Unit 7: Molecular biology and genetics
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DNA replication
The fact that DNA is a self-replicating molecule and can make copies of itself
is the basis of all life forms. It is the essence of what life is. Indeed, according
to Richard Dawkins in his book The Selfish Gene, DNA is the life form on planet
Earth, perpetuating itself by using its code to direct the building of living
organisms. Those best adapted organisms will survive and their DNA will
proliferate in their descendants.
On successful completion of this topic you will:
•• be able to carry out experimental techniques involving manipulating
DNA, RNA and protein (LO3).
To achieve a Pass in this unit you will need to show that you can:
•• explain the process of DNA replication (3.1)
•• safely perform techniques to isolate DNA and mRNA (3.2)
•• describe the polymerase chain reaction (3.3).
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Unit 7: Molecular biology and genetics
1DNA
Every time a cell divides to produce new cells its DNA is copied. Each molecule
of DNA undergoes semi-conservative replication. Put very simply, the DNA
unwinds and unzips to expose nucleotide bases. DNA polymerases catalyse the
addition of activated DNA nucleotides, according to complementary base-pairing
rules, to make two new identical molecules of DNA, each one containing one
old strand and one new strand. Hence each new molecule contains half of the
original molecule.
Before DNA synthesis begins the original strands are separated and the synthesis
of the daughter strands begins at the replication fork at a site called an origin
of replication where a replisome is assembled from many proteins. The initiation
complex that is formed attracts DNA polymerases.
Synthesis of the new strands is called elongation and is aided by the proteins in
the replisome.
Lastly the termination site replicates (see Figures 7.3.1 and 7.3.2).
Figure 7.3.1: The DNA replication fork.
Because both daughter strands are
synthesised in the 5’ to 3’ direction, the
DNA complementary to the lagging
strand is synthesised in small fragments
called Okazaki fragments. These
fragments are then joined together.
3’
5’
5’
3’ 5’
3’
5’
3’
Most recently
synthesized
DNA
5’
3’ 5’
5’
3’
3’
3’
3’
5’
Figure 7.3.2: Enzymes involved
in DNA replication.
3’
5’
Leading strand
Lagging strand with
Okzaki fragments
5’
Enzyme
Primase
synthesises RNA
DNA polymerase III
extends RNA primer
into Okazaki fragments
Next Okazaki fragment
is synthesised
DNA polymerase I
uses nick translation
to replace RNA primer
with DNA
Ligase seals the nick
7.3: DNA replication
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Unit 7: Molecular biology and genetics
Key terms
Lagging strand: The DNA strand that
is synthesised (during replication)
in a 5’ to 3’ direction away from the
replication fork in short, Okazaki
fragments that are then joined.
Leading strand: The DNA strand that
is synthesised (during replication) with
no or few interruptions, in a 5’ to 3’
direction towards the replication fork.
Primers: Short, single-stranded
sequences of DNA or RNA, usually of
around 10–20 bases long.
The replisome
The replisome consists of many proteins, including helicase, gyrase/
topoisomerase, primase, DNA polymerases, RNAse H and ligase. One DNA
polymerase complex synthesises the lagging strand and another synthesises the
leading strand. There are also factors, called replication proteins, that protect both
the unstable single-stranded unwound leading and lagging strands from making
hydrogen bonds with themselves and forming hairpins.
Helicase
Helicase causes the hydrogen bonds between complementary base pairs to
break and so catalyses the separation of the two parental strands that will act as
templates for synthesis of the daughter molecules. Helicase moves along the DNA
in a 3’ to 5’ direction.
Gyrase
Gyrase (a form of topoisomerase) unwinds the resulting supercoil that forms
upstream of the section of unwound DNA.
DNA polymerases
DNA polymerases catalyse the elongation phase of replication.
Clamp proteins
Clamp proteins help keep the DNA polymerases attached to the leading and
lagging strands and make sure the process proceeds at a suitably fast rate.
Priming
In eukaryotic cells a DNA-dependent RNA polymerase creates an RNA primer, of
about 10 bases long, on both the newly separated leading and lagging strands,
once for the leading strand and once per Okazaki fragment (about 1000 base pairs
long) on the lagging strand. The RNA primer attached to its DNA template is called
A-form DNA. (Normal DNA is called B-form DNA.)
In prokaryotes primase creates an RNA primer at the beginning of the newly
separated leading and lagging strands. DNA polymerase enzymes cannot bind
directly to single-stranded DNA and these primers provide a short chain of
nucleotides that give the correct configuration to allow the active site of DNA
polymerase to fit on and begin elongation.
Elongation
The leading and lagging strands are anti-parallel. In the leading strand nucleotide
synthesis (catalysed by DNA polymerase epsilon in eukaryotes and by DNA
polymerase III in prokaryotes) proceeds in the 5’ to 3’ direction (3’ to 5’ direction on
the template strand) and makes a continuous complementary strand. Synthesis
of the other strand in the opposite direction cannot occur at the same time so
replication of the lagging strand is discontinuous. It involves making short
discrete nucleotide chains, called Okazaki fragments, that are then joined by
DNA repair enzymes, such as DNA polymerase I and ligase, so it is not made in one
continuous strand.
7.3: DNA replication
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Unit 7: Molecular biology and genetics
Activity: Semidiscontinuous and
semi-conservative
1 Explain why the replication
of DNA is described as semidiscontinuous.
2 Explain why the replication
of DNA is described as semiconservative.
3 Research and write an illustrated
account to show how Meselson
and Stahl’s experiment confirmed
that DNA replication is semiconservative.
4 Make a 3D poster showing how a
piece of DNA replicates.
This can only happen once a sufficient length of DNA has been unwound so
replication of this strand lags behind that of the leading strand.
RNAse H enzymes remove the unstable RNA primers from the newly synthesised
fragments and replace them with DNA fragments.
DNA ligase (aided by polymerase I in prokaryotes) enzyme connects the Okazaki
fragments, closing the gaps between their sugar-phosphate backbones by
catalysing the formation of phosphodiester bonds.
Proofreading enzymes correct any mistakes due to insertion of incorrect bases.
Case study: Investigation to find if the replisome moves along
the DNA molecule
You probably envisage the replisome moving along the DNA molecule. In 2000 Katherine Lemon
and Alan Grossman, at the Massachusetts Institute of Technology (MIT), carried out an experiment
using the bacteria Bacillus subtilis. They tagged the replisomes with a green fluorescent protein
and used microscopy to observe its position in the cell during DNA replication. The replisome was
always in the same position.
•• What can you conclude from this investigation – does the replisome move along the DNA
molecule or is the DNA fed through the replisome?
The polymerase chain reaction (PCR)
First developed in 1983 by Kary Mullis, the polymerase chain reaction (PCR) is a
way of amplifying small amounts of DNA in a laboratory for analysis. It is similar
to DNA replication that happens in cells but it can only be used to amplify short
lengths of DNA – up to 40-kilo base pairs – not whole chromosomes. Figure 7.3.3
summarises the process.
•• Taq DNA polymerase is obtained from a thermophilic bacterium, Thermus
aquaticus, so that the temperature does not have to be lowered to 37 °C at any
stage, and this speeds up the process.
•• Heat (95 °C), rather than helicase, causes the DNA strands to separate.
•• The temperature is reduced to 55 °C and DNA primers, complementary to
the 3’ ends of each strand of the DNA, are added to anneal at the ends of the
separated chains and initiate DNA polymerase activity.
•• Now DNA polymerase and a supply of activated DNA nucleotides (ATP, GTP,
CTP and TTP) are added, the temperature is raised to 72 °C and the DNA is
replicated.
•• This one cycle has doubled the DNA. It can be repeated, increasing the DNA
exponentially.
This process used to be lengthy, as it involved using water baths and timers, but it
is now carried out in a PCR thermocycler that adjusts temperatures as necessary.
Portfolio activity: PCR
reaction
Carry out PCR amplification of a DNA
sample.
7.3: DNA replication
Activity: How many cycles?
1 How many PCR cycles does it take to amplify one length of DNA into (a) 1 million lengths
(b) 2 million lengths?
2 If it takes 8.5 seconds for one PCR cycle, how long does it take to amplify one length of DNA to
1 million copies of it?
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Unit 7: Molecular biology and genetics
Activity: Comparing DNA replication and PCR
Compare the process of DNA replication with the polymerase chain reaction. You may want to use annotated diagrams or a table of information.
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2 Heat to 95 °C –
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1 Double-stranded
DNA sample
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Pre-implantation genetic
testing technician
Molecular biologist
7.3: DNA replication
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PCR
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3 Add primers and
reduce temperature to 55 °C
to allow primers to anneal
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Figure 7.3.3: The polymerase
chain reaction (PCR).
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4 Raise temperature to 72 °C
DNA polymerase binds
and extends primers
using free
nucleotides
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Kacper works in a hospital laboratory, using the PCR reaction for pre-implantation genetic testing.
If both parents carry a recessive allele, for example, for cystic fibrosis, they may choose to have
IVF. Eggs are fertilised and grown in vitro until they reach the eight-cell stage. Without damaging
the embryos, one cell can be taken and its DNA extracted and amplified using PCR. It can then be
tested to see if it has normal alleles for the CFTR gene. One or two healthy embryos will then be
implanted into the mother’s uterus.
Patience is a molecular biologist who carries out genetic testing for several conditions including
cystic fibrosis, coeliac disease, Down’s syndrome and HIV at a private pathology company. This
involves extracting DNA from biological samples and amplifying it using the PCR. She begins each
day by checking the worklist on the lab computer to see which tests have to be carried out that
day. She then plans the day, according to how many tests and how long each one will take, so that
she can get them all done. It takes a long time to extract DNA for HPV (human papilloma virus)
assay so she extracts these first.
She has A levels in biology, chemistry and maths and a degree in biomedical science. She hopes
to become a registered clinical scientist, which will take about six years and involves passing an
exam so she can register with the Health Professions Council. This will open up many more career
opportunities for her.
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Unit 7: Molecular biology and genetics
Checklist
At the end of this topic guide you should be familiar with the following ideas:
 DNA is a self-replicating molecule and is duplicated during the S phase of the cell cycle,
before the cell divides
 replisomes, complexes of many proteins, most acting as enzymes, direct the DNA replication,
which is semi-conservative and semi-discontinuous
 as a molecule of DNA begins to unwind it forms the replication fork where unpaired
nucleotides are exposed and can bind to complementary activated nucleotides – this process
starts at the 3’ end of the template strand and is continuous on one strand (the leading
strand), starting where an RNA primer has been added
 on the other strand (the lagging strand), antiparallel to the leading strand, new DNA is
synthesised in short Okazaki fragments, each started by an RNA primer that is then displaced,
and ligase enzyme then joins the fragments together
 proofreading enzymes check for errors
 the polymerase chain reaction is a useful laboratory technique for augmenting small amounts
of smaller lengths of DNA for forensic or clinical analysis.
Acknowledgements
The publisher would like to thank the following for their kind permission to reproduce their
photographs:
Corbis: MedicalRF.com
All other images © Pearson Education
We are grateful to the following for permission to reproduce copyright material:
Figure 7.3.1: The DNA replication fork, from Molecular biology of the cell, 5th ed. Alberts et al.
Copyright 2008 from Molecular Biology of the Cell, Fifth Edition by Alberts et al. Reproduced
by permission of Garland Science/Taylor & Francis LLC; Figure 7.3.2: Enzymes involved in DNA
replication, from Genes by Benjamin Lewin, published by OUP, 1997. Used by permission; Figure
7.3.3: The polymerase chain reaction (PCR), from OCR A2 Biology, Pearson. Used with permission of
Pearson Education Ltd.
Every effort has been made to trace the copyright holders and we apologise in advance for any
unintentional omissions. We would be pleased to insert the appropriate acknowledgement in any
subsequent edition of this publication.
7.3: DNA replication
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