Molecular Biology
Done By:
Reem Ghazal
Corrected By:
Reem Ghazal
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Restriction endonucleases• Endonucleases are enzymes that degrade
DNA within the molecule.
• Restriction endonucleases : Bacterial enzymes that recognize and cut (break) the phosphodiester bond between nucleotides at specific sequences (4- to 8-bp restriction sites) generating restriction fragments.
Restriction endonuclease
Restriction site
Restriction fragments-The nuclease degrades
nucleic acids. - Breaks them down into
different fragments **examples on similar
enzymes: -A) lipases: degrades
lipids.-B) esterases: breaks
ester bonds down.
Restriction: means that these enzymes
restrict the growth of bacterial phages/
viruses.
The enzyme cleaves within the restriction
site; generating restriction fragments.
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Palindromic sequences• The sequences recognized by restriction
endonucleases—their sites of action—read the same from left to right as they do from right to left (on the complementary strand).
- Palindromic sequences: the sequences of both the top and bottom strands are exactly the same.- In general, the restrictionendonucleases recognise restriction sites that arecomposed of palindromic sequences.
They recognize specific sequences• The enzyme EcoRI recognizes and cuts within the
sequence (GAATTC).
The DNA stays intact The DNA is cut
into two pieces
Restriction endonucleases are very specific; so if there was a single
change in thesequence within the restriction site, the
restriction endonucleases
won’t cleave within the restriction site.
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Cuts and number of fragments
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• Restriction endonucleases can cut the same DNA strand at
several locations generating multiple restriction fragments
of different lengths.
• What if a location on one strand is not recognized?
DNA sequences can differ and are not identical but highly similar. The sequenceswithin DNA can
differ by one nucleotide/ base.Note: notice that the lengths of the
fragments candiffer or be
exactly the same.
Heterozygous: having two different alleles.
Polymorphism: different DNA sequences among individuals
or groups.
Poly —> multiple, morph —> shape
So; we are talking about multiple shapes of DNA
fragments.
** in the example, the two alleles aredifferent in a
restriction site , so we say that the person isheterozygous for this
specific restriction site.
Variation in the length of fragments: polymorphism.
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DNA polymorphisms• Individual variations in DNA sequence (genetic
variants) may create or remove restriction-enzyme recognition sites generating different restriction fragments.
• Remember:• Our cells are diploid.
• Alleles can be homozygous or heterozygous at any DNA location or sequence.
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• The presence of different DNA forms in individuals generates a restriction fragment length polymorphism, or RFLP.
• Individuals can generate restriction fragments of variable lengths. This is known as molecular fingerprinting.
• These can be detected by gel electrophoresis by itself or along with Southern blotting.
** if we add the same endonucleasesto the DNA of two individuals,
fragments of different lengths will be generated; due to the differences in DNA sequences for both individuals,
this is what we call molecular fingerprinting. Each individual has a special specific molecular fingerprint,
except for identical twins.
Restriction fragment length polymorphismRFLP: restriction fragment length polymorphism.
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Gel electrophoresis only
Homozygous
individual for B
Homozygo
us individual
for A
Heterozyg
ous (A/B)
there are twopossible alleles on aparticular location of
the DNA of these individuals (allele A
and allele B, depending on the
sequence).
Here are three individuals.
The person has the exact allele on both
chromosomes(homozygous for this particular location). So
if a certain endonuclease is
added, it will recognise the two restriction sites on both chromosomes, and will make two cuts on both chromosomes.
If we do gel electrophoresis, they will run according to
size; so the largest (on both chromosomes since they have the
same size) will run the slowest as one band as
in:
** DNA fragments that have similar lengths will get mixed together and
run within the same band mixed. So in the
first individual, we have the generation of six
fragments but in the gel, they look as three
bands (because they have similar lengths).
Homozygous
individual for A
Homozygous
individual for BHeterozygous
(A/B)
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In the second individual, we have generation of eight
fragments, but in the gel there are four bands. (Fragments of
similar lengths will run within the same band ).
And in the third individual, we have the generation of 7
fragments but in the gel, there are 4 bands.
Homozygous
individual for B
Homozygous
individual for A
Heterozygous
(A/B)
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Electrophoresis then Southern blotting• Only DNA fragments that hybridize to the probe
are detected.
W
e
l
l
s
Note: the size of the
DNA detected DNA
fragment reflects its
size, not the size of
the probe
Southern blotting: separating DNA
fragments according to the size, then
transferring them from the gel to a membrane, then adding a probe.
The probe will bind to the fragment, and
will recognise the whole long
fragment,. Theslowest will
appear at the top of gel/ membrane.
The person homozygous for
allele A will only have one
band.
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RFLP in the clinic• RFLP can be used as diagnostic tools.
• For example, if a mutation that results in the development of a disease also causes the generation of distinctive RFLP fragments, then we can tell:
• if the person is diseased as a result of this mutation
• from which parent this allele is inherited 11It can be used
with forensic medicine too; because each individual has
their own molecular
fingerprint.
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Example 1: Disease detection by RFLP(sickle cell anemia)
• Sickle cell anemia is caused by a mutation in one nucleotide (base) in the globin gene that is responsible for making hemoglobin.
• The position of this nucleotide happens to be within a restriction site.
• Individuals can be have
• Homozygous with two normal alleles (designated as A)
• Heterozygous or carriers of one normal allele and one mutated allele (designated as AS)
• Homozygous for the mutated allele, or affected (designated as S)
Normal allele
Mutated allele
Site of
mutationRestriction
sites
Probe
1.15 Kb 0.2 Kb
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1) if a person has a normal allele, the restriction endonuclease will make 3 cuts (the black arrows) and the probe will recognise a
fragment that has 1.15 kB length.
2) If a person has mutation, the restriction endonuclease will not
be able to make a cut at the same locations and the probewill recognise a fragment that
has a length of 1.35 kB.
1.15 Kb 0.2 Kb
Mutation: Both alleles (both chromosomes) must have GTG instead of GAG.
** a carrier (of the disease) would have one normal allele (GAG) and the other allele (GTG).
S stands for sickle.Hb (hemoglobin) is the
oxygen carrier in blood.
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1) If a person has two normal alleles, the restriction endonuclease will cut and if we do southern blotting, the probe will
recognise the fragment that has a length of 1.15 kB.
2) If a person has mutation on both alleles, the restriction endonuclease will not cut at sites of mutation (blue colour) but will cut at the (site of the black arrow at the left)
and probe will recognise 1.35 kB length.
3) If a person is a carrier, the probe will recognise the long
fragment (1.15 kB) and the short fragment (1.35 kB long).
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Example 2: Paternity testingThe DNA of children must come from both parents. So if we do rflp, all DNA
restriction fragments that appear in the child’s
genetic profile must come from both parents
collectively.
The test: we take the DNA of both parents and from
children, then we add restriction endonuclease,
and it will make cuts everywhere; generating
fragments that are specific for each child (molecular
fingerprint).
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We compare the lengths of the child’s DNA fragments to the length/size of mother and
father’s DNA fragments 15
Results:D1: 50% of her fragments are from the mother and the other 50% are from the
father; she is a daughter of both parents.
D2: the daughter of both parents (for the same explanation).
S1: 50% of his fragments are from the father but! The sizes of other 50% of fragments are not the same as the
mother’s; so he is the son of the father but not the mother.
S2: none of his fragments have the same size as the mother’s and father’s;
he is adopted.
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Example 3: Forensics
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RFLP is an old technique (probably not used anymore) that can be used in crimes to
identify the murderer. student club 2020
if there is a blood stain in a crime scene, you can isolate the DNA from this stain and you can do RFLP again
(fragmentation, separation by electrophoresis, transfer to a membrane and you add a probe that is specific to certain
DNA fragments) and then you can see the profile.
** to know the murderer, we must have a total match (the unknown DNA profile will match the suspect’s profile).
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Real cases
We compare the genetic profile of these individualswith the unknown
profile at the crime scene. (Comparing fragments and their
sizes/ lengths).
The first case: the murderer is suspect
1.
Second case: suspect 1 is the culprit.
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