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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