D1.

1 DNA replication
Continuity and change—Molecules
Standard level and higher level: 2 hours
Additional higher level: 2 hours
D1.1 DNA replication
Continuity and change—Molecules
Standard level and higher level: 2 hours
Additional higher level: 2 hours

SL and HL
D1.1.1—DNA replication as production of exact copies of DNA with identical base sequences
Students should appreciate that DNA replication is required for reproduction and for growth and tissue
replacement in multicellular organisms.
D1.1.2—Semi-conservative nature of DNA replication and role of complementary base pairing
Students should understand how these processes allow a high degree of accuracy in copying base
sequences.
D1.1.3—Role of helicase and DNA polymerase in DNA replication
Limit to the role of helicase in unwinding and breaking hydrogen bonds between DNA strands and the
general role of DNA polymerase.
D1.1.4—Polymerase chain reaction and gel electrophoresis as tools for amplifying and separating DNA
Students should understand the use of primers, temperature changes and Taq polymerase in the
polymerase chain reaction (PCR) and the basis of separation of DNA fragments in gel electrophoresis.
D1.1.5—Applications of polymerase chain reaction and gel electrophoresis
Students should appreciate the broad range of applications, including DNA profiling for paternity and
forensic investigations.
Additional higher level
D1.1.6—Directionality of DNA polymerases
Students should understand the difference between the 5' and 3' terminals of strands of nucleotides and
that DNA polymerases add the 5' of a DNA nucleotide to the 3' end of a strand of nucleotides.
D1.1.7—Differences between replication on the leading strand and the lagging strand
Include the terms “continuous”, “discontinuous” and “Okazaki fragments”. Students should know that
replication has to be initiated with RNA primer only once on the leading strand but repeatedly on the
lagging strand.
D1.1.8—Functions of DNA primase, DNA polymerase I, DNA polymerase III and DNA ligase in replication
Limit to the prokaryotic system.
D1.1.9—DNA proofreading
Limit to the action of DNA polymerase III in removing any nucleotide from the 3' terminal with a
mismatched base, followed by replacement with a correctly matched nucleotide.
 Double helix structure of DNA

“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic material.”
Watson & Crick
 Nucleotide structure

Phosphate:
Links
neighboring Base:
sugars Four types occur in
DNA
• Adenine
Sugar:
• Guanine
Deoxyribose
• Cytosine
in DNA
• Thymine
Directionality of 5¢

DNA PO4

You need to number the 5¢ CH2

base
carbons! ... it matters! O
4¢ 1¢
C
Nucleotide 3¢ 2¢
PO4
O
–O P O

N base O base
5¢ CH2
5¢ CH2 O
O
4¢ 1¢
4¢ Deoxyribose 1¢
3¢ 2¢
OH
3¢
2¢
OH 3¢
 What is DNA Replication?
Parent Replicated
chromosome chromosome

Chromatid

DNA replication is the process in Centromere

which DNA is replicated to produce
an exact copy of itself.
DNA replication is carried out in Parent DNA
preparation for cell division.
Replication is achieved by a single G C A T
strand of the DNA molecule being
used as a template for a new
complementary strand.
– The replicated DNA consists of one
original strand of DNA, and a newly
synthesized strand. DNA that will
become
– DNA replication is thus said to be separate
semi-conservative. chromatids
Meselson -Stahl
 DNA Replication

2. Elongation
DNA replication A new DNA strand is made
consists of three stages:
using the
original as a template.
1. Unwinding. 3. Termination
The DNA strand DNA synthesis is
is unwound to completed, and
allow the
replication to new DNA
occur. molecule
assumes its
double
helix structure.
 Control of DNA Replication
5' 3'

DNA replication is
highly regulated.
Each stage of replication Helicase
is controlled by enzymes.
RNA polymerase

DNA polymerase III

DNA polymerase III

DNA polymerase I

3' 5'
5' DNA ligase
3'
 DNA Replication

Origins of
replication
Replication
Bubbles:
Hundreds of
replicating
bubbles
(Eukaryotes).
Single replication
fork (bacteria).
 DNA Replication

Strand Separation:
1. Helicase: enzyme which
catalyze the unwinding and
separation (breaking H-
Bonds) of the parental
double helix.

2. Single-Strand Binding
Proteins: proteins which
attach and help keep the
separated strands apart. Replication
fork
 DNA Replication

Origins of replication
Replication Forks: hundreds of Y-shaped regions of
replicating DNA molecules where new
strands are growing.
3’

Parental DNA Molecule

5’ Replication
Fork
3’

5’
 DNA Replication
3.- Primase: enzyme that polymerizes (synthesizes) the RNA
Primer. Before new DNA strands can form, there must be small
pre-existing RNA primers present to start the addition of new
nucleotides (DNA Polymerase ).
DNA polymerases add nucleotides to a chain or polymer of
nucleotides being assembled on a template strand, but they
cannot initiate the process. There has to be a 3ʹ terminal to
which a free nucleotide can be added.

5’ 3’

RNA
5’
Primer
 DNA Replication

DNA primase is a type of RNA polymerase. During DNA

replication it assembles a chain of about 10 RNA nucleotides
on the template strand, to provide a site where DNA
polymerase III can bind and start adding nucleotides to the 3ʹ
end of a DNA strand.

On the leading strand an RNA primer is only needed once, but

primers are inserted every 100–200 nucleotides along the
lagging strand.
 Replication:
Elongation

Free nucleotides Parent strand

The unwound DNA strands are used to construct of DNA is
(parent strands) are used the new DNA strand used as a
template.
as a template.
New DNA is formed by
adding free nucleotides.
The free nucleotides are
joined together by the The new
enzyme DNA polymerase (daughter) strand
of DNA.
III.
The new strand is called
the daughter strand.
 DNA Replication
In DNA replication new strands can only be assembledd in a
5ʹto 3ʹ direction by DNA polymerases . The two strands formed
when helicase separates the DNA are antiparallel, their 5ʹ to 3ʹ
directions are opposite. DNA polymerase using these strands as
templates for assembling new strands moves in opposite
directions, always 5ʹ to 3ʹ.

5’ 3’
5’
RNA
Nucleotides DNA Polymerase III Primer
 DNA Replication

Synthesis of the new DNA Strands:

4.- DNA Polymerase III: with a RNA primer in place,

DNA Polymerase III (enzyme) catalyze the synthesis
of a new DNA strand in the 5’ to 3’ direction.

5’ 3’

RNA
5’
DNA Polymerase III Primer
Nucleotide
 Nucleotide Matching

Free nucleotides are matched A pairs with T

with their corresponding
T pairs with A
template bases following the
G pairs with C
base pairing rule
C pairs with G
The nucleotides are present
as deoxynucleoside Parent
Parent
triphosphates. strand strand

Hydrolysis of these provides

the energy for incorporating
the nucleotide into the
DNA strand.
Two new daughter
strands forming
 Energy of Replication
Where does energy for bonding usually come from?

energy

ATP ADP AMP

modified nucleotide
 DNA Replication
Leading Strand: synthesized as a single polymer in
the 5’ to 3’ direction.

5’ 3’
5’
RNA
Nucleotides DNA Polymerase III Primer
 DNA Replication

Lagging Strand: also synthesized in the 5’ to 3’

direction, but discontinuously against overall
direction of replication.

Leading Strand
5’ 3’

3’ 5’
DNA Polymerase III RNA Primer
5’ 3’

3’ 5’
Lagging Strand
 Leading & Lagging strands
Lagging strand
• Okazaki fragments
• joined by ligase 5¢
agments
Okazak
i f r 5¢ 3¢
5¢
RNA 3¢ RNA

3¢ Lagging strand
3¢ 5¢
RNA

ligase
3¢
5¢ growing Leading strand
replication fork
3¢ RNA 5¢

3¢
Limits of DNA polymerase III DNA polymerase III
• can only build onto 3¢ end
of an existing DNA strand Leading strand
• continuous synthesis
 The Leading Strand
5 3'
Enzymes can build new '

Overall direction
of replication
DNA strands only in the
5’ to 3’ direction.
One strand, the leading
strand, is synthesized as Replication
fork
a continuous strand.
DNA polymerase III

The parent strand Helicase splits

provides a 'template' and unwinds the
for synthesis of the double-stranded
s
si

new strand
he

DNA
nt
sy

The leading strand is

of

3' synthesized continuously in

n

5'
io

5' the 5' to 3' direction by DNA

ct
ire

polymerase III.
D
 The Lagging Strand
RNA polymerase
The second new strand, 5' 3' Makes a short RNA
primer which is later
called the lagging

Overall direction
removed.
strand, is constructed in

of replication
fragments. DNA polymerase III extends
the RNA primer with short
The fragments are later lengths of complementary
joined together. DNA to make Okazaki
fragments.
Helicase splits and
unwinds the double-
stranded DNA

D
ire
RNA

ct
io
primer

n
of
sy
nt
he
si
New complementary

s
strand is synthesized
3'
discontinuously in 5'
fragments 1000-2000 bp
long
 DNA Replication

Okazaki Fragments: series of short segments on

the lagging strand.

DNA
Okazaki Fragment Polymerase
RNA
Primer
5’ 3’

3’ 5’
Lagging Strand
 Okazaki Fragments
5' 3'

Overall direction
of replication
DNA polymerase I
digests the RNA
primer and replaces
it with DNA.

DNA ligase joins

neighboring
fragments
together into
longer strands.

The lagging strand is

D syn
ire th
formed in fragments

ct es
io is
(called Okazaki

n
3'

of
fragments) which are
5'
later joined together.
3'
 DNA Replication

5. DNA polymerase I : once the DNA strand is

completely synthesized the primers are removed
by and DNA nucleotides replaced the primer.

DNA
Okazaki Fragment Polymerase
RNA
Primer
5’ 3’

3’ 5’
Lagging Strand
 DNA Replication

6. DNA ligase: a linking enzyme that catalyzes the

formation of a covalent bond from the 3’ to 5’ end of
joining stands.

Example: joining two Okazaki fragments together.

DNA ligase
Okazaki Fragment 1 Okazaki Fragment 2
5’ 3’

3’ 5’
Lagging Strand
 Replication:
Termination The double
strands of DNA
When DNA synthesis is coil up into a
helix
completed, the process is said to
be terminated.
Each new DNA molecule contains
one strand of original DNA, and
one strand of newly synthesized
DNA. Chromosome (2
chromatids)

The two new strands of DNA coils

up into a helix as a chromosome.
The newly formed chromosome
will later split into two Parent strand

Daughter strand
chromatids.
 DNA Amplification

Using the technique called Polymerase Chain Reaction

(PCR), researchers are able to create vast quantities of
DNA identical to trace samples. This process is also
known as DNA amplification.
A crime scene
DNA sequencing (body tissue samples)

DNA profiling/fingerprinting
A single viral particle
Gene cloning (from an infection)

Transformation
Making artificial genes

Fragments of DNA from

a long extinct animal
 PCR Equipment
Amplification of DNA can be carried out with simple-to-use
PCR machines called thermal cyclers
 Steps in the PCR Process
Separate Strands
Separate the target DNA strands
by heating at 98°C for 5
minutes

The laboratory process Add Reaction Mix

Repeat for about
called the polymerase Add primers (short RNA strands
25 cycles
that provide a starting sequence
chain reaction or PCR for DNA replication), nucleotides Repeat cycle of
involves the following (A, T, G and C) and DNA
polymerase enzyme.
heating and
cooling until
steps enough copies of
the target DNA
have been
Incubate produced.
Cool to 60°C and incubate for a few
minutes. During this time, primers
attach to single-stranded DNA. DNA
polymerase synthesizes
complementary strands.
 Polymerase Chain Reaction
PCR No. of target
cycles DNA strands
Original
1 2
DNASample
2 4
3 8
Replication of DNA 4 16

ocurrs at an 5
6
32
64
exponential rate 7 128
Cycle 1
and can make 8
9
256
512
literally billions of 10 1024

copies in only a 11
12
2048
4096
few hours. 13 8192
Cycle 2 14 16 384
15 32 768
16 65 536
17 131 072
18 262 144
19 524 288

Cycle 3 20 1 048 576

21 2 097 152
22 4 194 304
23 8 388 608
24 16 777 216
25 33 554 432
 The Process of PCR

A DNA sample called

the target DNA is
obtained

DNA is denatured (DNA strands

are separated) by heating the
sample for 5 minutes at 98°C

Primers (short strands of

mRNA) are annealed (bonded)
Primer annealed
to the DNA
 The Process of PCR

The sample is cooled to

60°C. A thermally stable
DNA polymerase enzyme
binds to the primers on each
side of the exposed DNA
strand.
This enzyme synthesizes a
complementary strand of
DNA using free nucleotides.

After one cycle, there are now

two copies of the original
sample.
 Gel Electrophoresis
A technique known as gel Wells into which Sample

electrophoresis can be Cathode samples to be analyzed

are placed.
used to separate large
molecules (including Buffer
nucleic acids or proteins)
on the basis of their size,
electric charge, and other Plastic
physical properties. Frame

To prepare DNA for

electrophoresis, the DNA is Anode Gel
often cut up into smaller
pieces. Called a restriction
digest, and it produces a range
of DNA of different lengths.

To carry out electrophoresis,

the DNA samples are placed in DNA fragments, shown
wells and covered with a buffer symbolically above, move
towards the positive terminal
solution that gradually (smaller fragments move faster Buffer
dissolves them into solution. than longer ones). solution
 Analyzing DNA

By applying an electric
field to the solution, the Wells: Holes created
in the gel with a
molecules move towards comb.
one or other electrode -ve terminal
depending on the charge
on the molecule itself. DNA
solutions: Large DNA markers:
A mixture of DNA
DNA is negatively charged Mixtures of
fragments
molecules with
because the phosphates different sizes known molecular
have a negative charge. of DNA weights. They
fragments are are used to
loaded into
Molecules of different estimate the
each well. sizes of the DNA
sizes (molecular weights) DNA fragments in the
become separated fragments: The sample lanes.
(spread out) on the gel matrix acts Small
as a seive for fragments
gel surface. the DNA
molecules.
These can be visualized
by applying dyes or
+ve
radio-labeled probes. terminal
Tray: Contains the set
gel.
 DNA Profiling
DNA profiling (DNA fingerprinting) is a technique for
genetic analysis, which identifies the variations found
in the DNA of every individual.
The profile refers to the distinctive
pattern of DNA restriction fragments
or PCR products which is used to
identify an individual.

DNA profiling does not determine a base

sequence for a sample but merely sorts
variations in base sequences.
Only one in a billion (i.e. a thousand
million) persons is likely to have an
identical DNA profile, making it a useful
tool for forensic investigations and
paternity analysis.
 Visualizing the Profile

DNA fragments (PCR product after endonuclease

digestion) visualized under UV light after staining with
ethidium bromide and migration in an agarose
electrophoresis gel.
 Uses of DNA Profiling

DNA profiling can be used for

investigating:
the presence of a particular gene,
such as cystic fibrosis) in a family.
genetic relatedness of different
organisms
e.g. checking on pedigree in stock
breeding programs.
e.g. checking that captive
populations of endangered species
are not inbred.
 Forensic DNA Evidence
DNA fingerprints from tissue
samples can be used as
evidence in the same way
traditional fingerprinting is
used.
Which DNA fingerprint from
the three suspects matches
that of the tissue sample
submitted as evidence?
Why would the DNA from the
victim be included in this test?