D1.1 DNA replication Flashcards
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.
A replica is an exact copy of something. DNA replication is the synthesis of new strands of DNA with precisely the same base sequence as the original strands.
replication of a DNA molecule yields two identical daughter molecules
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G
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G
A C
T G
G
TI
A A
G
C
G
G
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一
G
1
G
Organisms replicate all their DNA before cell division, so both daughter cells have the entire genome. Unicellular organisms reproduce by cell division. In multicellular organisms, cell division is part of three processes:
* growth, which requires extra body cells to be produced
* replacement of damaged tissues
* reproduction, to provide cells that develop into gametes. DNA replication is thus an example of continuity rather than change. Base sequences pass unchanged from cell to cell in a multicellular organism and from generation to generation by reproduction.
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.
Replication starts with separation of a parent DNA molecule into two single strands, by breaking hydrogen bonds between the bases. Each single strand is then used as a template for the assembly of a new polymer of nucleotides.
black = original strands
mid-grey and light grey = new strands
This is called semi-conservative replication because each of the DNA molecules produced has one new strand and one strand conserved from the parent molecule.
The two DNA molecules produced by replication are identical in base sequence to each other and to the original parent molecule. This is due to complementary base pairing. Adenine only pairs with thymine and cytosine only pairs with guanine. The consequence is that each new strand is complementary to the template strand on which it was made and identical to the other template strand. Complementary base pairing ensures a high degree of accuracy in the copying of base sequences-it is very rare for the wrong base to be inserted.
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.
A replication fork is the site where a parent
DNA molecule is separated into two single strands, each of which is used as a template for the synthesis of a new strand.
The replication fork gradually moves along the parent molecule. The changes that occur at a replication fork are carried out by multiple enzymes working together. The roles of two types of enzyme, helicase and DNA, are shown here.
Stage 1
Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds.
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Stage 2
DNA polymerase links nucleotides together to form new strands, using the pre-existing strands as templates. Each nucleotide in the new strand has the base that is complementary to the base of the nucleotide on the template strand.
Complementary base-pairing ensures that the new strands assembled on each template strand are identical in base sequence to the other template strand.
Stage 3
The daughter DNA molecules each rewind into a double helix.
free nucleotides
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.
PCR (polymerase chain reaction) is used for copying
DNA artificially. It is carried out in small tubes called
eppendorfs, which are loaded into a thermocycler (a
PCR machine). The eppendorfs contain these things:
* DNA sample-this usually contains more DNA than
just the length of DNA molecule that is to be copied.
* Taq DNA polymerase a special type of heat-stable
DNA polymerase. This enzyme is obtained from
Thermus aquaticus, a bacterium adapted to life in
hot springs. It allows high temperatures to be used,
speeding up replication.
* Primers-short DNA strands that bind to DNA in the
sample after it has been split into single strands by heat.
Primers are made with the base sequence needed to
bind at the point where DNA polymerase should attach
to the DNA and start copying. Two primers are needed,
one for each of the two single strands formed when the
double-stranded DNA in the sample is split.
* DNA nucleotides-for assembling the new strands.
PCR happens by a repeated cycle of temperature
changes:
negative DNA sample DNA moves through positive
electrode placed in well the gel towards the
e l e c t r o d e
positive electrode
electrophoresis tank gel fluid
The mesh of polymers in the gel restricts movement,
with small molecules moving faster than larger ones. Gel
electrophoresis therefore separates DNA on the basis
of size of molecule. When the smallest molecules have
nearly reached the positive end of the gel, the current is
switched off and a stain is used to reveal DNA in the gel.
DNA molecules of the same length form a band in the gel.
Five or more different samples are run side-by-side in lanes
across a gel, starting from separate wells. This allows the
pattern of bands to be compared. An example is shown.
a b s c d e
DNA is h e a t e d to
95°C to separate
the two strands.
The temperature is increased to 73°C,
which encourages Taq DNA polymerase
to replicate both strands, starting at the
primer, producing two double-stranded
copies of the original DNA.
The temperature is reduced to 53°C,
which allows primers to bind to
both strands of the DNA next to the
sequence that is to be copied.
There are twice as many copies of the desired DNA base
sequence after each cycle of replication. This increase is
called DNA amplification. By the end of
30-40 rounds of PCR, taking just a few hours, there
could b e more than a billion copies of a desired
sequence in a 0.2 ml eppendorf.
Gel electrophoresis is a method of separating mixtures
of positively or negatively charged macromolecules. The
mixture is placed in a well near one end of a thin sheet of
gel. The gel is a mesh of polymers of an inert material such
as agarose, with fluid-filled spaces between the polymers.
An electric field is applied to the gel by attaching
Gel electrophoresis is particularly useful for separating
human DNA with short tandem repeats. This DNA occurs
electrodes at both ends. Charged macromolecules are
attracted to one or other of the electrodes. DNA moves
towards the positive electrode (the anode) because
phosphate groups in DNA molecules are negatively
charged. The wells into which DNA is loaded are
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at particular positions in the human genome, where
short sequences of three to five bases are repeated. The
number of repeats is very variable between individuals.
For example, at one position on chromosome 7, the
sequence GATA is repeated between 6 and 15 times.
Each different number of repeats forms a separate band
therefore at the end with the negative electrode.
during 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.
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.
PCR and gel electrophoresis are used together to
generate DNA profiles (genetic fingerprints).
1. A DNA sample is taken from a person.
2. Primers are used that promote the simultaneous
amplification of about 15 different short tandem repeats
(STRs) by the polymerase chain reaction.
3. The amplified STRs are separated by gel
electrophoresis, generating a pattern of bands that is
very likely to be unique to the individual.
DNA profiling has a range of applications:
Forensic investigations provide evidence for use in
court cases. DNA profiling can provide very strong
evidence of guilt if a suspect’s DNA profile matches that
of DNA from the crime scene or from a victim’s body.
Only tiny quantities of DNA are needed for profiling,
which could come from a single human hair or traces of
a body fluid.
Paternity tests are used to test whether a man is the
father of a child. DNA profiles for the child, their mother
and the man are needed. All bands in the child’s profile
will also be in the profile of the mother or true father. If
the child has one or more bands not in the mother’s or
the man’s profile, someone else must be the father.
Example: the Enderby double murder case
The first DNA profiles to be used in a forensic
investigation are shown on the image of the
electrophoresis gel on page 40.
Key:
a = hair roots from the first victim,
b = mixed semen and vaginal fluids from the first victim,
c = blood of second victim,
d = vaginal swab from second victim,
e = semen stain on second victim,
s = blood of prime suspect.
Two bands in track b indicated by arrows must be from
DNA in the culprit’s semen but are not present in DNA
from the prime suspect, who was not guilty despite
having confessed to the murders.
D1.1.6—Directionality of DNA polymerasese
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.
The directionality of DNA is explained in Section
Al.2.11. All the nucleotides in a single strand of DNA
are oriented in the same way. This is because the
strand was assembled by DNA polymerase, which
always adds the phosphate of a free nucleotide to
the deoxyribose of the nucleotide at the growing
end of the chain. The direction of replication is 5’
to 3’, because the phosphate group is the 5’ side
of a nucleotide and the deoxyribose is the 3’ end of
the chain.
nucleotides
are not a d d e d to
the 5’ terminal
- T
［
DNA polymerase III
links free nucleotides
to the deoxyribose
at the 3’ terminal
A
free nucleotid
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.
The two strands of a DNA molecule are antiparallel
because they run in opposite directions. The same
enzymes are used to assemble chains of DNA
nucleotides on the two strands and it always begins
with assembly of an RNA primer, but there are also
some significant differences. The two strands are
known as the leading and lagging strands.
Leading strand
An RNA primer is assembled at the start of the leading
strand. As DNA polymerase Ill moves in the same
direction as the replication fork, it can then assemble
any length of new strand. Replication is continuous.
Lagging strand
a new strand of DNA has already been assembled.
fork with another RNA primer. A series of short lengths
about 200 nucleotides long in eukaryotes. Replication
is therefore discontinuous.
DNA polymerase Ill moves away from the replication
fork, adding nucleotides to the growing chain, but it
soon reaches the previous RINA primer, beyond which
Replication has to be reinitiated close to the replication
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of DNA strand are assembled on the lagging strand.
They are known as Okazaki fragments and are only
D1.1.8—Functions of DNA primase, DNA polymerase I, DNA polymerase III and DNA ligase in replication
Limit to the prokaryotic system.
Semi-conservative replication is carried out by a complex system of enzymes. There are differences between
prokaryotes and eukaryotes in the mechanism of replication, though the basic principles are the same. The system
3’5 5’3’
used in prokaryotes is shown below.
On the leading
6. DNA ligase seals up
the nick by making
another sugar- -
phosphate bond
5. DNA polymerase I removes
the RNA primer and replaces it
with DNA, but leaves a nick
because it cannot make a
3 to 5 b o n d
4. DNA polymerase Ill moves
along the lagging strand away
from the replication fork
assembling a strand of DNA
nucleotides, until it reaches
another primer
3. DNA polymerase Il binds - -
to the lagging strand at the 3’ end
of an RNA primer and adds al
DNA nucleotide to it
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2. DNA primase adds a primer
—a short length of RNA .-
attached by base pairing
to the lagging strand
-
1. Helicase unwinds
the double helix and
separates the two
s t r a n d s
7. DNA primase adds a primer
to the 3’ end of the leading
strand, allowing DNA polymerase
Il to bind and start assembling a
strand of DNA nucleotides
Okazaki
fragments
–RNA
primer
8. DNA polymerase moves
along the leading strand in
t h e s a m e direction as t h e
replication fork, so it assembles
a strand of DNA continuously
Direction of
movement of
the replication
fork
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.
DNA polymerase Il usually adds nucleotides correctly
according to the rules of complementary base pairing,
but occasionally an error is made. For example, a free
nucleotide with the base G might be paired with T in the
template strand
G Correct base pairing with
3 hydrogen bonds
G Mispairing with only 1
hydrogen bond
When DNA polymerase Ill recognizes a base mismatch
b e t w e e n t h e last n u c l e o t i d e it h a s a d d e d a n d t h e b a s e o n
the template strand, it excises the incorrect nucleotide,
moves back along the template strand by one nucleotide
and re-inserts a nucleotide with the correct base. This
process is known as DNA proofreading. It greatly
reduces the frequency of mutation during replication.
