D1.
1 DNA
REPLICATION
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GUIDING QUESTIONS
1. How is new DNA produced?
2. How has knowledge of DNA replication enabled applications in biotechnology?
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D1.1.1—DNA REPLICATION AS
PRODUCTION OF EXACT COPIES OF DNA
WITH IDENTICAL BASE SEQUENCES
DNA replication is the production of new strands of
DNA with base sequences identical to existing
strands.
DNA replication is required for two biological processes.
1. Reproduction—offspring need copies of the base
sequences of their parents, so parents must replicate
their DNA to reproduce.
2. Growth and tissue replacement in multicellular
organisms—each cell in a multicellular organism needs a
full set of the organism’s base sequences. This means that
before a cell divides into two daughter cells, it must
replicate all of its DNA. Cell division is needed for
growth and to replace cells in tissues where they have
been lost.
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(HL: DNA REPLICATION
OCCURS DURING THE S
PHASE OF INTERPHASE IN
THE CELL CYCLE.)
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D1.1.2—SEMI-CONSERVATIVE NATURE OF DNA REPLICATION
AND ROLE OF COMPLEMENTARY BASE PAIRING
When DNA is replicated, the two strands of the
double helix must separate.
Both original strands are used as templates
to guide the polymerization of a new strand.
The new strands are formed by adding
nucleotides one by one and linking them together
along a DNA molecule.
When replication is complete, there are two DNA
molecules, both composed of an original
strand and a newly synthesized strand.
DNA replication is referred to as semi-
conservative.
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The base sequence on the template strand determines
the base sequence on the new strand.
Only a nucleotide carrying a base that is
complementary to the next base on the template
strand can successfully be added to the new 6
The rule that one base always pairs with another is
called complementary base pairing.
It ensures that the two DNA molecules resulting from
DNA replication are identical in their base sequences
to the parent molecule. Complementary base
pairing ensures a high degree of accuracy when
new strands are assembled on a template
strand.
It also makes it possible to check the base sequence
that has been assembled, recognize any mispairing,
then cut out and replace the incorrect nucleotides.
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D1.1.3—ROLE OF HELICASE
AND DNA POLYMERASE IN
DNA REPLICATION
Helicase and DNA Polymerase are two enzymes
responsible for DNA Replication.
Helicase separates the two strands of a DNA
molecule so that they can each act as a template
for the formation of a new strand.
As it moves along a DNA molecule, helicase
breaks hydrogen bonds between bases,
unwinding the DNA double helix.
DNA Polymerase assembles new strands of DNA,
using the two original strands as templates. DNA
polymerase brings complementary nucleotides
into the position where hydrogen bonds
could form with the nitrogen bases on the
template strands.
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D1.1.4—POLYMERASE CHAIN REACTION
AND GEL ELECTROPHORESIS AS TOOLS
FOR AMPLIFYING AND SEPARATING DNA
•PCR or Polymerase Chain Reaction is a method used by scientists
to produce millions of copies of a sequence DNA.
•This makes it possible to study the DNA further, without the risk of using
up a limited sample.
•This can be completed in two hours and means that scientists can quickly
amplify DNA.
•DNA from very small samples such as semen, blood, tissues, or even from
specimens that have been dead for a long time can be amplified using
PCR.
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HTTPS://WWW.YOUTUBE.COM/WATCH?
V=8ABG9BEPDVC&T=33S
Mixtures of DNA base sequences can be loaded
into the PCR machine, but only selected
sequences are amplified.
Primers added at the start of cycling select
which sequences are copied. A primer is a short
single strand of DNA designed to bind to
the DNA at the point where the selected
base sequence for replication begins.
Taq DNA polymerase and DNA nucleotides
with each of the four bases are needed for PCR.
Taq DNA polymerase is extracted from bacteria
living in hot springs. The temperatures of these
springs can be as high as 80°C. Enzymes in most
organisms would rapidly denature, but these are
adapted to high temperatures.
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A
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Gel electrophoresis is used to separate DNA
molecules by length.
The separation is done in a sheet of gel.
In the gel, close to one end are rectangular holes
called wells.
The gel is placed in a shallow tank with electrodes at
both ends. An electrolyte solution is poured over the
gel to cover it. One DNA sample is pipetted into each
well.
A voltage is applied across the electrodes to create
an electric field. This makes charged molecules move
through the gel. DNA has negative charges, so it
moves towards the positive anode. The gel is
therefore orientated in the electrophoresis tank with
the wells close to the negative cathode.
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The gel consists of a mesh of filaments that resists
https://learn.genetics.utah.edu/content/labs/gel/
https://www.sumanasinc.com/webcontent/animations/content/gelel
ectrophoresis.html
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The gel is treated with a •The result is a picture of bands
dye to make DNA visible. of DNA.
DNA molecules of the
same length will have
moved the same distance,
so they form a visible band
in a lane.
The number of bands in a
lane indicates how many
different lengths of DNA
were in the sample.
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The right-hand lane is usually
used to create a “ladder”. This
is done by placing a mixture of
DNA fragments of known length
in the right-hand well.
The ladder is used to estimate
the lengths of bands in other
lanes on the gel.
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D1.1.5—APPLICATIONS OF
POLYMERASE CHAIN
REACTION AND GEL
ELECTROPHORESIS
Applicati
on of PCR
Not in
syllabus
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DNA PROFILING
DNA profiling is the process where a specific DNA
pattern, called a profile, is obtained from a person.
The general procedure includes:
1) The isolation of the DNA from an evidence sample
containing DNA of unknown origin, and generally at a
later time, the isolation of DNA from a sample (e.g.,
blood) from a known individual (suspect and victim).
2) Amplification of the DNA sample using PCR.
3) The breakdown of the DNA sample into fragments
using enzymes known as restriction enzymes
(endonucleases) which cut DNA at specific
sequences.
4) Separation of DNA fragments using gel
electrophoresis.
5) The comparison and interpretation of the test
results from the unknown and known samples to
determine whether the known individual is not the source
of the DNA or is included as a possible source of the DNA.
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•DNA profiling can be used in
criminal investigations (murder
or rape) when there is DNA left
at the scene of the crime. A DNA
sample can be taken from the
suspect, profiled and compared
to the DNA found at the scene.
•One difficulty of DNA profiling
can be that cross contamination
of the DNA sample could alter
the results.
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Who is the father?
DNA profiling can be used for
paternity testing, to identify
who the father is of a child.
DNA profiles of the child, the
child’s mother and the man who
might be the father are
compared.
If any bands in the child’s profile
do not occur in the profile of
either the mother or the man,
another person must be the
father.
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B
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D
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NOS: RELIABILITY IS ENHANCED BY
INCREASING THE NUMBER OF MEASUREMENTS
IN AN EXPERIMENT OR TEST.
In DNA profiling, increasing the number of markers used reduces
the probability of a false match.
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D1.1.6—DIRECTIONALITY OF
DNA POLYMERASES (HL
ONLY)
Terminal nucleotides either have a
sugar or a phosphate group that has
not been used to form a covalent bond
with another nucleotide.
• One terminus of a strand is the 3′ end
because the site available for a link to
another nucleotide is the third carbon
of the sugar in the terminal nucleotide.
• The other terminus of the strand is
the 5′ end because the available site is
the phosphate group of the nucleotide
and that is linked to the fifth carbon
atom of the sugar. 24
However many nucleotides
are linked together into a
strand, there will always be a
5′ end and a 3′ end.
The direction in which
DNA polymerase works is
always 5′ to 3′—the 5′
phosphate of a free
nucleotide is linked to the 3′
end of the growing strand.
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D1.1.7—DIFFERENCES BETWEEN
REPLICATION ON THE LEADING STRAND
AND THE LAGGING STRAND (HL ONLY)
DNA polymerase can only assemble new strands in a 5′to 3′
direction. The two strands formed when helicase separates the
DNA are antiparallel—their 5′ to 3′ directions are opposite.
DNA polymerase using these strands as templates for
assembling new strands moves in opposite directions—
always 5′ to 3′.
• On one strand, DNA polymerase adds nucleotides moving
towards the replication fork. Replication is continuous on this
strand so it is completed more quickly. This is called the leading
strand.
• On the other strand, DNA polymerase adds nucleotides moving
away from the replication fork. They must be added in a
series of lengths as the replication fork exposes more of the
template strand and DNA can bind again. These short lengths
of new DNA strand are called Okazaki fragments. Having to
restart replication by DNA polymerase repeatedly makes the
process discontinuous and relatively slow. This is called the
lagging strand. 27
D1.1.8—FUNCTIONS OF DNA PRIMASE, DNA
POLYMERASE I, DNA POLYMERASE III AND
DNA LIGASE IN REPLICATION (HL ONLY)
Many enzymes are involved in DNA replication. Eukaryotic DNA
replication involves 11 different DNA polymerases. We will study
replication in Prokaryotic DNA.
1. DNA Helicase
2. DNA Primase
3. DNA Polymerase III
4. DNA Polymerase I
5. DNA Ligase
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https://www.youtube.com/watch?v=TNKWgcFPHqw&t=8s
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GROWTH IN LIVING ORGANISMS INCLUDES
REPLICATION OF DNA. EXPLAIN DNA
REPLICATION. (8 MARKS)
D1.1.9—DNA
PROOFREADING (HL ONLY)
DNA polymerases replicate DNA with great fidelity. Where errors
are made they are usually corrected, preventing mutations.
There are multiple methods for doing this, the earliest of which is
called “proofreading” and happens during replication immediately
after a nucleotide with a mismatched base has been added to the
growing chain.
In prokaryotes proofreading is done by DNA polymerase III.
When DNA polymerase recognizes a base mismatch between the
last nucleotide it has added to the growing chain and the base on
the template strand, it excises the incorrect nucleotide,
moves back along the template strand by one nucleotide
and inserts a nucleotide with the correct base.
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