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DP IB Biology: HL Your notes

DNA Replication
Contents
DNA Replication
Electrophoresis & PCR
Electrophoresis & PCR: Applications
Mechanism of DNA Replication

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DNA Replication
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Importance of DNA Replication
The replication of DNA is semi-conservative and depends on complementary base pairing
Semi-conservative means that one strand of the 'parent' DNA is kept in the 'daughter' molecule
This is called the template strand
The other half is determined by the code on the template strand and is built up from free nucleotides in
the nuclear space around the chromosomes
This takes place in the nucleus
If an adenine is the next exposed base on the original strand, a thymine nucleotide is added and
vice versa
If a cytosine is the next exposed base on the original strand, a guanine nucleotide is added and
vice versa
Nucleotides are added one by one to the new strand according to the rules of complementary
base-pairing
Hydrogen bonds can only form between the template strand and the new strand if the correct bases
are paired up
Therefore, the new DNA molecule has kept half of the parent DNA and then used this to create a new,
daughter strand
DNA replication is important in multicellular organisms for many reasons, such as:
Growth
Replacement of old/damaged cells and tissues
Reproduction

Examiner Tips and Tricks

Make sure you don’t confuse ‘parent cell’ with ‘parent organism’. A parent cell is any cell in the body
that divides into two cells and the terminology is used to refer to the ‘original’ cell that the DNA
came from before it was split and replicated semi-conservatively.

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Semi-Conservation Replication
DNA polymerase links nucleotides together to form a new strand, using the pre-existing strand as a Your notes
template
Before a (parent) cell divides, it needs to copy the DNA contained within it
This is so that the two new (daughter) cells produced will both receive the full copies of the
parental DNA
The DNA is copied via a process known as semi-conservative replication (half the DNA is kept)
The process is called so because in each new DNA molecule produced, one of the polynucleotide
DNA strands (half of the new DNA molecule) is from the original DNA molecule being copied
The other polynucleotide DNA strand (the other half of the new DNA molecule) has to be newly
created by the cell

Semi-conservative DNA replication diagram

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Your notes

Semi-conservative replication of DNA

The importance of keeping one original DNA strand

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It ensures there is genetic continuity with a high degree of accuracy between generations of cells
In other words, it ensures that the new cells produced during cell division inherit all their genes Your notes
with the correct sequence of DNA bases from their parent cells

Crick and Watson proposed semi-conservative replication

As part of their discovery of the double-helix structure of DNA, Crick and Watson made a hypothesis
about how DNA copies during cell growth
They proposed a semi-conservative model, but had not provided the evidence
This was provided by two later scientists, Meselson and Stahl, in another award-winning piece of
research
Analysis of Meselson and Stahl’s results gave the necessary support for Crick & Watsons' hypothesis
of semi-conservative replication of DNA

Helicase & DNA Polymerase

DNA replication occurs in preparation for mitosis, when DNA must be doubled before the parent cell
can divide to produce two genetically identical daughter cells
The enzyme helicase first unwinds the DNA, by flattening out its helical structure
Analogy - think about untwisting a rope ladder
Helicase then causes the hydrogen bonds to break between pairs of bases, exposing bases on either
strand
Analogy - unzipping a zipper
Each of these single polynucleotide DNA strands acts as a template for the formation of a new strand
made from free nucleotides that are attracted to the exposed DNA bases by base pairing

Function of helicase and DNA polymerase diagram

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Your notes

Helicase and DNA polymerase work together to replicate each strand of DNA
DNA polymerase links nucleotides together to form a new strand, using the pre-existing strand as a
template
Following the action of helicase, the template strand is exposed and new nucleotides are joined
together by DNA polymerase, which catalyses condensation reactions to form a new strand
These reactions occur between the deoxyribose sugar and phosphate groups of adjacent
nucleotides within the new strands, creating the sugar-phosphate backbone of the new DNA
strands
DNA polymerase always builds a new DNA strand in the same direction; the 5' to 3' direction
DNA polymerase will always attach to the 3’ end of the original template strand and read the
strand in a 3' to 5' direction
Because the two DNA strands are antiparallel, this means that the new strand is built in the 5' to
3' direction
Hydrogen bonds then form between the complementary base pairs of the template and new
DNA strands
This method of replicating DNA is known as semi-conservative replication because half of the original
DNA molecule is kept (conserved) in each of the two new DNA molecules

Overview of DNA replication diagram

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Your notes

The combined actions of helicase and DNA polymerase create new complementary DNA strands

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Electrophoresis & PCR

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Electrophoresis & PCR
Gel electrophoresis
Gel electrophoresis is a technique used widely in the analysis of DNA, RNA, and proteins
During electrophoresis, the molecules are separated with an electric current according to their size
or mass and their net (overall) charge
This separation occurs because of:
The electrical charge molecules carry:
Positively charged molecules will move towards the cathode (negative pole), whereas
negatively charged molecules will move towards the anode (positive pole), e.g. DNA is
negatively charged due to the phosphate groups and thus, when placed in an electric current,
the molecules move towards the anode
The different sizes of the molecules:
Different sized molecules move through the gel (agarose for DNA and polyacrylamide for
proteins) at different rates. The tiny pores in the gel result in smaller molecules moving quickly,
whereas larger molecules move slowly
The type of gel:
Different gels have different sized pores that affect the speed at which the molecules can
move through the gel

DNA separation
DNA can be collected from almost anywhere on the body, e.g. the root of a hair or saliva from a cup.
After collection, DNA must be prepared for gel electrophoresis so that the DNA can be sequenced or
analysed for genetic profiling (fingerprinting)
To prepare the fragments, scientists must first increase (amplify) the number of DNA molecules by the
Polymerase Chain Reaction (PCR)
Then restriction (DNA-cutting) enzymes are used to chop the DNA into fragments

Method
To separate the DNA fragments in gel electrophoresis:

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1. Create an agarose gel plate in a tank. Wells (a series of small rectangular holes) are cut into the gel
at one end
Your notes
2. Submerge the gel in an electrolyte solution (a salt solution that conducts electricity) in the tank
3. Load (insert) the DNA fragments into the wells using a micropipette
4. Apply an electrical current to the tank. The negative electrode must be connected to the end of
the plate with the wells as the DNA fragments will then move towards the anode (positive pole) due
to the attraction between the negatively charged phosphates of DNA and the anode
5. DNA fragments with a smaller mass (i.e. shorter DNA fragments) will move faster and further from
the wells than the larger fragments
6. The fragments are not visible so must be transferred onto absorbent paper or nitrocellulose which
is then heated to separate the two DNA strands
7. Probes are then added to develop a visual output, either:
A radioactive label (eg. a phosphorus isotope), which causes the probes to emit radiation that
makes the X-ray film go dark, creating a pattern of dark bands
A fluorescent stain or dye (eg. ethidium bromide), which fluoresces (shines) when exposed to
ultraviolet (UV) light, creating a pattern of coloured bands
Gel electrophoresis diagram

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Your notes

The process of electrophoresis

Examiner Tips and Tricks

Remember gel electrophoresis is the separation of molecules according to their size and charge
(negatively charged DNA molecules move to the positive pole). Examiners like to ask questions
about gel electrophoresis, so make sure you understand each of the different steps in the process.

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is a common molecular biology technique used in most applications
of gene technology
For example, it is used in DNA profiling (e.g. identification of criminals and determining paternity) or
genetic engineering

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In the COVID-19 pandemic, PCR has been used in routine diagnostic testing to amplify small
amounts of viral RNA
It can be described as the in vitro method of DNA amplification Your notes
It is used to produce large quantities of specific fragments of DNA or RNA from very small quantities
(even just one molecule of DNA or RNA)
Using PCR, scientists can produce billions of identical copies of the DNA or RNA samples within a
few hours, these can then be used for analysis
The requirements of PCR
Each PCR reaction requires:
The target DNA or RNA that is being amplified
It's important that the whole genome is not required to be copied - only specific sections that
vary from one individual to another
These sections are identified by adding a primer sequence that binds to them
DNA polymerase - the enzyme used to build the new DNA or RNA strand. The most commonly used
polymerase is Taq polymerase as it comes from a thermophilic bacterium Thermus aquaticus
This means it does not denature at the high temperature involved during the first stage of the
PCR reaction
Free nucleotides - used in the construction of the DNA or RNA strands
Buffer solution - to provide the optimum pH for the reactions to occur in

The key stages of PCR

The PCR process involves three key stages per cycle
In each cycle the DNA is doubled (so in a standard run of 20 cycles a million DNA molecules are
produced)
The PCR process occurs in a piece of specialist equipment called a thermal cycler, which
automatically provides the optimal temperature for each stage and controls the length of time spent
at each stage
The three stages are:
1. Denaturation – the double-stranded DNA is heated to 95°C which breaks the hydrogen bonds
that bond the two DNA strands together
2. Annealing – the temperature is decreased to between 50 - 60°C so that primers can anneal to the
ends of the single strands of DNA

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3. Elongation / Extension – the temperature is increased to 72°C for at least a minute, as this is the
optimum temperature for Taq polymerase to build the complementary strands of DNA to produce
the new identical double-stranded DNA molecules Your notes
Each whole cycle takes a few minutes, so 30 cycles can take just a few hours and can generate
231 (over 1 billion) copies of a gene from a single DNA molecule, by exponential amplification
DNA amplification during PCR diagram

Target DNA sequences can be copied exponentially by PCR to generate billions of copies in a short time

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Electrophoresis & PCR: Applications

Your notes
Electrophoresis & PCR: Applications
DNA profiling
DNA profiling (genetic fingerprinting) enables scientists to identify suspects of a crime and identify
corpses
Every person (apart from identical twins) has repeating, short, non-coding regions of DNA (20 to 50
bases long) that are unique to them
These regions of DNA are called VNTRs (Variable Number Tandem Repeats)
DNA profiling involves using gel electrophoresis to separate VNTR fragments according to length to
create a pattern of bands that is unique to every individual, sometimes called the genetic fingerprint
To create a DNA profile from the DNA being tested scientists complete the following in sequence:
1. Obtain the DNA, which can be extracted from the root of a hair, a spot of blood, semen or saliva
2. Increase the quantity of DNA by using PCR to produce large quantities of the required fragment of
DNA from very small samples (even just one molecule of DNA or RNA).
3. Use restriction endonucleases to cut the amplified DNA molecules into fragments
4. Separate the fragments using gel electrophoresis
5. Add radioactive or fluorescent probes that are complementary and therefore bind to specific
DNA sequences
6. X-ray images are produced or UV light is used to produce images of the fluorescent labels glowing
7. These images contain patterns of bars (the DNA profile) which are then analysed and compared

Use of DNA profiling in Paternity Investigations

Every person inherits their DNA VNTRs from both their mother and their father
A man may sometimes deny being the father of a child to evade parenting responsibilities
A woman may not know for sure which of her recent sexual partners is the father of a child
A child may wish to know definitively who his/her father is to be aware of possible inherited illnesses
that might affect him/her in future
DNA profiles of the mother and child are compared, along with the profile of the alleged father (all
three are needed)

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Patterns of bands are compared on all three genetic profiles

Any band that appears in the child's profile must show in either the mother's or father's profiles; if Your notes
not, the alleged true father is a different man

Worked Example
Who’s the Father? – Use the DNA profiles of all 6 people shown to work out who the child’s father is

DNA profiles of a child, a mother and 4 possible fathers

Remember, any band showing in the child's profile must be present in the mother OR father's profile,
OR both. If not, that man is not the child's father.
Answer:
Step 1: Look at the child's first DNA band (labelled 1)
The mother possesses this same band, so the child could have inherited that DNA from its mother. It
is therefore needless to look at whether any of the men possess that band
Step 2: Look at the child's second DNA band (labelled 2)

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The mother does not possess this band, so the child must have inherited it from its father. Only men
B and D possess this band, so men A and C are eliminated
Your notes
Step 3: Look at the child's third DNA band (labelled 3)
As with band 1, the mother possesses this same band, so the child could have inherited that DNA
from its mother. It is therefore needless to look at whether any of the men possess that band
Step 4: Look at the child's fourth DNA band (labelled 4)
The mother does not possess this band, so the child must have inherited it from its father. Only men
A, B and C possess this band, but A and C have already been eliminated
Step 5: Conclude that B is the father
Step 6: Look for supporting evidence from band 6
The mother does not possess this band, and the only man who possesses it is B. This reinforces the
conclusion that Man B is the child's father

Use of DNA profiling in Forensic Investigations

DNA profiling has been used by forensic scientists to identify suspects of crimes
Samples of body cells or fluids (e.g. blood, saliva, hair, semen) are taken from the crime scene or
victims body (e.g. rape victims)
DNA is removed and profiled
The profile is compared to samples from the suspect (or criminal DNA database), victim and
people with no connection to the crime (control samples)
Care must be taken to avoid contamination of the samples
DNA profiling can also be used in forensics to identify bodies or body parts that are unidentifiable (e.g.
too badly decomposed or parts remaining after a severe fire)
DNA profiling from a crime scene can also eliminate innocent people whose DNA may happen to
appear there

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Your notes

Using DNA profiling in criminal investigations. Suspect 3 has the most fragments in common with the
crime scene DNA so it is likely that Suspect 3 is the culprit.

Examiner Tips and Tricks

In the exam, you will be expected to interpret the results of gel electrophoresis experiments used to
separate DNA fragments. For example, you will be given a few different genetic fingerprints and will
have to match the victim to the crime or determine the parents of children. In these questions, you
need to look for the most bands in common or a combination of parents' fingerprints that covers all
the child's bands.

NOS: Reliability is enhanced by increasing the number of

measurements in an experiment or test

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Increasing the reliability of scientific conclusions is a very important aspect of all experimentation, but
this is particularly important when evidence is being collected for use in a court of law
Your notes
It is almost impossible to be 100% certain that any evidence provided is absolutely correct
In DNA profiling, reliability can be increased by increasing the number of VNTR markers being
observed and compared
The more VNTRs used, the lower the chance of a false match

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Mechanism of DNA Replication

Your notes
Directionality of DNA Polymerase
Similar to transcription and translation, DNA replication must occur in the 5’ to 3’ direction
DNA polymerase only works in a 5’ to 3’ direction, adding nucleotides to the 3’ end of a strand of
nucleotides
DNA nucleotides have a phosphate bonded to the 5’ carbon of the deoxyribose sugar
When DNA polymerase adds a new nucleotide to extend the DNA strand, the 5’ phosphate group of the
incoming DNA nucleotide bonds to the free 3’ -OH group on the growing strand

DNA nucleotide structure diagram

DNA nucleotides have a phosphate bonded to the 5’ carbon of the pentose sugar
5' and 3' ends of a DNA strand diagram

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Your notes

When DNA polymerase adds a new nucleotide, the 5’ phosphate group of the incoming nucleotide
bonds to the free 3’ -OH group on the growing strand

The Leading & Lagging Strand

Double-stranded DNA consists of two antiparallel strands
This means that one strand runs from 5' to 3', while the other strand runs from 3' to 5'
During DNA replication, the two strands are ‘unzipped’ and DNA polymerase moves along each
template strand linking nucleotides together to form a new strand

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Crucially, DNA polymerase can only add new nucleotides in a 5’ to 3’ direction

As the template strands are antiparallel, replication needs to proceed in opposite directions Your notes
As the replication fork opens up in one direction only, each new strand is synthesised differently
The leading strand is made continuously, following the fork as it opens
The lagging strand is made discontinuously, in short fragments, away from the fork
These fragments are called Okazaki fragments
As more template strand is exposed, new fragments are created
Okazaki fragments are later joined together by DNA ligase to form a continuous complementary
DNA strand
Before new DNA nucleotides can be added to the new DNA strand, first an RNA primer must be added
to create a binding point for DNA polymerase III
The RNA primer only has to be added once on the leading strand but several are needed on the
lagging strand to initiate each fragment
Difference between replication on the lagging and leading strands of
DNA diagram

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Your notes

During DNA replication, synthesis of the leading strand is continuous but synthesis of the the lagging
strand is discontinuous in small fragments (not all the enzymes involved are shown)

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Enzymes in DNA Replication

DNA replication is carried out by a complex system of enzymes working as a team Your notes

Helicase unwinds the DNA double helix at the replication fork

Helicase then causes the hydrogen bonds between the two strands to break so that they can
separate
Single-stranded binding proteins keep the separated strands apart whilst the template strand is
copied
DNA primase generates a short RNA primer on the template strands
Providing an initiation point for DNA polymerase III to add new nucleotides
A number of polymerases are involved in DNA replication, each with different functions
Two of these polymerases are
DNA polymerase III, which starts replication next to the RNA primer linking nucleotides in a 5’
to 3’ direction to form a new strand
DNA polymerase I, which removes the RNA primers on the leading and lagging strands and
replaces it with DNA
DNA ligase joins up the Okazaki fragments by catalysing the formation of sugar-phosphate bonds

Proofreading Replicated DNA

Each time a human cell replicates it requires 3 billion new base pairs to be synthesised in order to fully
replicate the genome
The copying process is not 100% perfect and mistakes do occur, these are called mutations
Mutations can be harmful to the functioning of the new cell and lead to diseases such as cancer
In prokaryotes, in order to reduce mistakes during replication the enzyme DNA polymerase III acts as a
proof-reader of the new daughter strand of DNA
It can recognise incorrect DNA nucleotides in the daughter strand
It reverses direction in order to remove the incorrect nucleotide from the 3' end of this strand
The correct nucleotide is then inserted and the polymerase III enzyme continues replication

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