’
3’
5’
3’
5’
3’
3’
3’
3’
5’
5’
3’
3’
5’
3’
5’
3’
3’
5’
Step 6: Heat to 72 oC again. The DNA polymerase
copies the DNA again. The process is copied over
and over again for roughly 20-30 cycles.
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DNA and
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Genome
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Requirements for PCR
Sequence specific primers – these are
designed by the scientist and can be
manufactured by a machine.
The sequence for primers can be designed
by looking at the published genome
sequences.
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DNA and
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Genome
1.
2.
3.
4.
Primers
Supply of nucleotides
pH buffer
Mg2+ - DNA polymerase
co-factor (makes the
polymerase work
better)
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DNA and
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Genome
Uses of PCR
1. DNA Profiling
PCR helps to rapidly
identify people. Specific
areas of DNA known to
vary between individuals
is amplified. Giving
different sized fragments
in different people.
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2. Disease detection
DNA sequences that are
known to indicate
certain genetic
disorders or diseases
are amplified using PCR
for the purposes of
diagnosis.
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3. Archeological analysis
Ancient DNA, degraded
over the years, can be
amplified and used in
archaeological,
paleontological and
evolutionary research.
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5. Population studies
Analysis of human or
other species’ population
genetics can be rapidly
performed using PCR
analysis.
6. Sequencing DNA
sequences can be worked
out.
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DNA and
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Genome
Key concepts
• Small sections of DNA can be replicated in vitro using the
PCR.
• PCR manipulates the natural process of DNA replication.
• PCR is now an automated technique widely used in many
areas of research and industry.
• PCR requires template DNA, Taq polymerase, dideoxynucleic acids with each of the four DNA bases, Mg2+,
primers and a buffer.
• PCR involves continuous and repeated cycles of heating and
cooling.
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DNA and
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Genome